Multipartite luciferase peptides and polypeptides

Abstract

To provide tools that can be used to detect and monitor colocalization and / or interaction of molecules, macromolecules, and molecular complexes.SOLUTION: Provided herein are compositions and methods for the assembly of tripartite or multipartite bioluminescent complexes. In particular embodiments, a bioluminescent complex is formed upon the interaction of three or more peptide and / or polypeptide components.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 941,255, filed November 27, 2019, which is hereby incorporated by reference in its entirety.

[0002] Provided herein are bioluminescent polypeptides and compositions and methods for the assembly of tripartite or multipartite bioluminescent complexes. In certain embodiments, bioluminescent complexes are formed when three or more peptide and / or polypeptide components interact. [Background technology]

[0003] Biological processes and analyte detection depend on the colocalization and interactions between molecules, macromolecules, and molecular complexes. To understand such processes and to develop techniques and compounds to manipulate them for research, clinical, and other practical applications, it is necessary to have tools available to detect and monitor these colocalizations / interactions. In particular, studying these interactions under physiological conditions (e.g., at normal expression levels to monitor protein interactions) or within complex sample matrices (e.g., blood samples, environmental samples) requires high sensitivity. Summary of the Invention

[0004] Provided herein are bioluminescent polypeptides and compositions and methods for the assembly of trimolecular or multimolecular bioluminescent complexes. In certain embodiments, bioluminescent complexes are formed when three or more peptide and / or polypeptide components interact.

[0005] Experiments conducted during the development of embodiments herein demonstrate the assembly of bioluminescent complexes capable of producing luminescence in the presence of an appropriate substrate (e.g., coelenterazine or a coelenterazine analog substrate) from complementary polypeptide(s) and peptide(s) that collectively span the length (or greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or more) of the luciferase-based sequence (or collectively comprise at least 40% or more (e.g., greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or more) sequence identity to the luciferase-based sequence). In some embodiments, the "complementary" polypeptide(s) and peptide(s) are separate molecules that each correspond to a portion of the luciferase-based sequence. Due to structural complementarity, they assemble to form a bioluminescent complex.

[0006] Further experiments were carried out in the development of embodiments herein to develop monomeric bioluminescent polypeptides with improved properties (eg, stability, luminescence, etc.).

[0007] In some embodiments, the complementary polypeptide(s) and peptide(s) are fragments of a luciferase-based sequence that assemble to form a bioluminescent complex. In some embodiments, the fragments collectively comprise the full length of the luciferase-based sequence. In some embodiments, the fragments collectively comprise at least 75% (e.g., more than 75% in length, more than 80% in length, more than 85% in length, more than 90% in length, more than 95% in length, or more) of the full length of the luciferase-based sequence.

[0008] In some embodiments, the complementary polypeptide(s) and peptide(s) are variants of portions of a luciferase-based sequence that each contain at least 40% (e.g., greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or more) sequence identity to the corresponding portion of the luciferase-based sequence and assemble to form a bioluminescent complex. In some embodiments, the complementary polypeptide(s) and peptide(s) collectively comprise at least 40% (e.g., greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or more) sequence identity to the entire luciferase-based sequence and are variants of a portion of the luciferase-based sequence that assemble to form a bioluminescent complex. In some embodiments, the fragments collectively comprise the full length of the luciferase-based sequence. In some embodiments, the complementary polypeptide(s) and peptide(s) collectively comprise at least 75% (e.g., greater than 75% length, greater than 80% length, greater than 85% length, greater than 90% length, greater than 95% length, or more) of the full length of the luciferase-based sequence.

[0009] Examples of luciferase-based sequences include SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:788, and SEQ ID NO:789. Some embodiments herein provide polypeptide components that are fragments of a luciferase-based sequence (e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:788, and SEQ ID NO:789) or variants thereof (e.g., greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% sequence identity), as well as one or more complementary peptide(s) and / or polypeptide(s) that collectively span the remainder of the luciferase-based sequence. For example, if the luciferase-based sequence is 170 amino acid residues in length, exemplary polypeptide components can be, e.g., 102, 124, 133, or 148 amino acids in length, and the 1, 2, 3, 4, 5, or more complementary peptides can represent the remaining 68, 46, 37, or 22 amino acids. In some embodiments, each polypeptide component individually comprises at least 40% (e.g., greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or more) sequence identity to the corresponding portion of the luciferase-based sequence.

[0010] In some embodiments, (a) a polypeptide component comprising 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to a polypeptide fragment of SEQ ID NO:788 or SEQ ID NO:789, and (b) one or more complementary peptides, dipeptides, tripeptides, and the like that collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to the complementary portion of SEQ ID NO:788 or SEQ ID NO:789. Provided herein are systems or kits comprising a coelenterazine or coelenterazine analog substrate and / or a polypeptide, wherein in the presence of the coelenterazine or coelenterazine analog substrate, the bioluminescent signal generated by a bioluminescent complex assembled from the polypeptide component and one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides is substantially increased compared to the bioluminescent signal generated by the polypeptide component or one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides and the coelenterazine substrate alone. In some embodiments, the polypeptide components comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 790, and one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 794.In some embodiments, the polypeptide components comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO:791, and one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO:795. In some embodiments, the polypeptide components comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO:792, and one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO:796. In some embodiments, the polypeptide components comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO:793, and one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO:797. In some embodiments, the polypeptide components comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO:790, and one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO:798.In some embodiments, the polypeptide components comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO:791, and one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO:799. In some embodiments, the polypeptide components comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO:792, and one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO:800. In some embodiments, the polypeptide components comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO: 793, and the one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO: 801. In some embodiments, the bioluminescent signal is substantially increased when the polypeptide components associate with the one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides. In some embodiments, the polypeptide components and / or one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides comprise amino acid sequences that are not naturally occurring sequences or fragments thereof.In some embodiments, the polypeptide components and / or one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides comprise unnatural amino acids, amino acid analogs, and / or peptoid amino acids. In some embodiments, the polypeptide components and / or one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides are present as fusions with one or more additional amino acid sequences. In some embodiments, the additional amino acid sequences are selected from the group consisting of a protein of interest, an interacting element, a co-localization element, and a binding moiety. In some embodiments, the additional amino acid sequence is a binding moiety selected from the group consisting of an antibody (polyclonal, monoclonal, and / or recombinant antibody), an antibody fragment, Protein A, the Ig-binding domain of Protein A, Protein G, the Ig-binding domain of Protein G, Protein A / G, the Ig-binding domain of Protein A / G, Protein L, the Ig-binding domain of Protein L, Protein M, the Ig-binding domain of Protein M, an oligonucleotide probe, a peptide nucleic acid, a DARPin, an aptamer, an affimer, a purified protein (either the analyte itself or a protein that binds to the analyte), and an analyte-binding domain(s) of a protein. In some embodiments, the additional amino acid sequence is a first interacting polypeptide configured to form a complex with a second interacting polypeptide upon contact of the first and second interacting polypeptides. In some embodiments, the additional amino acid sequence is a first co-localization polypeptide configured to co-localize with a second co-localization polypeptide within a subcellular compartment, cell, tissue, or organism. In some embodiments, the additional amino acid sequence is a protein of interest and a potential drug target. In some embodiments, provided herein are bioluminescent complexes comprising a polypeptide component of a system or kit described herein and one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides.

[0011] In some embodiments, provided herein are systems or kits comprising two or more peptide, dipeptide, tripeptide, and / or polypeptide components that collectively have 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 788 or SEQ ID NO: 789, wherein in the presence of coelenterazine or a coelenterazine analog substrate, the bioluminescent signal generated by the bioluminescent complex is substantially increased when compared to the bioluminescent signal generated by the polypeptide or one or more complementary peptides and the coelenterazine substrate alone. In some embodiments, the system or kit includes a polypeptide component having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 790, and one or more complementary peptides, dipeptides, and / or tripeptides that collectively have 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 794. In some embodiments, the polypeptide comprises 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 791, and one or more complementary peptides collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 795. In some embodiments, the polypeptide comprises 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 792, and one or more complementary peptides collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 796.In some embodiments, the polypeptide comprises 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 793, and one or more complementary peptides collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 797. In some embodiments, the polypeptide comprises 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 790, and one or more complementary peptides collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 798. In some embodiments, the polypeptide comprises 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 791, and one or more complementary peptides collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 799. In some embodiments, the polypeptide comprises 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 792, and one or more complementary peptides collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 800.In some embodiments, the polypeptide comprises 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 793, and the one or more complementary peptides collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 801. In some embodiments, a bioluminescent signal is substantially increased when the polypeptide associates with the one or more complementary peptides. In some embodiments, the polypeptide and / or one or more complementary peptides comprise an amino acid sequence that is not a naturally occurring sequence or a fragment thereof. In some embodiments, the polypeptide and / or one or more complementary peptides comprise unnatural amino acids, amino acid analogs, and / or peptoid amino acids. In some embodiments, the polypeptide and / or one or more complementary peptides are present as a fusion with one or more additional amino acid sequences. In some embodiments, the additional amino acid sequence is selected from the group consisting of a protein of interest, an interacting element, a co-localization element, and a binding moiety. In some embodiments, the additional amino acid sequence is a binding moiety selected from the group consisting of an antibody (polyclonal, monoclonal, and / or recombinant antibody), an antibody fragment, Protein A, the Ig-binding domain of Protein A, Protein G, the Ig-binding domain of Protein G, Protein A / G, the Ig-binding domain of Protein A / G, Protein L, the Ig-binding domain of Protein L, Protein M, the Ig-binding domain of Protein M, an oligonucleotide probe, a peptide nucleic acid, a DARPin, an aptamer, an affimer, a purified protein (either the analyte itself or a protein that binds to the analyte), and an analyte-binding domain(s) of a protein. In some embodiments, the additional amino acid sequence is a first interacting polypeptide that is configured to form a complex with a second interacting polypeptide upon contact of the first interacting polypeptide and the second interacting polypeptide.In some embodiments, the additional amino acid sequence is a first co-localization polypeptide configured to co-localize with a second co-localization polypeptide within a subcellular compartment, cell, tissue, or organism. In some embodiments, the additional amino acid sequence is a protein of interest and a potential drug target. In some embodiments, provided herein are bioluminescent complexes comprising two or more peptide, dipeptide, tripeptide, and / or polypeptide components of a system or kit described herein.

[0012] In some embodiments, (a) (i) polypeptide components that comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to a polypeptide fragment of SEQ ID NO:788 or SEQ ID NO:789; (ii) polypeptide components that collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to the complementary portion of SEQ ID NO:788 or SEQ ID NO:789. and (iii) a coelenterazine or coelenterazine analog substrate, and (b) detecting luminescence, wherein a higher level of luminescence compared to the level of luminescence produced by the polypeptide components and coelenterazine or coelenterazine analog alone indicates the formation of a bioluminescent complex of the polypeptide components and the one or more complementary peptides. In some embodiments, the polypeptide components and one or more of the first and second peptides are expressed intracellularly, exogenously added to the cell, and / or added to the sample. In some embodiments, (i) the polypeptide component comprises 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 790, and one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 794. or (ii) the polypeptide components collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO: 791 and one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides have 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO: 795.or (iii) the polypeptide components collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 792 and one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides have 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 796. , 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 793, or (iv) the polypeptide components comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 793 and one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides have 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, or (v) the polypeptide components collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO:790 and one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides have 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or more) sequence identity to SEQ ID NO:798. %, 90%, 95%, or more) sequence identity to SEQ ID NO:791; or (vi) the polypeptide components comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO:791 and one or more complementary peptides, dipeptides, tripeptides and / or polypeptides have 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO:799.or (vii) the polypeptide components comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO:792 and one or more complementary peptides, dipeptides, tripeptides and / or polypeptides have 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO:800. or (viii) the polypeptide components comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO:793 and one or more complementary peptides, dipeptides, tripeptides and / or polypeptides collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO:801.

[0013] In some embodiments, provided herein are methods that include (a) combining (i) two or more peptide, dipeptide, tripeptide, and / or polypeptide components that collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to the entire length of SEQ ID NO: 788 or SEQ ID NO: 789, and (ii) a coelenterazine or coelenterazine analog substrate, and (b) detecting luminescence, wherein a higher level of luminescence compared to the level of luminescence produced by the peptide, dipeptide, tripeptide, and / or polypeptide components and the coelenterazine or coelenterazine analog indicates the formation of a bioluminescent complex of the peptide and polypeptide components. In some embodiments, the polypeptide components and one or more of the first and second peptides may be expressed intracellularly, exogenously added to the cell, and / or added to the sample. In some embodiments, (i) the polypeptide components comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO: 790 and one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO: 794, or (ii) the polypeptide components , (iii) the polypeptide components comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO:791, and one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO:795; or (iv) the polypeptide components comprise 40% or more (e.g., 40%,or (iv) the polypeptide components comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO: 793, and one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO: 796; or 797, (v) the polypeptide components comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO: 790, and one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO: 790; or (vi) the polypeptide components comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO: 790. 798, (vi) the polypeptide components comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO: 791; or (vi) the polypeptide components comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO: 791. 799, or (vii) the polypeptide components comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 792; or (vii) the polypeptide components comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 792.or more) sequence identity to SEQ ID NO: 800, wherein one or more complementary peptides, dipeptides, tripeptides and / or polypeptides collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 800, or (viii) the polypeptide components comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 793. 801, wherein one or more complementary peptides, dipeptides, tripeptides and / or polypeptides collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO: 801.

[0014] In some embodiments, a method for detecting an interaction between a first molecular entity and a second molecular entity includes: (a) tagging the first molecular entity with a first peptide, dipeptide, or tripeptide tag; (b) tagging the second molecular entity with a second peptide, dipeptide, or tripeptide tag; (c) combining the tagged first molecular entity and the tagged second molecular entity and / or allowing the tagged first molecular entity and the tagged second molecular entity to contact each other; and (d) adding one or more peptide, dipeptide, tripeptide, and / or polypeptide components, wherein the first peptide, dipeptide, or tripeptide tag, the second peptide, dipeptide, or tripeptide tag, and the second peptide, dipeptide, or tripeptide tag are combined. Provided herein are methods comprising: (a) adding a coelenterazine or coelenterazine analog substrate to a bioluminescent complex, wherein the coelenterazine or coelenterazine analog substrate and the peptide, dipeptide, tripeptide, and / or polypeptide components collectively comprise an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to the entirety of SEQ ID NO: 788 or 789 and are capable of assembling to form a bioluminescent complex; (b) adding a coelenterazine or coelenterazine analog substrate; and (c) detecting a luminescent signal produced by the bioluminescent complex, wherein the magnitude of the luminescent signal correlates with the strength of the interaction between the first molecular entity and the second molecular entity. In some embodiments, the first molecular entity and / or the second molecular entity is a protein of interest or a peptide of interest, and tagging comprises creating a fusion of the first molecular entity and / or the second molecular entity with a first tag and / or a second tag. In some embodiments, the first molecular entity and / or the second molecular entity is a small molecule, and tagging comprises linking the first molecular entity and / or the second molecular entity directly or indirectly to the first tag and / or the second tag.In some embodiments, one of the first molecular entity and the second molecular entity is a drug or drug candidate, and the other is a drug target or drug target candidate, and the bioluminescent signal indicates binding of the drug or drug candidate to the other, which is the drug target or drug target candidate. In some embodiments, combining the tagged first molecular entity and the tagged second molecular entity comprises expressing one or both in a cell and / or adding one or both to a cell.

[0015] In some embodiments, a method for detecting an interaction in a cell between a first protein or peptide entity and a second protein or peptide entity comprises: (a) expressing in the cell a fusion comprising the first protein or peptide entity and a first peptide, dipeptide, or tripeptide tag comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to a first portion of SEQ ID NO: 788 or 789; (b) expressing in the cell a fusion comprising the second protein or peptide entity and a second peptide, dipeptide, or tripeptide tag comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to a second portion of SEQ ID NO: 788 or 789; (c) expressing in the cell a fusion comprising the second protein or peptide entity and a second peptide, dipeptide, or tripeptide tag comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to a third portion of SEQ ID NO: 788 or 789. and expressing in a cell one or more peptide, dipeptide, tripeptide, and / or polypeptide components comprising an amino acid sequence having 40% or more (e.g., 80%, 85%, 90%, 95%, or more) sequence identity to the entirety of SEQ ID NO: 788 or 789, wherein the first tag, the second tag, and the components collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to the entirety of SEQ ID NO: 788 or 789, to form the first protein or peptide entity. and a second protein or peptide entity to generate a bioluminescent complex; (d) adding a coelenterazine or coelenterazine analog substrate to the cell; and (e) detecting a luminescent signal generated by the bioluminescent complex, wherein the magnitude of the luminescent signal correlates with the strength of the interaction between the first protein or peptide entity and the second protein or peptide entity.

[0016] In some embodiments, a method of detecting co-localization of a first molecular entity and a second molecular entity comprises: (a) tagging the first molecular entity with a first peptide, dipeptide, or tripeptide tag comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to a first portion of SEQ ID NO: 788 or 789; (b) tagging the second molecular entity with a first peptide, dipeptide, or tripeptide tag comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to a second portion of SEQ ID NO: 788 or 789; (c) combining the tagged first molecular entity and the tagged second molecular entity in the same system; (d) adding one or more peptide, dipeptide, tripeptide, and / or polypeptide components to the system, wherein the components have 40% or more sequence identity with a third portion of SEQ ID NO: 788 or 789. (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity, and the first tag, the second tag, and the components collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to the entirety of SEQ ID NO: 788 or 789, and the first peptide tag, the second peptide tag, and the components are complementary to the first molecular entity and the second molecular entity. (e) adding a coelenterazine or coelenterazine analog substrate to the system, and (f) detecting a luminescent signal produced by the bioluminescent complex, wherein the presence of a luminescent signal above background indicates colocalization of the first molecular entity and the second molecular entity in the system and / or the magnitude of the luminescent signal correlates with the amount of colocalization of the first molecular entity and the second molecular entity in the system.Provided herein are methods comprising: In some embodiments, the system comprises a cell, tissue, organ, whole organism, and / or biochemical non-cellular sample; In some embodiments, the first molecular entity and / or the second molecular entity is a protein of interest or a peptide of interest, and tagging comprises creating a fusion of the first molecular entity and / or the second molecular entity with a first tag and / or a peptide tag; In some embodiments, the first molecular entity and / or the second molecular entity is a small molecule, and tagging comprises directly or indirectly linking the first molecular entity and / or the second molecular entity to the first tag and / or the second tag; In some embodiments, combining the tagged first molecular entity and the tagged second molecular entity comprises expressing one or both in the system and / or adding one or both to the system;

[0017] In some embodiments, a method for detecting co-localization within a cell of a first protein or peptide entity and a second protein or peptide entity comprises: (a) expressing in the cell a fusion comprising the first protein or peptide entity and a first peptide, dipeptide, or tripeptide tag comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to a first portion of SEQ ID NO: 788 or 789; (b) expressing in the cell a fusion comprising the second protein or peptide entity and a second peptide, dipeptide, or tripeptide tag comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to a second portion of SEQ ID NO: 788 or 789; (c) expressing in the cell a fusion comprising the second protein or peptide entity and a second peptide, dipeptide, or tripeptide tag comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to a third portion of SEQ ID NO: 788 or 789; and expressing in a cell one or more peptide, dipeptide, tripeptide, or polypeptide components having 40% or more (e.g., 85%, 90%, 95%, or more) sequence identity to the entirety of SEQ ID NO: 788 or 789, wherein the first tag, the second tag, and the components collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to the entirety of SEQ ID NO: 788 or 789, and wherein the first tag, the second tag, and the components are integrated into the first protein or (d) adding a coelenterazine or coelenterazine analog substrate to the cell; and (e) detecting a luminescent signal produced by the bioluminescent complex, wherein the presence of a luminescent signal above background indicates co-localization of the first protein or peptide entity and the second protein or peptide entity within the cell, and / or the magnitude of the luminescent signal is indicative of co-localization of the first protein or peptide entity and the second protein or peptide entity within the cell.Provided herein are methods comprising said detecting, which correlates with the amount of co-localization of said first protein or peptide entity and said second protein or peptide entity in a system.

[0018] In some embodiments, there is provided a method for detecting a target molecule, wherein the target molecule exhibits a first antigen, epitope, or sequence and a distinct second antigen, epitope, or sequence, the method comprising: (a) contacting a sample containing the target molecule with (i) a first primary binding moiety that recognizes the first antigen, epitope, or sequence and (ii) a second primary binding moiety that recognizes the second antigen, epitope, or sequence, and allowing the first and second primary binding moieties to bind to the first and second antigens, epitopes, or sequences; (b) contacting the sample with a first primary binding moiety that recognizes the first antigen, epitope, or sequence and a second primary binding moiety that recognizes the second antigen, epitope, or sequence; with (i) a first secondary binding moiety conjugated to a first tag and (ii) a second secondary binding moiety conjugated to a second tag, wherein the first secondary binding moiety recognizes the first primary binding moiety, the second secondary binding moiety recognizes the second primary binding moiety, and the first or second tag has an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to the first and second moieties of SEQ ID NO: 788 or 789. (c) allowing the first and second secondary binding moieties to bind to the first and second primary binding moieties; (d) contacting the sample with one or more peptide, dipeptide, tripeptide, and / or polypeptide components having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to a third portion of SEQ ID NO: 788 or 789, wherein the first tag, the second tag, and the components are 788 or 789, collectively comprising 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity with the entirety of SEQ ID NO: 788 or 789, wherein the first tag, the second tag, and the components are configured to generate a bioluminescent complex upon interaction; (d) contacting the sample with coelenterazine or a coelenterazine analog substrate; and (e) detecting a luminescent signal generated by the bioluminescent complex;Provided herein are methods comprising detecting, wherein the presence of a luminescent signal above background indicates the presence of a target molecule and / or the magnitude of the luminescent signal correlates with the amount of the target molecule in the sample. In some embodiments, the binding moieties are independently selected from the group consisting of antibodies (polyclonal, monoclonal, and / or recombinant antibodies), antibody fragments, Protein A, the Ig-binding domain of Protein A, Protein G, the Ig-binding domain of Protein G, Protein A / G, the Ig-binding domain of Protein A / G, Protein L, the Ig-binding domain of Protein L, Protein M, the Ig-binding domain of Protein M, oligonucleotide probes, peptide nucleic acids, DARPins, aptamers, affimers, purified proteins (either the analyte itself or a protein that binds to the analyte), and analyte-binding domain(s) of a protein. In some embodiments, the target molecule is a protein, nucleic acid, or small molecule. In some embodiments, the sample is in vitro or in vivo.

[0019] In some embodiments, a method of detecting a target molecule, wherein the target molecule exhibits a first antigen, epitope, or sequence and a distinct second antigen, epitope, or sequence, comprises: (a) contacting a sample with (i) a first binding moiety conjugated to a first tag and (ii) a second binding moiety conjugated to a second tag, wherein the first secondary binding moiety recognizes the first antigen, epitope, or sequence, the second binding moiety recognizes the second antigen, epitope, or sequence, and the first tag is represented by SEQ ID NO: 788 or 789, wherein the second tag comprises an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to a first portion of SEQ ID NO: 788 or 789; (b) allowing the first and second binding moieties to bind to the first and second antigens, epitopes, or sequences; (c) contacting the sample with a third tag having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to a first portion of SEQ ID NO: 788 or 789; and contacting the first tag, the second tag, and the component with a peptide, dipeptide, tripeptide, or polypeptide component having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to the entirety of SEQ ID NO: 788 or 789, wherein the first tag, the second tag, and the component collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to the entirety of SEQ ID NO: 788 or 789, and Provided herein are methods comprising: (a) contacting the second tag and the component, wherein the second tag and the component are configured to generate a bioluminescent complex upon interaction; (d) contacting the sample with a coelenterazine or coelenterazine analog substrate; and (e) detecting a luminescent signal generated by the bioluminescent complex, wherein the presence of a luminescent signal above background indicates the presence of a target molecule and / or the magnitude of the luminescent signal correlates with the amount of the target molecule in the sample.In some embodiments, the binding moieties are independently selected from the group consisting of antibodies (polyclonal, monoclonal, and / or recombinant antibodies), antibody fragments, Protein A, the Ig-binding domain of Protein A, Protein G, the Ig-binding domain of Protein G, Protein A / G, the Ig-binding domain of Protein A / G, Protein L, the Ig-binding domain of Protein L, Protein M, the Ig-binding domain of Protein M, oligonucleotide probes, peptide nucleic acids, DARPins, aptamers, affimers, purified proteins (either the analyte itself or a protein that binds to the analyte), and analyte-binding domain(s) of a protein. In some embodiments, the target molecule is a protein, nucleic acid, or small molecule. In some embodiments, the sample is an in vitro sample, an in vivo sample, or a biochemical sample.

[0020] In some embodiments, provided herein are peptides, dipeptides, tripeptides, and / or polypeptides listed in Table 1, Table 9, or Table 10. In some embodiments, a single peptide, dipeptide, tripeptide, or polypeptide listed in Table 1, Table 9, or Table 10 is provided (e.g., as a reagent, as a tag, etc.). In some embodiments, a pair (two) or set (e.g., two, three, four, five, or more) of peptides, dipeptides, tripeptides, and / or polypeptides listed in Table 1, Table 9, or Table 10 are provided. In particular, pairs or sets of peptides, dipeptides, tripeptides, and / or polypeptides are provided that are complementary and capable of forming a bioluminescent complex upon interacting with each other (e.g., facilitated, non-facilitated).

[0021] In some embodiments, the polypeptide components are 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) of the polypeptides of SEQ ID NOs: 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 117, 119, 121, 123, 125, 127, 129, 131, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638 , 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 72 7, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 802, 804, 806, 808, 813, 815, or 829. In some embodiments, 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) of the amino acids in SEQ ID NOs: 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943 , 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 117, 119, 121, 123, 125, 127, 129, 131, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630,632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 733, 734, 735, 736, 737, 738, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 770, 772, 773, 774, 775, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 34, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 802, 804, 806, 808, 813, 815, or 829, provided by one or more peptides or dipeptides capable of forming a bioluminescent complex. In some embodiments, SEQ ID NOs: 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 157, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 19, 121, 123, 125, 127, 129, 131, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 65 4, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726 , 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 802, 804, 806, 808, 813, 815,or 829, having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to one of SEQ ID NOs: 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 117, 119, 121, 123, 125, 127, 129, 131, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640 , 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 72 Suitable fragments of the polypeptides of 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 802, 804, 806, 808, 813, 815, or 829 are provided. In some embodiments, such fragments are capable of forming bioluminescent conjugates with combinations of suitable peptides, dipeptides, tripeptides, polypeptides, etc., provided herein.

[0022] In some embodiments, the peptide components comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to one of SEQ ID NOs: 900-907. In some embodiments, a polypeptide comprising 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to one of SEQ ID NOs: 900-907 is provided by one or more peptides, dipeptides, tripeptides, polypeptides, etc. that are capable of forming a bioluminescent complex.

[0023] In some embodiments, provided herein are peptides, dipeptides, tripeptides, and / or polypeptides having at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to one or more of the peptides, dipeptides, tripeptides, and / or polypeptides listed in Table 1, Table 9, or Table 10. In some embodiments, a single peptide, dipeptide, tripeptide, or polypeptide is provided (e.g., as a reagent, as a tag, etc.) having at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to one or more of the peptides, dipeptides, tripeptides, and / or polypeptides listed in Table 1, Table 9, or Table 10. In some embodiments, provided are pairs (two) or sets (e.g., two, three, four, five, or more) of peptides, dipeptides, tripeptides, and / or polypeptides that have at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity with one or more of the peptides, dipeptides, tripeptides, and / or polypeptides listed in Table 1, Table 9, or Table 10. In particular, provided are pairs or sets of peptides, dipeptides, tripeptides, and / or polypeptides that are complementary and capable of forming a bioluminescent complex upon interacting (e.g., facilitated, unfacilitated) with each other.

[0024] In some embodiments, provided herein are polypeptides comprising 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to one of SEQ ID NOs: 790, 791, 792, or 793. In some embodiments, the polypeptides are provided alone or as pairs / sets with complementary peptide(s), dipeptide(s), and / or tripeptides for formation of a bioluminescent complex. In some embodiments, provided are fusions of the polypeptides herein with proteins of interest, interacting elements, co-localization elements, etc. In some embodiments, nucleic acids and vectors encoding the polypeptides and fusions thereof are provided.

[0025] In some embodiments, provided herein are peptides comprising SEQ ID NOs: 817, 818, 819, 13, 15, 23, or 25. In some embodiments, provided herein are peptides comprising 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to one of SEQ ID NOs: 817, 818, 819, 13, 15, 23, or 25. In some embodiments, peptides are provided alone or as pairs / sets with complementary polypeptides and / or other peptide(s), dipeptide(s), and / or tripeptides for formation of a bioluminescent complex. In some embodiments, provided are fusions of the peptides herein with proteins of interest, interacting elements, co-localization elements, etc. In some embodiments, provided are nucleic acids and vectors encoding the peptides and fusions thereof. In some embodiments, molecules of interest and / or proteins of interest are tagged with the peptides herein.

[0026] In some embodiments, provided herein are β6-7-like dipeptides comprising SEQ ID NOs: 817 and 818. In some embodiments, provided herein are β6-7-like dipeptides having 40% or greater (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater) sequence identity to SEQ ID NOs: 817 and 818. In some embodiments, dipeptides are provided alone or as pairs / sets with complementary polypeptides and / or other peptide(s), dipeptide(s), and / or tripeptides for formation of a bioluminescent complex. In some embodiments, nucleic acids and vectors encoding dipeptides and fusions thereof are provided. In some embodiments, molecules of interest and / or proteins of interest are tagged with the dipeptides herein.

[0027] In some embodiments, provided herein are β7-8-like dipeptides comprising SEQ ID NOs: 818 and 819. In some embodiments, provided herein are β7-8-like dipeptides having 40% or greater (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater) sequence identity to SEQ ID NOs: 818 and 819. In some embodiments, the dipeptides are provided alone or as pairs / sets with complementary polypeptides and / or other peptide(s), dipeptides, and / or tripeptides for the formation of bioluminescent complexes. In some embodiments, the dipeptides are provided alone or as pairs / sets with complementary polypeptides and / or other peptide(s), dipeptides, and / or tripeptides for the formation of bioluminescent complexes. In some embodiments, nucleic acids and vectors encoding the dipeptides and fusions thereof are provided. In some embodiments, the molecule of interest and / or protein of interest is tagged with a dipeptide herein.

[0028] In some embodiments, provided herein is a β8-9-like dipeptide comprising SEQ ID NO: 819 / 23 or 819 / 25. In some embodiments, provided herein is a β8-9-like dipeptide having 40% or greater (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater) sequence identity to SEQ ID NO: 819 / 23 or 819 / 25. In some embodiments, the dipeptide is provided alone or as a pair / set with complementary polypeptides and / or other peptide(s), dipeptide(s), and / or tripeptides for the formation of a bioluminescent complex. In some embodiments, the dipeptide is provided alone or as a pair / set with complementary polypeptides and / or other peptide(s), dipeptide(s), and / or tripeptides for the formation of a bioluminescent complex. In some embodiments, nucleic acids and vectors encoding the dipeptides and fusions thereof are provided. In some embodiments, the molecule of interest and / or protein of interest is tagged with a dipeptide herein.

[0029] In some embodiments, provided herein are β9-10-like dipeptides comprising SEQ ID NO: 23 / 13, 23 / 15, 25 / 13, or 25 / 15. In some embodiments, provided herein are β8-9-like dipeptides having 40% or greater (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater) sequence identity to SEQ ID NO: 23 / 13, 23 / 15, 25 / 13, or 25 / 15. In some embodiments, dipeptides are provided alone or as pairs / pairs with complementary polypeptides and / or other peptide(s), dipeptides, and / or tripeptides for the formation of bioluminescent complexes. In some embodiments, dipeptides are provided alone or as pairs / pairs with complementary polypeptides and / or other peptide(s), dipeptides, and / or tripeptides for the formation of bioluminescent complexes. In some embodiments, nucleic acids and vectors encoding the dipeptides and fusions thereof are provided, hi some embodiments, molecules of interest and / or proteins of interest are tagged with the dipeptides herein.

[0030] In some embodiments, provided herein are β6-8-like tripeptides comprising SEQ ID NOs: 817-819. In some embodiments, provided herein are β6-8-like tripeptides having 40% or greater (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater) sequence identity to SEQ ID NOs: 817-819. In some embodiments, the tripeptides are provided alone or as pairs / sets with complementary polypeptides and / or other peptide(s), dipeptide(s), and / or tripeptides for the formation of bioluminescent complexes. In some embodiments, the tripeptides are provided alone or as pairs / sets with complementary polypeptides and / or other peptide(s), dipeptide(s), and / or tripeptides for the formation of bioluminescent complexes. In some embodiments, nucleic acids and vectors encoding the tripeptides and fusions thereof are provided. In some embodiments, the molecule of interest and / or protein of interest is tagged with the tripeptide herein.

[0031] In some embodiments, provided herein are β7-9-like tripeptides comprising SEQ ID NO: 818 / 819 / 23 or 818 / 819 / 25. In some embodiments, provided herein are β7-9-like tripeptides having 40% or greater (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater) sequence identity to SEQ ID NO: 818 / 819 / 23 or 818 / 819 / 25. In some embodiments, the tripeptides are provided alone or as pairs / pairs with complementary polypeptides and / or other peptide(s), dipeptides, and / or tripeptides for the formation of a bioluminescent complex. In some embodiments, the tripeptides are provided alone or as pairs / pairs with complementary polypeptides and / or other peptide(s), dipeptides, and / or tripeptides for the formation of a bioluminescent complex. In some embodiments, nucleic acids and vectors encoding the tripeptides and fusions thereof are provided, hi some embodiments, molecules of interest and / or proteins of interest are tagged with the tripeptides herein.

[0032] In some embodiments, provided herein are β8-10-like tripeptides comprising SEQ ID NOs: 819 / 23 / 13, 819 / 23 / 15, 819 / 25 / 13, or 819 / 25 / 15. In some embodiments, provided herein are β7-9-like tripeptides having 40% or greater (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater) sequence identity to SEQ ID NOs: 819 / 23 / 13, 819 / 23 / 15, 819 / 25 / 13, or 819 / 25 / 15. In some embodiments, the tripeptides are provided alone or as pairs / pairs with complementary polypeptides and / or other peptide(s), dipeptide(s), and / or tripeptides for formation of a bioluminescent complex. In some embodiments, tripeptides are provided alone or in pairs / sets with complementary polypeptides and / or other peptide(s), dipeptides, and / or tripeptides for the formation of bioluminescent complexes. In some embodiments, tripeptides are provided alone or in pairs / sets with complementary polypeptides and / or other peptide(s), dipeptides, and / or tripeptides for the formation of bioluminescent complexes. In some embodiments, nucleic acids and vectors encoding the tripeptides and fusions thereof are provided. In some embodiments, molecules of interest and / or proteins of interest are tagged with the tripeptides herein.

[0033] In some embodiments, provided herein are peptides comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO:23 and less than 100% sequence identity to SEQ ID NO:6 and SEQ ID NO:9, wherein the bioluminescent signal generated in the presence of a coelenterazine or coelenterazine analog substrate when the peptide is contacted with a second peptide consisting of SEQ ID NO:25 and a polypeptide complement consisting of SEQ ID NO:17, SEQ ID NO:21, or SEQ ID NO:302 is substantially increased compared to the bioluminescent signal generated by the peptide and the coelenterazine or coelenterazine analog substrate alone. In some embodiments, the bioluminescent signal is substantially increased when the peptide is associated with the second peptide and the polypeptide complement. In some embodiments, the peptide exhibits one or more enhanced traits compared to the peptides of SEQ ID NO:6 and / or SEQ ID NO:9, wherein the traits are selected from affinity for a second peptide and polypeptide complement, or enhanced expression, solubility, stability, and / or bioluminescent activity when combined with a second peptide and polypeptide complement. In some embodiments, the amino acid sequence is not a naturally occurring protein (e.g., not SEQ ID NO:1), not a variant version of a naturally occurring protein (e.g., not SEQ ID NO:3), not a fragment of a naturally occurring protein (e.g., not SEQ ID NOs:5-7), and not a fragment of a variant version of a naturally occurring protein (e.g., not one of SEQ ID NOs:8-10). In some embodiments, the amino acid sequence contains unnatural amino acids, amino acid analogs, and / or peptoid amino acids. In some embodiments, the peptide is chemically conjugated to a linker, a reactive moiety, a detection element (e.g., a fluorophore), an interaction / binding element, etc.

[0034] In some embodiments, provided herein is a fusion polypeptide (e.g., genetic fusion (or alternatively, chemical conjugation or synthetically produced)) comprising a peptide described in the previous paragraph and an additional amino acid sequence or compound (e.g., a small molecule drug). In some embodiments, the additional amino acid sequence is selected from the group consisting of a protein of interest, an interacting element, a co-localization element, and / or a binding moiety. In some embodiments, the additional amino acid sequence is a binding moiety selected from the group consisting of an antibody (polyclonal, monoclonal, and / or recombinant antibody), an antibody fragment, Protein A, an Ig-binding domain of Protein A, Protein G, an Ig-binding domain of Protein G, Protein A / G, an Ig-binding domain of Protein A / G, Protein L, an Ig-binding domain of Protein L, Protein M, an Ig-binding domain of Protein M, a peptide nucleic acid, a DARPin, an Affimer, a purified protein (e.g., an analyte or a protein that binds to an analyte), and an analyte-binding domain(s) of a protein. In some embodiments, the additional amino acid sequence is a first interacting polypeptide configured to form a complex with a second interacting polypeptide upon contact of the first and second interacting polypeptides. In some embodiments, the additional amino acid sequence is a first co-localizing polypeptide configured to co-localize with a second co-localizing polypeptide within a subcellular compartment, cell, tissue, or organism. In some embodiments, the additional amino acid sequence is a protein of interest and a potential drug target.

[0035] In some embodiments, provided herein are peptides comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO:25 and less than 100% sequence identity to SEQ ID NO:7 and SEQ ID NO:10, wherein when the peptide is contacted with a second peptide consisting of SEQ ID NO:23 and a polypeptide complement consisting of SEQ ID NO:17, SEQ ID NO:21, or SEQ ID NO:302, the bioluminescent signal generated in the presence of a coelenterazine or coelenterazine analog substrate is substantially increased compared to the bioluminescent signal generated by the peptide and the coelenterazine or coelenterazine analog substrate alone. In some embodiments, the bioluminescent signal is substantially increased when the peptide is associated with the second peptide and the polypeptide complement. In some embodiments, the peptide exhibits one or more enhanced traits compared to the peptides of SEQ ID NO:7 and / or SEQ ID NO:10, wherein the traits are selected from affinity for a second peptide and polypeptide complement, or enhanced expression, solubility, stability, and bioluminescence activity when combined with a second peptide and polypeptide complement. In some embodiments, the amino acid sequence is not a naturally occurring protein (e.g., not SEQ ID NO:1), not a variant version of a naturally occurring protein (e.g., not SEQ ID NO:3), not a fragment of a naturally occurring protein (e.g., not SEQ ID NOs:5-7), and not a fragment of a variant version of a naturally occurring protein (e.g., not one of SEQ ID NOs:8-10). In some embodiments, the amino acid sequence contains unnatural amino acids, amino acid analogs, and / or peptoid amino acids. In some embodiments, the peptide is chemically conjugated to a linker, a reactive moiety, a detection element (e.g., a fluorophore), an interaction / binding element, etc.

[0036] In some embodiments, provided herein are fusion polypeptides (e.g., genetic fusions, synthetically produced fusions, chemical conjugates, enzymatic conjugates, etc.) comprising a peptide described in the previous paragraph and an additional amino acid sequence. In some embodiments, the additional amino acid sequence is selected from the group consisting of a protein of interest, an interacting element, a co-localization element, and a binding moiety. In some embodiments, the additional amino acid sequence is a binding moiety independently selected from the group consisting of an antibody (polyclonal, monoclonal, and / or recombinant antibody), an antibody fragment, Protein A, an Ig-binding domain of Protein A, Protein G, an Ig-binding domain of Protein G, Protein A / G, an Ig-binding domain of Protein A / G, Protein L, an Ig-binding domain of Protein L, Protein M, an Ig-binding domain of Protein M, a peptide nucleic acid, a DARPin, an Affimer, a purified protein (either the analyte itself or a protein that binds to the analyte), and an analyte-binding domain(s) of a protein. In some embodiments, the additional amino acid sequence is a first interacting polypeptide configured to form a complex with a second interacting polypeptide upon contact of the first and second interacting polypeptides. In some embodiments, the additional amino acid sequence is a first co-localizing polypeptide configured to co-localize with a second co-localizing polypeptide within a subcellular compartment, cell, tissue, or organism. In some embodiments, the additional amino acid sequence is a protein of interest and a potential drug target.

[0037] In some embodiments, the first peptide comprises (a) an amino acid sequence having more than 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) but less than 100% sequence identity with SEQ ID NO:25 and less than 100% sequence identity with SEQ ID NO:7 and SEQ ID NO:10, and (b) a first peptide comprising more than 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) but less than 100% sequence identity with SEQ ID NO:23, and less than 100% sequence identity with SEQ ID NO:24. Provided herein are compositions comprising SEQ ID NO:6, SEQ ID NO:9, and a second peptide comprising an amino acid sequence having less than 100% sequence identity to SEQ ID NO:29, wherein when the first peptide is contacted with the second peptide and a polypeptide complement consisting of SEQ ID NO:17, SEQ ID NO:21, or SEQ ID NO:302, the bioluminescent signal produced in the presence of coelenterazine or a coelenterazine analog substrate is substantially increased compared to the bioluminescent signal produced by the first peptide and / or the second peptide and the coelenterazine substrate alone. In some embodiments, the bioluminescent signal is substantially increased when the first peptide is associated with the second peptide and the polypeptide complement. In some embodiments, the first peptide exhibits one or more enhanced traits compared to the peptides of SEQ ID NO:7 and / or SEQ ID NO:10, and the second peptide exhibits one or more enhanced traits compared to the peptides of SEQ ID NO:6, SEQ ID NO:9, and SEQ ID NO:29, wherein the traits are selected from affinity for the second peptide and its polypeptide complement, or enhanced expression, solubility, stability, and bioluminescence activity when combined with the second peptide and its polypeptide complement. In some embodiments, the amino acid sequence of the first and / or second peptide is not a naturally occurring protein or fragment thereof. In some embodiments, the amino acid sequence of the first and / or second peptide contains unnatural amino acids, amino acid analogs, and / or peptoid amino acids.

[0038] In some embodiments, provided herein are compositions comprising a fusion polypeptide comprising the first and second peptides described in the previous paragraph and an additional amino acid sequence. In some embodiments, the additional amino acid sequence is selected from the group consisting of a protein of interest, an interacting element, a co-localization element, and a binding moiety. In some embodiments, the additional amino acid sequence is a binding moiety independently selected from the group consisting of an antibody (polyclonal, monoclonal, and / or recombinant antibody), an antibody fragment, Protein A, the Ig-binding domain of Protein A, Protein G, the Ig-binding domain of Protein G, Protein A / G, the Ig-binding domain of Protein A / G, Protein L, the Ig-binding domain of Protein L, Protein M, the Ig-binding domain of Protein M, a peptide nucleic acid, a DARPin, an Affimer, a purified protein (either the analyte itself or a protein that binds to the analyte), and an analyte-binding domain(s) of a protein. In some embodiments, the additional amino acid sequence is a first interacting polypeptide configured to form a complex with a second interacting polypeptide upon contact of the first interacting polypeptide and the second interacting polypeptide. In some embodiments, the additional amino acid sequence is a first co-localization polypeptide configured to co-localize with a second co-localization polypeptide in a subcellular compartment, cell, tissue, or organism, hi some embodiments, the additional amino acid sequence is a protein of interest and a potential drug target.

[0039] In some embodiments, provided herein is a polypeptide comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO: 17, SEQ ID NO: 21, or SEQ ID NO: 302 and less than 100% sequence identity to SEQ ID NO: 5 and SEQ ID NO: 8, wherein when the polypeptide is contacted with a first peptide consisting of SEQ ID NO: 23 and a second peptide consisting of SEQ ID NO: 25, the bioluminescent signal generated in the presence of a coelenterazine or coelenterazine analog substrate is substantially increased compared to the bioluminescent signal generated by the peptides and the coelenterazine or coelenterazine analog substrate alone. In some embodiments, the bioluminescent signal is substantially increased when the polypeptide is associated with the first and second peptides. In some embodiments, the polypeptide exhibits one or more enhanced traits compared to the polypeptide of SEQ ID NO:5 and / or SEQ ID NO:8, wherein the traits are selected from affinity for the first and / or second peptide, or enhanced expression, solubility, stability, and / or bioluminescence activity when combined with the first and second peptides. In some embodiments, the amino acid sequence is not a naturally occurring protein (e.g., not SEQ ID NO:1), is not a variant version of a naturally occurring protein (e.g., not SEQ ID NO:3), is not a fragment of a naturally occurring protein (e.g., not SEQ ID NOs:5-7), and is not a fragment of a variant version of a naturally occurring protein (e.g., not one of SEQ ID NOs:8-10). In some embodiments, the amino acid sequence contains unnatural amino acids, amino acid analogs, and / or peptoid amino acids.

[0040] In some embodiments, provided herein are fusion polypeptides (e.g., genetic fusions, synthetically produced fusions, chemical conjugates, enzymatic conjugates, etc.) comprising a polypeptide described in the previous paragraph and an additional amino acid sequence, nucleic acid sequence, or other fused or added molecule. In some embodiments, the additional sequence or other molecule is selected from the group consisting of a protein of interest, an interacting element, a co-localization element, and a binding moiety. In some embodiments, the additional sequence or other molecule is a binding moiety selected from the group consisting of an antibody (polyclonal, monoclonal, and / or recombinant antibody), an antibody fragment, Protein A, an Ig-binding domain of Protein A, Protein G, an Ig-binding domain of Protein G, Protein A / G, an Ig-binding domain of Protein A / G, Protein L, an Ig-binding domain of Protein L, Protein M, an Ig-binding domain of Protein M, a peptide nucleic acid, a DARPin, an Affimer, a purified protein (either the analyte itself or a protein that binds to the analyte), and an analyte-binding domain(s) of a protein. In some embodiments, the additional sequence or other fused or added molecule is a first interacting polypeptide configured to form a complex with a second interacting polypeptide upon contact of the first and second interacting polypeptides. In some embodiments, the additional sequence or other fused or added molecule is a first co-localizing polypeptide configured to co-localize with a second co-localizing polypeptide within a subcellular compartment, cell, tissue, or organism. In some embodiments, the additional sequence or other fused or added molecule is a protein of interest and a potential drug target.

[0041] In some embodiments, provided herein is a polypeptide comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO: 17, SEQ ID NO: 21, and / or SEQ ID NO: 302 and less than 100% sequence identity to SEQ ID NO: 5 and SEQ ID NO: 8, wherein when the polypeptide is contacted with a first peptide consisting of SEQ ID NO: 23 and a second peptide consisting of SEQ ID NO: 25, the bioluminescent signal generated in the presence of a coelenterazine or coelenterazine analog substrate is substantially increased compared to the bioluminescent signal generated by the peptides and the coelenterazine or coelenterazine analog substrate alone. In some embodiments, the bioluminescent signal is substantially increased when the polypeptide is associated with the first and second peptides. In some embodiments, the polypeptide exhibits enhanced one or more traits compared to the polypeptide of SEQ ID NO:5 and / or SEQ ID NO:8, wherein the traits are selected from affinity for the first and / or second peptide, or enhanced expression, solubility, stability, and / or bioluminescent activity when combined with the first and second peptide. In some embodiments, the amino acid sequence is not a naturally occurring protein (e.g., not SEQ ID NO:1), is not a variant version of a naturally occurring protein (e.g., not SEQ ID NO:3), is not a fragment of a naturally occurring protein (e.g., not SEQ ID NOs:5-7), and is not a fragment of a variant version of a naturally occurring protein (e.g., not one of SEQ ID NOs:8-10). In some embodiments, the amino acid sequence contains unnatural amino acids, amino acid analogs, and / or peptoid amino acids.

[0042] In some embodiments, provided herein are fusion polypeptides (e.g., genetic fusions, synthetically produced fusions, chemical conjugates, enzymatic conjugates, etc.) comprising a peptide described in the previous paragraph and an additional amino acid sequence. In some embodiments, the additional amino acid sequence is selected from the group consisting of a protein of interest, an interacting element, a co-localization element, and a binding moiety. In some embodiments, the additional amino acid sequence is a binding moiety independently selected from the group consisting of an antibody (polyclonal, monoclonal, and / or recombinant antibody), an antibody fragment, Protein A, an Ig-binding domain of Protein A, Protein G, an Ig-binding domain of Protein G, Protein A / G, an Ig-binding domain of Protein A / G, Protein L, an Ig-binding domain of Protein L, Protein M, an Ig-binding domain of Protein M, a peptide nucleic acid, a DARPin, an Affimer, a purified protein (either the analyte itself or a protein that binds to the analyte), and an analyte-binding domain(s) of a protein. In some embodiments, the additional amino acid sequence is a first interacting polypeptide configured to form a complex with a second interacting polypeptide upon contact of the first and second interacting polypeptides. In some embodiments, the additional amino acid sequence is a first co-localizing polypeptide configured to co-localize with a second co-localizing polypeptide within a subcellular compartment, cell, tissue, or organism. In some embodiments, the additional amino acid sequence is a protein of interest and a potential drug target.

[0043] In some embodiments, provided herein is a β9 / β10-like dipeptide comprising an amino acid sequence having more than 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) but less than 100% sequence identity with SEQ ID NO: 35 and less than 100% sequence identity with SEQ ID NO: 205 and SEQ ID NO: 206, wherein when the peptide is contacted with a polypeptide complement consisting of SEQ ID NO: 17, SEQ ID NO: 21, or SEQ ID NO: 302, the bioluminescent signal generated in the presence of a coelenterazine or coelenterazine analog substrate is substantially increased compared to the bioluminescent signal generated by the peptide and the coelenterazine or coelenterazine analog substrate alone. In some embodiments, the dipeptide (e.g., β9 / β 10 β-like dipeptides) can be used in conjunction with the polypeptide components described herein (e.g., β-like dipeptides). 1~8 In other embodiments, the dipeptide (e.g., β9 / β-like polypeptide) associates (e.g., forms a bioluminescent complex) without facilitation (e.g., by an interacting element). 10 -like dipeptides) and polypeptide components described herein (e.g., β 1~8 In some embodiments, the peptides of SEQ ID NO: 205 and / or SEQ ID NO: 206 exhibit one or more enhanced traits compared to the peptides of SEQ ID NO: 205 and / or SEQ ID NO: 206, wherein the traits are selected from affinity for the polypeptide complement, or enhanced expression, solubility, stability, and / or bioluminescent activity when combined with the polypeptide complement. In some embodiments, the amino acid sequence is not a naturally occurring protein or fragment thereof. In some embodiments, the amino acid sequence contains unnatural amino acids, amino acid analogs, and / or peptoid amino acids.

[0044] In some embodiments, provided herein are fusion polypeptides (e.g., genetic fusions, synthetically produced fusions, chemical conjugates, enzymatic conjugates, etc.) comprising a β9 / β10-like dipeptide described herein and an additional amino acid sequence. In some embodiments, the additional amino acid sequence is selected from the group consisting of a protein of interest, an interacting element, a co-localization element, and a binding moiety. In some embodiments, the additional amino acid sequence or other fused or attached molecule is a binding moiety selected from the group consisting of an antibody (polyclonal, monoclonal, and / or recombinant antibody), an antibody fragment, Protein A, the Ig-binding domain of Protein A, Protein G, the Ig-binding domain of Protein G, Protein A / G, the Ig-binding domain of Protein A / G, Protein L, the Ig-binding domain of Protein L, Protein M, the Ig-binding domain of Protein M, a peptide nucleic acid, a DARPin, an Affimer, a purified protein (either the analyte itself or a protein that binds to the analyte), and an analyte-binding domain(s) of a protein. In some embodiments, the additional amino acid sequence or other fused or added molecule is a first interacting polypeptide configured to form a complex with a second interacting polypeptide upon contact of the first and second interacting polypeptides. In some embodiments, the additional amino acid sequence or other fused or added molecule is a first co-localizing polypeptide configured to co-localize with a second co-localizing polypeptide within a subcellular compartment, cell, tissue, or organism. In some embodiments, the additional amino acid sequence or other fused or added molecule is a protein of interest and a potential drug target.

[0045] In some embodiments, provided herein are nucleic acids and / or vectors encoding the peptides, polypeptides, and / or fusion polypeptides described herein. In some embodiments, provided herein are cells expressing nucleic acids and / or vectors encoding the peptides, polypeptides, and / or fusion polypeptides described herein. In some embodiments, provided herein are synthetic production of the peptides, polypeptides, and / or fusion polypeptides described herein. In some embodiments, the peptides, polypeptides, and / or fusion polypeptides described herein are chemically conjugated to additional moieties (e.g., interacting elements, co-localizing elements, proteins of interest, molecules of interest, etc.).

[0046] In some embodiments, the polypeptide comprises: (a) a polypeptide comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO:17, SEQ ID NO:21, or SEQ ID NO:302 and less than 100% sequence identity to SEQ ID NO:5 and SEQ ID NO:8; (b) a first peptide comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO:23 and less than 100% sequence identity to SEQ ID NO:6 and SEQ ID NO:9; and (c) a polypeptide comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO:25. Provided herein is a bioluminescent conjugate comprising a second peptide comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity with SEQ ID NO:7 and SEQ ID NO:10, wherein the bioluminescent conjugate produces substantially increased bioluminescence in the presence of a coelenterazine or coelenterazine analog substrate compared to the coelenterazine or coelenterazine analog substrate in the presence of the polypeptide alone, the first peptide alone, the second peptide alone, and any two of the polypeptide, the first peptide, and the second peptide. In some embodiments, the first peptide is a first peptide tag, and the second peptide is a second peptide tag, and the first and second peptide tags are each linked to a moiety independently selected from the group consisting of a molecule of interest, a peptide of interest, a protein of interest, an interacting element, a co-localization element, or a binding moiety. In some embodiments, the first peptide tag or the second peptide tag is linked to a drug or a drug candidate, and the other peptide tag is linked to a drug target or a drug target candidate, and the intensity of bioluminescence from the bioluminescent complex correlates with the affinity of the drug or drug candidate for the drug target or drug target candidate.In some embodiments, a first peptide tag is linked to a first interacting element and a second peptide tag is linked to a second interacting element, wherein the intensity of bioluminescence from the bioluminescent complex correlates with the affinity of the first interacting element for the second interacting element under the conditions being tested (e.g., in some embodiments, the combination of the first peptide, second peptide, polypeptide component, and substrate does not form a bioluminescent complex (and does not produce significant light output (e.g., above background)) in the absence of interaction between the interacting elements). In some embodiments, a first peptide tag is linked to a first co-localization element and a second peptide tag is linked to a second co-localization element, wherein substantially increased bioluminescence indicates co-localization, but not necessarily interaction, of the first co-localization element and the second co-localization element under the conditions being tested.

[0047] In some embodiments, the peptides and polypeptides provided herein are not fragments of a larger (e.g., pre-existing) protein, hi other embodiments, one or more peptides and / or polypeptides provided herein are fragments of a larger (e.g., pre-existing) protein.

[0048] In some embodiments, (a) a first peptide comprising (i) an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO:23 and less than 100% sequence identity to SEQ ID NO:6 and SEQ ID NO:9; (ii) a first peptide comprising 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO:25; (iii) a second peptide comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity with SEQ ID NO: 17, SEQ ID NO: 21, or SEQ ID NO: 302, and less than 100% sequence identity with SEQ ID NO: 7 and SEQ ID NO: 10;

[0010] Provided herein are methods comprising combining (a) a polypeptide component comprising an amino acid sequence having a sequence identity of at least 100% (or more) with SEQ ID NO:5 and SEQ ID NO:8, wherein the first peptide tag, the second peptide tag, and the polypeptide component are configured to generate a bioluminescent complex upon interaction of the first and second molecular entities, and (iv) a coelenterazine or coelenterazine analog substrate, and (b) detecting luminescence, wherein a higher level of luminescence compared to the level of luminescence generated by the polypeptide component and coelenterazine or coelenterazine analog alone indicates formation of a bioluminescent complex of the polypeptide component and the first and second peptides. In some embodiments, one or more of the polypeptide component and the first and second peptides are expressed intracellularly, exogenously added to a cell, and / or added to a sample.

[0049] In some embodiments, a method of detecting an interaction between a first molecular entity and a second molecular entity comprises: (a) tagging the first molecular entity with a first peptide tag comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO:23 and less than 100% sequence identity to SEQ ID NO:6 and SEQ ID NO:9; (b) tagging the second molecular entity with a first peptide tag comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO:23 and less than 100% sequence identity to SEQ ID NO:6 and SEQ ID NO:9; (c) tagging the first molecular entity with a second peptide tag comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity with SEQ ID NO: 25 and less than 100% sequence identity with SEQ ID NO: 7 and SEQ ID NO: 10; (d) combining the tagged first molecular entity and the tagged second molecular entity; (e) combining the first molecular entity with a second peptide tag comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity with SEQ ID NO: 25 and less than 100% sequence identity with SEQ ID NO: 7 and SEQ ID NO: 10; and (e) adding a polypeptide component comprising an amino acid sequence having 0% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity and less than 100% sequence identity with SEQ ID NO:5 and SEQ ID NO:8, wherein the first peptide tag, the second peptide tag, and the polypeptide component are configured to generate a bioluminescent complex upon interaction of the first molecular entity and the second molecular entity; (e) adding coelenterazine or a coelenterazine analog substrate; and (f) detecting a luminescent signal generated by the bioluminescent complex, wherein the magnitude of the luminescent signal correlates (e.g., is proportional, is directly proportional, etc.) with the number, strength, favorability, and / or stability of the interaction(s) between the first molecular entity and the second molecular entity. In some embodiments, the catalytic efficiency, substrate turnover, and / or specific activity of the resulting bioluminescent complex correlates (e.g., is proportional, directly proportional, etc.) with the number, strength, favorability, and / or stability of the interaction(s) between the first and second molecular entities.In some embodiments, the first molecular entity and / or the second molecular entity is a protein or peptide of interest, and tagging comprises creating a fusion (or synthetic conjugation) of the first molecular entity and / or the second molecular entity with a first peptide tag and / or a second peptide tag. In some embodiments, the first molecular entity and / or the second molecular entity is a small molecule, and tagging comprises directly or indirectly linking the first molecular entity and / or the second molecular entity to the first peptide tag and / or the second peptide tag. In some embodiments, one of the first molecular entity and the second molecular entity is a drug or a drug candidate, and the other is a drug target or a drug target candidate, and the bioluminescent signal indicates binding of the drug or drug candidate to the other, which is the drug target or drug target candidate. In some embodiments, combining the tagged first molecular entity and the tagged second molecular entity comprises expressing one or both in a cell and / or adding one or both to a cell. In some embodiments, combining the tagged first molecular entity and the tagged second molecular entity is performed in vitro, in a non-cellular sample, etc. In some embodiments, the affinity of a drug or drug candidate for a drug target or drug target candidate is determined by diluting an unlabeled drug target or drug target candidate into a system using the systems and methods herein. In some embodiments, two or more of steps (a)-(f) are performed simultaneously. In some embodiments, two or more of steps (a)-(f) are performed separately.

[0050] In some embodiments, a method for performing a competitive assay for detecting an interaction between a first molecular entity and a second molecular entity comprises: (a) (i) a tracer comprising a first molecular entity tagged with a first peptide tag comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO:23 and less than 100% sequence identity to SEQ ID NO:6 and SEQ ID NO:9; (ii) a sequence (iii) a second molecular entity tagged with a second peptide tag comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity with SEQ ID NO:25 and less than 100% sequence identity with SEQ ID NO:7 and SEQ ID NO:10; (iv) a coelenterazine or coelenterazine analog substrate; (v) a second molecular entity tagged with a second peptide tag comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity with SEQ ID NO:17, SEQ ID NO:21, or SEQ ID NO:302; Provided herein are methods comprising: (a) combining a polypeptide component comprising an amino acid sequence having at least 5%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more sequence identity with SEQ ID NO:5 and SEQ ID NO:8, and less than 100% sequence identity with SEQ ID NO:5 and SEQ ID NO:8; and (b) a sample suspected of containing an untagged first molecular entity, wherein the first peptide tag, the second peptide tag, and the polypeptide component form a bioluminescent complex configured to generate a bioluminescent signal in the presence of coelenterazine or a coelenterazine analog substrate; (c) detecting the bioluminescent signal generated by the bioluminescent complex; and (d) comparing the bioluminescent signal generated in the presence of the sample with a control bioluminescent signal generated in the absence of the sample, wherein a decrease in the bioluminescent signal indicates the presence or amount of the untagged first molecular entity in the sample. In some embodiments, the first molecular entity is a small molecule or peptide (e.g., a drug or drug candidate).In some embodiments, the second molecular entity is a drug target or drug target candidate (eg, a protein).

[0051] In some embodiments, a method for detecting an interaction in a cell between a first protein, peptide, or molecular entity and a second protein, peptide, or molecular entity comprises: (a) a fusion comprising the first protein, peptide, or molecular entity and a first peptide tag comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO:23 and less than 100% sequence identity to SEQ ID NO:6 and SEQ ID NO:9. (b) expressing in the cell (or adding to a cell or other system (e.g., a non-cellular sample)) the second protein, peptide, or molecular entity (e.g., a genetic fusion, a synthetic fusion, a chemical conjugation, etc.), and (c) forming a fusion protein comprising the second protein, peptide, or molecular entity and a second peptide tag comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO:25 and less than 100% sequence identity to SEQ ID NO:7 and SEQ ID NO:10. (c) expressing in the cell (or adding to the cell or other system (e.g., non-cellular sample) a compound (e.g., genetic fusion, synthetic fusion, chemical conjugation, etc.) of SEQ ID NO: 17, SEQ ID NO: 21, or SEQ ID NO: 302) that has 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity with SEQ ID NO: 17, SEQ ID NO: 21, or SEQ ID NO: 302 and less than 100% sequence identity with SEQ ID NO: 5 and SEQ ID NO: 8. (d) expressing (or adding to a cell or other system (e.g., a non-cellular sample)) the first peptide tag, the second peptide tag, and the polypeptide components configured to generate a bioluminescent complex upon interaction of the first protein, peptide, or molecular entity and the second protein, peptide, or molecular entity; (d) adding coelenterazine or a coelenterazine analog substrate to the cell; and (e) detecting a luminescent signal generated by the bioluminescent complex.Provided herein are methods comprising detecting, wherein the magnitude of the luminescent signal correlates with the strength of the interaction between the first protein, peptide, or molecular entity and the second protein, peptide, or molecular entity. In some embodiments, two or more of steps (a) through (e) are performed simultaneously. In some embodiments, two or more of steps (a) through (e) are performed separately.

[0052] In some embodiments, a method for detecting co-localization of a first molecular entity and a second molecular entity comprises: (a) tagging the first molecular entity with a first peptide tag comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO:23 and less than 100% sequence identity to SEQ ID NO:6 and SEQ ID NO:9; (b) tagging the second molecular entity with a first peptide tag comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO:23 and less than 100% sequence identity to SEQ ID NO:6 and SEQ ID NO:9; (c) combining the tagged first molecular entity and the tagged second molecular entity in the same system; (d) adding a polypeptide component to the system, wherein the polypeptide component is selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 45, SEQ ID NO: 50, SEQ ID NO: 55, SEQ ID NO: 60, SEQ ID NO: 65, SEQ ID NO: 70, SEQ ID NO: 75, SEQ ID NO: 80, SEQ ID NO: 85, SEQ ID NO: 90, SEQ ID NO: 95, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 21, or SEQ ID NO: 302 and an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity with SEQ ID NO: 5 and SEQ ID NO: 8, wherein the first peptide tag, the second peptide tag, and the polypeptide components are configured to generate a bioluminescent complex upon co-localization of the first molecular entity and the second molecular entity. (e) adding a coelenterazine or coelenterazine analog substrate to the system, and (f) detecting a luminescent signal produced by the bioluminescent conjugate, wherein the presence of a luminescent signal above background indicates colocalization of the first molecular entity and the second molecular entity within the system, and / or the magnitude of the luminescent signal correlates with the amount of colocalization of the first molecular entity and the second molecular entity within the system. In some embodiments, the system comprises a cell, tissue, organ, or whole organism.In some embodiments, the first molecular entity and / or the second molecular entity is a protein of interest or a peptide of interest, and tagging comprises creating a fusion (e.g., genetic fusion, synthetic fusion, chemical conjugation, enzymatic conjugation, etc.) of the first molecular entity and / or the second molecular entity with the first peptide tag and / or the second peptide tag. In some embodiments, the first molecular entity and / or the second molecular entity is a small molecule, and tagging comprises directly or indirectly linking the first molecular entity and / or the second molecular entity to the first peptide tag and / or the second peptide tag. In some embodiments, combining the tagged first molecular entity and the tagged second molecular entity is performed in vitro, in a non-cellular sample, etc. In some embodiments, combining the tagged first molecular entity and the tagged second molecular entity comprises expressing one or both in a system and / or adding one or both to a system. In some embodiments, two or more of steps (a)-(f) are performed simultaneously. In some embodiments, two or more of steps (a)-(f) are performed independently.

[0053] In some embodiments, a method for detecting intracellular co-localization of a first protein, peptide, or molecular entity and a second protein, peptide, or molecular entity includes: (a) co-localizing within the cell a fusion comprising the first protein or peptide entity and a first peptide tag comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO:23 and less than 100% sequence identity to SEQ ID NO:6 and SEQ ID NO:9; (b) expressing in the cell a fusion comprising the second protein or peptide entity and a second peptide tag comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO:25 and less than 100% sequence identity to SEQ ID NO:7 and SEQ ID NO:10; (c) expressing in the cell a fusion comprising the second protein or peptide entity and a second peptide tag comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO:17, SEQ ID NO:21, and / or SEQ ID NO:302. , 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity with SEQ ID NO:5 and SEQ ID NO:8, wherein the first peptide tag, the second peptide tag, and the polypeptide component are configured to generate a bioluminescent complex upon co-localization of the first protein or peptide entity and the second protein or peptide entity; (d) adding coelenterazine or a coelenterazine analog substrate to the cell; and (e) detecting a luminescent signal generated by the bioluminescent complex, wherein presence of a luminescent signal above background indicates co-localization of the first protein or peptide entity and the second protein or peptide entity within the cell and / or wherein the magnitude of the luminescent signal correlates with the amount of co-localization of the first protein or peptide entity and the second protein or peptide entity in the system.In some embodiments, two or more of steps (a)-(e) are performed simultaneously. In some embodiments, two or more of steps (a)-(e) are performed independently.

[0054] In some embodiments, provided herein are kits that include (a) a first binding moiety conjugated to a first peptide tag comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO:23 and less than 100% sequence identity to SEQ ID NO:6 and SEQ ID NO:9, and (b) a second binding moiety conjugated to a second peptide tag comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO:25 and less than 100% sequence identity to SEQ ID NO:7 and SEQ ID NO:10. In some embodiments, the first and second binding moieties are independently selected from the group consisting of antibodies (polyclonal, monoclonal, and / or recombinant antibodies), antibody fragments, Protein A, the Ig-binding domain of Protein A, Protein G, the Ig-binding domain of Protein G, Protein A / G, the Ig-binding domain of Protein A / G, Protein L, the Ig-binding domain of Protein L, Protein M, the Ig-binding domain of Protein M, oligonucleotide probes, peptide nucleic acids, DARPins, aptamers, affimers, purified proteins (either the analyte itself or a protein that binds to the analyte), and analyte-binding domain(s) of a protein. In some embodiments, the first and second binding moieties are primary binding moieties configured to bind to an antigen, epitope, or sequence of the same target entity. In some embodiments, the first and second binding moieties are secondary binding moieties configured to bind to an antigen, epitope, or sequence of the primary binding moiety. In some embodiments, the kit further comprises a polypeptide reagent comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO: 17, SEQ ID NO: 21, and / or SEQ ID NO: 302 and less than 100% sequence identity to SEQ ID NO: 5 and SEQ ID NO: 8. In some embodiments, the kit further comprises coelenterazine or a coelenterazine analog.

[0055] In some embodiments, there is provided a method for detecting a target molecule, wherein the target molecule exhibits a first antigen, epitope, or sequence and a distinct second antigen, epitope, or sequence, the method comprising: (a) contacting a sample containing the target molecule with (i) a first primary binding moiety that recognizes the first antigen, epitope, or sequence and (ii) a second primary binding moiety that recognizes the second antigen, epitope, or sequence, and allowing the first and second primary binding moieties to bind to the first and second antigens, epitopes, or sequences; (b) contacting the sample with a first primary binding moiety that recognizes the first antigen, epitope, or sequence and a second primary binding moiety that recognizes the second antigen, epitope, or sequence; contacting the polypeptide with (i) a first secondary binding moiety conjugated or fused to a first peptide tag and (ii) a second secondary binding moiety conjugated or fused to a second peptide tag, wherein the first secondary binding moiety recognizes the first primary binding moiety, the second secondary binding moiety recognizes the second primary binding moiety, and the first or second peptide tag has 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO:23. and allowing the first and second secondary binding moieties to bind to the first and second primary binding moieties; (a) contacting the first and second peptide tags with a first or second primary binding moiety and allowing the first and second secondary binding moieties to bind to the first and second primary binding moieties; and (b) contacting the first and second peptide tags with a first or second primary binding moiety and allowing the first and second secondary binding moieties to bind to the first and second primary binding moieties; (c) contacting the first and second peptide tags with a first or second primary binding moiety and allowing the first and second secondary binding moieties to bind to the first and second primary binding moieties; and (d) contacting the first and second secondary binding moieties with a first or second primary binding moiety and allowing the first and second secondary binding moieties to bind to the first and second primary binding moieties. c) contacting the sample with a polypeptide component having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO:17, SEQ ID NO:21, and / or SEQ ID NO:302 (and less than 100% sequence identity to SEQ ID NO:5 and SEQ ID NO:8), wherein the first peptide tag, the second peptide tag, and the polypeptide component are configured to generate a bioluminescent complex upon interaction;Provided herein are methods comprising: (d) contacting the sample with coelenterazine or a coelenterazine analog substrate; and (e) detecting a luminescent signal produced by the bioluminescent conjugate, wherein the presence of a luminescent signal above background indicates the presence of a target molecule and / or the magnitude of the luminescent signal correlates with the amount of the target molecule in the sample. In some embodiments, the binding moieties are independently selected from the group consisting of an antibody (polyclonal, monoclonal, and / or recombinant antibody), an antibody fragment, Protein A, an Ig-binding domain of Protein A, Protein G, an Ig-binding domain of Protein G, Protein A / G, an Ig-binding domain of Protein A / G, Protein L, an Ig-binding domain of Protein L, Protein M, an Ig-binding domain of Protein M, an oligonucleotide probe, a peptide nucleic acid, a DARPin, an aptamer, an affimer, a purified protein (either the analyte itself or a protein that binds to the analyte), and an analyte-binding domain(s) of a protein. In some embodiments, the target molecule is a protein, peptide, nucleic acid, chemical, or drug. In some embodiments, the sample is in vitro or in vivo.

[0056] In some embodiments, a method of detecting a target molecule, wherein the target molecule exhibits a first antigen, epitope, or sequence and a distinct second antigen, epitope, or sequence, comprises: (a) contacting the sample with (i) a first binding moiety conjugated or fused to a first peptide tag and (ii) a second binding moiety conjugated or fused to a second peptide tag, wherein the first binding moiety recognizes the first antigen, epitope, or sequence and the second binding moiety recognizes the second antigen, epitope, or sequence; The first or second peptide tag comprises an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO:23 (and less than 100% sequence identity to SEQ ID NO:6 and SEQ ID NO:9), and the other of the first or second peptide tag comprises an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO:25 (and (c) contacting the sample with an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity with SEQ ID NO:17, SEQ ID NO:21, and / or SEQ ID NO:302 (and less than 100% sequence identity with SEQ ID NO:5 and SEQ ID NO:8), and allowing the first and second binding moieties to bind to the first and second antigens, epitopes, or sequences; (d) contacting the sample with a polypeptide component having a coelenterazine or coelenterazine analog substrate, wherein the first peptide tag, the second peptide tag, and the polypeptide component are configured to generate a bioluminescent complex upon interaction; (d) contacting the sample with a coelenterazine or coelenterazine analog substrate; and (e) detecting a luminescent signal generated by the bioluminescent complex, wherein the presence of a luminescent signal above background indicates the presence of a target molecule, and / or the magnitude of the luminescent signal isThe methods provided herein include detecting, which correlates with the amount of a target molecule in the sample. In some embodiments, the binding moieties are independently selected from the group consisting of antibodies (polyclonal, monoclonal, and / or recombinant antibodies), antibody fragments, Protein A, the Ig-binding domain of Protein A, Protein G, the Ig-binding domain of Protein G, Protein A / G, the Ig-binding domain of Protein A / G, Protein L, the Ig-binding domain of Protein L, Protein M, the Ig-binding domain of Protein M, oligonucleotide probes, peptide nucleic acids, DARPins, aptamers, affimers, purified proteins (either the analyte itself or a protein that binds to the analyte), and analyte-binding domain(s) of a protein. In some embodiments, the target molecule is a protein, peptide, nucleic acid, chemical, or drug. In some embodiments, the sample is in vitro or in vivo.

[0057] In some embodiments, (a) a peptide component comprising an amino acid sequence having (i) 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity with SEQ ID NO:35 and less than 100% sequence identity with SEQ ID NO:205 and SEQ ID NO:206; (ii) 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity with SEQ ID NO:17, SEQ ID NO:21, and / or SEQ ID NO:302 and less than 100% sequence identity with SEQ ID NO:5 and SEQ ID NO:8; and (iii) a coelenterazine or coelenterazine analog substrate, wherein the peptide and polypeptide components are configured to generate a bioluminescent complex upon interaction, and (b) detecting luminescence, wherein a higher level of luminescence compared to the level of luminescence generated by the polypeptide component and coelenterazine or coelenterazine analog alone indicates formation of a bioluminescent complex of the polypeptide component with the peptide. In some embodiments, the peptide is a fusion (e.g., genetic fusion, synthetic fusion, chemical conjugate, enzymatic conjugate, etc.) with a first interacting element, and the polypeptide component is a fusion (e.g., genetic fusion, synthetic fusion, chemical conjugate, enzymatic conjugate, etc.) with a second interacting element, wherein the peptide and polypeptide components form a bioluminescent complex upon interaction of the interacting elements, but do not form a bioluminescent complex in the absence of interaction between the interacting elements. In some embodiments, the peptide and polypeptide components form a bioluminescent complex in the absence of facilitation (eg, by an interacting element).In some embodiments, the peptide is a fusion or conjugate (e.g., genetic fusion, synthetic fusion, chemical conjugate, enzymatic conjugate, etc.) with a protein, peptide, or molecule of interest (e.g., not an interacting element), and / or the polypeptide component is a fusion or conjugate (e.g., genetic fusion, synthetic fusion, chemical conjugate, enzymatic conjugate, etc.) with a protein, peptide, or molecule of interest (e.g., not an interacting element). In some embodiments, the peptide and polypeptide components form a bioluminescent complex upon co-localization (e.g., within a sample, within a cell, within a tissue, within a subject, etc.) without promotion by an interacting element. In some embodiments, the peptide and polypeptide components form a bioluminescent complex when promoted by an interacting element, but do not form a bioluminescent complex without promotion.

[0058] In some embodiments, provided herein are compositions comprising a monomeric polypeptide comprising an amino acid sequence having more than 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) but less than 100% sequence identity to SEQ ID NO: 788 or 789 and less than 100% sequence identity to SEQ ID NO: 1 or 3, wherein the polypeptide exhibits luminescence in the presence of coelenterazine or a coelenterazine analog substrate. In some embodiments, a peptide comprises an amino acid sequence having greater than 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or ranges therebetween) sequence identity to one or more of SEQ ID NOs: 780, 782, 784, 786, 802, 804, 806, 808, 813, 815, or 829. In some embodiments, the polypeptide further comprises an additional amino acid sequence. In some embodiments, fusion proteins of the polypeptides described herein are provided.

[0059] In some embodiments, provided herein are nucleic acids comprising a sequence encoding an amino acid sequence having more than 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) but less than 100% sequence identity to SEQ ID NO: 788 or 789, and less than 100% sequence identity to SEQ ID NO: 1 or 3, wherein the polypeptide exhibits luminescence in the presence of coelenterazine or a coelenterazine analog substrate. In some embodiments, the nucleic acid encodes an amino acid sequence having greater than 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or ranges therebetween) sequence identity to one or more of SEQ ID NOs: 780, 782, 784, 786, 802, 804, 806, 808, 813, 815, or 829. In some embodiments, nucleic acids encoding fusion proteins of the polypeptides described herein are provided.

[0060] In some embodiments, provided herein are methods for detecting bioluminescence, comprising contacting a monomeric bioluminescent polypeptide described herein with a coelenterazine or coelenterazine analog substrate and detecting luminescence.

[0061] In some embodiments, provided herein is a polypeptide comprising an amino acid sequence having greater than 40% sequence identity to SEQ ID NO: 15 linked to the N-terminus of an amino acid sequence having greater than 40% sequence identity to one of SEQ ID NOs: 17, 21, or 302, wherein the bioluminescent signal generated in the presence of coelenterazine or a coelenterazine analog substrate is substantially increased when the polypeptide is contacted with a peptide or polypeptide comprising the amino acid sequence of SEQ ID NO: 23, as compared to the bioluminescent signal generated by the polypeptide and the coelenterazine substrate alone. In some embodiments, provided are nucleic acids comprising sequences encoding the polypeptides described herein. In some embodiments, provided are fusions of the polypeptides described herein with additional amino acid sequences.

[0062] In some embodiments, provided herein are methods that include: (a) contacting a polypeptide comprising an amino acid sequence having greater than 40% sequence identity to SEQ ID NO: 15 linked to the N-terminus of an amino acid sequence having greater than 40% sequence identity to one of SEQ ID NOs: 17, 21, or 302, with a coelenterazine or coelenterazine analog substrate and a peptide or polypeptide comprising an amino acid sequence having greater than 40% sequence identity (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) to SEQ ID NO: 23; and (b) detecting luminescence.

[0063] In some embodiments, provided herein are systems that include: (a) a sensor polypeptide comprising a first amino acid sequence having at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 11 linked to an amino acid sequence that localizes to a specific cellular location; and (b) a protein of interest linked to a peptide tag comprising an amino acid sequence having at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 23, wherein a bioluminescent complex is formed between the polypeptide and the peptide tag upon colocalization within the specific cellular location. In some embodiments, the specific cellular location is selected from the plasma membrane, nucleus, mitochondria, and endoplasmic reticulum. In some embodiments, provided herein are methods comprising: (a) expressing a system described herein in a cell; (b) contacting the cell with coelenterazine or a coelenterazine analog substrate; and (c) detecting luminescence, wherein an increase in luminescence indicates formation of the bioluminescent complex and colocalization of the polypeptide and peptide tag. In some embodiments, the method comprises the following, further comprising the step of targeting the translocated protein of interest linked to a peptide tag to a specific cellular location.

[0064] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0065] [Figure 1]10 is a graph demonstrating that a polypeptide lacking the β9 and β10 portions (LgTrip 2098; SEQ ID NO: 17) exhibits reduced background luminescence compared to LgBiT (SEQ ID NO: 11) and is activated by complementation with peptides corresponding to β9 and β10. [Figure 2] 1 is a graph demonstrating activation of LgTrip 2098 (SEQ ID NO: 17) by distinct peptides corresponding to β9 and β10, respectively. [Figure 3] 1 is a graph showing the relative stability of exemplary LgTrip 2098 variants. [Figure 4] 1 is a graph showing the relative luminescence activity of the amino acid site saturation at position 42 of LgTrip 2098 variants. [Figure 5] 1 is a graph showing the relative stability of the amino acid change at position 42 of the LgTrip 2098 variant. [Figure 6] Graph showing the relative luminescence activity of amino acid changes at (A) position 4, (B) position 30, and (C) position 106 of LgTrip 2098 variants. [Figure 7A] 1 is a graph showing the relative luminescence activity of amino acid changes at position 101 of LgTrip 3092 variants. [Figure 7B] 1 is a graph showing the relative luminescence activity of amino acid changes at position 117 of LgTrip 3092 variants. [Figure 7C] 1 is a graph showing the relative luminescence activity of amino acid changes at position 127 of LgTrip 3092 variants. [Figure 7D] 1 is a graph showing the relative luminescence activity of amino acid changes at position 120 of LgTrip 3092 variants. [Figure 7E] 1 is a graph showing the relative luminescence activity of amino acid changes at position (E)126 of LgTrip 3092 variants. [Figure 8] 1 is a graph showing the relative stability of LgTrip 2098 (WT) (SEQ ID NO: 31), LgTrip 3092 (SEQ ID NO: 19), and LgBiT (SEQ ID NO: 11) at 37° C. [Figure 9] Graph showing the relative stability of LgTrip variants at (A) 42°C and (B) 60°C. [Figure 10A] Graph showing titration of SmTrip9 pep286 (SEQ ID NO: 37) in different LgTrip variants. [Figure 10B] Graph showing dilution of SmTrip10 pep86 (SEQ ID NO: 25) in different LgTrip variants. [Figure 10C] 1 is a graph showing the affinity of various LgTrip variants to SmTrip9 pep286 and SmTrip10 pep86. [Figure 11] Graph showing (A) stability (half-life) and (B) relative stability at 60° C. of various LgTrip variants. [Figure 12] Graphs showing the kinetic profiles of LgTrip variants SmTrip9 pep286 (SEQ ID NO: 37) and SmTrip10 pep86 (HiBiT, SEQ ID NO: 25) in the presence of NanoLuc (SEQ ID NO: 3) and compared to LgBiT (SEQ ID NO: 11) and SmTrip10 pep86 (HiBiT, SEQ ID NO: 25), analyzed in (A) TBS + 0.01% BSA and (B) NanoGlo® assay buffer. [Figure 13] Graphs showing the enhanced complementation of various trimolecular systems by rapamycin-induced FRB / FKBP complex formation: (A) SmTrip10 pep86 (SEQ ID NO: 25), SmTrip9 pep245 (SEQ ID NO: 23), and LgTrip 2098 (SEQ ID NO: 31), and (B) SmBiT (SEQ ID NO: 13), SmTrip9 pep245 (SEQ ID NO: 23), and LgTrip 2098 (SEQ ID NO: 31). [Figure 14] Graphs comparing the stability of LgBiT (SEQ ID NO: 11) and LgTrip 2098 (WT) (SEQ ID NO: 31) at 37° C. in (A) TBS+0.01% BSA and (B) Passive Lysis Buffer (PLB). [Figure 15] Graph comparing the stability of LgBiT (SEQ ID NO: 11), LgTrip 3546 (SEQ ID NO: 51), and NanoLuc (SEQ ID NO: 31) at 60° C. (A) Time course and (B) half-life. [Figure 16] Graphs comparing LgBiT (SEQ ID NO: 11), NanoLuc (SEQ ID NO: 3), and LgTrip 3546 (SEQ ID NO: 51) and LgTrip 2098 (WT) (SEQ ID NO: 31) in the presence of NaCl (A) and after 26 hours of exposure to NaCl (B). [Figure 17] Graphs comparing LgBiT (SEQ ID NO: 11), NanoLuc (SEQ ID NO: 3), and LgTrip 3546 (SEQ ID NO: 51) and LgTrip 2098 (WT) (SEQ ID NO: 31) variants (A) in the presence of urea and (B) after 26 hours of exposure to urea. [Figure 18] Graphs comparing LgBiT (SEQ ID NO: 11), NanoLuc (SEQ ID NO: 3), and LgTrip 3546 (SEQ ID NO: 51) and LgTrip 2098 (WT) (SEQ ID NO: 31) variants (A) at various pHs and (B) after 26 hours of exposure to different pHs. [Figure 19] 1 is a graph comparing the autoluminescence of LgBiT (SEQ ID NO: 11) and LgTrip 3546 (SEQ ID NO: 51). [Figure 20] Graphs comparing the luminescence of LgBiT (SEQ ID NO: 11) + SmTrip10 pep86 (HiBiT, SEQ ID NO: 25), LgBiT (SEQ ID NO: 11) + pep263 (SEQ ID NO: 35) (β9 / β10 dipeptide), and LgTrip 3546 (SEQ ID NO: 51) + pep263 (β9 / β10 dipeptide) (SEQ ID NO: 35): (A) RLU and (B) signal / background (S / B). [Figure 21]Enhanced complementation of LgTrip 2098 (SEQ ID NO: 31) and LgTrip 3546 (SEQ ID NO: 51) with SmTrip10 pep86 (SEQ ID NO: 25) and SmTrip9 pep245 (SEQ ID NO: 23), respectively: (A) Schematic of the assay system, (B) RLU, and (C) signal / background (S / B). [Figure 22] 1 is a graph and table comparing the affinity of various SmTrip10 sequences to LgTrip 3546 (SEQ ID NO: 51) and SmTrip9 pep286 (SEQ ID NO: 37). [Figure 23] 1 is a graph and table comparing the activation of LgTrip 2098 (SEQ ID NO: 31) and LgTrip 3546 (SEQ ID NO: 51) by standard orientation (pep263) (SEQ ID NO: 35) and reverse orientation (pep326) (SEQ ID NO: 179) dipeptides. [Figure 24] 1 is a graph and table showing activation of complementary polypeptides by dipeptides containing HiBiT or SmBiT sequences: a dipeptide having the HiBiT sequence pep263 (SEQ ID NO: 35) or a dipeptide having the SmBiT sequence pep274 (SEQ ID NO: 147). [Figure 25] (A) Graph showing the luminescence obtained by complementation of various combinations of polypeptide components (having additions or deletions compared to LgTrip3546) with SmTrip9 pep286 (SEQ ID NO: 37) and various β10-like peptides (SmTrip10 peptides). (B) Graph showing the luminescence obtained by complementation of LgTrip3546 (SEQ ID NO: 51) and SmTrip9 pep286 (SEQ ID NO: 37) with various β10-like peptides (SmTrip10 peptides). [Figure 26A] Graph showing luminescence produced by polypeptide / peptide combinations having overlap (relative to the base luciferase sequence) between the polypeptide component and a peptide corresponding to the β9 chain, or between β9 and β10-like peptides. [Figure 26B]Graph showing luminescence produced by polypeptide / peptide combinations having overlap (relative to the base luciferase sequence) between the polypeptide component and a peptide corresponding to the β9 chain, or between β9 and β10-like peptides. [Figure 26C] Graph showing luminescence produced by polypeptide / peptide combinations having overlap (relative to the base luciferase sequence) between the polypeptide component and a peptide corresponding to the β9 chain, or between β9 and β10-like peptides. [Figure 27] 1A and 1B show (A) titrations of various β9-like peptides (SmTrip9 peptides) in the presence of constant concentrations of LgTrip 3546 (SEQ ID NO: 51) and SmTrip10 pep86 (SEQ ID NO: 25), and (B) the luminescence obtained with dilutions of SmTrip10 pep86 (SEQ ID NO: 25) in the presence of constant concentrations of LgTrip 3546 (SEQ ID NO: 51) and various β9-like peptides (SmTrip9 peptides). [Figure 28] FIG. 1 shows a diagram and table showing the luminescence obtained with dilutions of various β10-like peptides (SmTrip 10 peptides) in the presence of constant concentrations of LgTrip 3546 (SEQ ID NO: 51) and SmTrip9 pep286 (SEQ ID NO: 37). [Figure 29] 1 shows a diagram and table showing the dilution of β9-like peptides (A) SmTrip9 pep286 (SEQ ID NO: 37) and (B) SmTrip9 pep287 (SEQ ID NO: 148) in the presence of fixed concentrations of various β10-like peptides (SmTrip10 peptides) and LgTrip 3546 (SEQ ID NO: 51) polypeptide components. [Figure 30] Graph showing the effect of construct orientation (β9-FKBP, FKBP-β9, β10-FKBP, FKBP-β10, β9-FRB, FRB-β9, β10-FRB, or FRB-β10) on promoted complementation in HEK293 cells. [Figure 31]Graph showing the effect of construct orientation (β9-FKBP, FKBP-β9, β10-FKBP, FKBP-β10, β9-FRB, FRB-β9, β10-FRB, or FRB-β10) on promoted complementation in E. coli cells. [Figure 32] 1 is a graph showing the calculated Kd values ​​of various β10-like peptides with LgTrip 3546 (SEQ ID NO: 51) and SmTrip9 pep286 (SEQ ID NO: 37). [Figure 33] 1 is a graph and table showing the luminescence from combinations of building blocks with different split positions between the polypeptide building block LgTrip 3546 (SEQ ID NO: 51) and a β9-like peptide. [Figure 34] 1 is a graph showing the luminescence of various LgTrip polypeptide components combined with components having sequence gaps and / or overlaps between them and SmTrip9 pep286 (SEQ ID NO: 37). [Figure 35] Graph showing luminescence in the presence of SmTrip10 pep86 (HiBiT, SEQ ID NO: 25) with combinations of NanoTrip components with gaps and / or overlaps in the sequence between the β9-like peptide (SmTrip9 peptide) and the polypeptide component LgTrip 3546 (SEQ ID NO: 51). [Figure 36] 1 is a table showing biochemical analysis of the effect of β9-like peptide (SmTrip9 peptide) length on β9-like peptide affinity and maximum light output by LgTrip 3546 (SEQ ID NO: 51) and SmTrip10 pep86. [Figure 37] 1 is a table showing biochemical analysis of the effect of β9-like peptide (SmTrip9 peptide) length on HiBiT affinity and maximum light output by LgTrip 3546 (SEQ ID NO: 51) and SmTrip10 pep86 (SEQ ID NO: 25). [Figure 38] 1 is a table showing the Kd and Bmax of β9-like SmTrip9 pep286 (SEQ ID NO: 37) point variants with LgTrip 3546 (SEQ ID NO: 51) and SmTrip10 pep86 (SEQ ID NO: 25). [Figure 39] 1 is a table showing the effect of various solubility tags on the affinity of β9-like peptides LgTrip 3546 (SEQ ID NO: 51) and SmTrip9 pep86 (SEQ ID NO: 25). [Figure 40] 1 is a table showing the effect of various C-terminal extension sequences on β9-like or β10-like peptide affinity and maximum light output. β9-like peptide dilutions (pep286 (SEQ ID NO: 37), pep292 (SEQ ID NO: 153), pep297 (SEQ ID NO: 157), pep302 (SEQ ID NO: 161)) and the β10-like peptide SmTrip10 pep86 (HiBiT, SEQ ID NO: 25) are shown. [Figure 41] Graph showing the effect of FRB-β10 construct linker length (15, 10, or 5 Gly / Ser residues), linker composition (containing or not containing Ala-Ile), inclusion of a hexahistidine tag, and β10 composition (SmTrip10 pep86 (SEQ ID NO: 25) or SmTrip10 pep289 (SEQ ID NO: 150)) on enhanced complementation with LgTrip 3546 (SEQ ID NO: 51) in E. coli lysates. [Figure 42] Graph showing the effect of FRB-β10 construct linker length (15, 10, or 5 Gly / Ser residues), linker composition (containing or not containing Ala-Ile), inclusion of a hexahistidine tag, and β10 composition (SmTrip10 pep86 (SEQ ID NO: 25) or SmTrip10 pep289 (SEQ ID NO: 150)) on enhanced complementation with LgTrip 3546 (SEQ ID NO: 51) in HEK lysates. [Figure 43] Graph showing the effect of shortening and lengthening the β9 sequence and construct orientation (β9-FKBP or FKBP-β9) on enhanced complementation with FRB-SmTrip10 pep86 (β10) (SEQ ID NO: 25) in E. coli lysates containing LgTrip 3546 (SEQ ID NO: 51). [Figure 44]Graph showing the effect of shortening and lengthening the β9 sequence and construct orientation (β9-FKBP or FKBP-β9) on enhanced complementation with FRB-SmTrip10 pep289 (β10) (SEQ ID NO: 150) in E. coli lysates containing LgTrip 3546 (SEQ ID NO: 51). [Figure 45] Graph showing the effect of shortening, lengthening the β9 sequence and construct orientation (β9-FKBP or FKBP-β9) on enhanced complementation with FRB-SmTrip10 pep86 (β10) (SEQ ID NO: 25) in E. coli lysates containing LgTrip 3546 (SEQ ID NO: 51). [Figure 46] Graph showing the effect of shortening, lengthening the β9 sequence and construct orientation (β9-FKBP or FKBP-β9) on enhanced complementation with FRB-SmTrip10 pep289 (β10) (SEQ ID NO: 150) in E. coli lysates containing LgTrip 3546 (SEQ ID NO: 51). [Figure 47] Graphs showing the effect of shortening or lengthening the β9 sequence and construct orientation (β9-FKBP or FKBP-β9) on the fold induction (promoted complementation / spontaneous complementation) by FRB-β10 (SmTrip10 pep86 (SEQ ID NO: 25) or SmTrip10 pep289 (SEQ ID NO: 150)) in E. coli lysates containing LgTrip 3546 (SEQ ID NO: 51) (Summary of Figures 45 and 46). [Figure 48] Graph showing the effect of shortening and lengthening the β9 sequence and construct orientation (β9-FKBP or FKBP-β9) on complementation promoted by FRB-SmTrip10 pep86 (SEQ ID NO: 25) in HEK293 lysates containing LgTrip 3546 (SEQ ID NO: 51). [Figure 49] Graph showing the effect of shortening and lengthening the β9 sequence and construct orientation (β9-FKBP or FKBP-β9) on complementation promoted by FRB-SmTrip10 pep289 (SEQ ID NO: 150) in HEK293 lysates (LgTrip 3546 (SEQ ID NO: 51)). [Figure 50] Graphs showing the effect of shortening or lengthening the β9 sequence and construct orientation (β9-FKBP or FKBP-β9) on the fold induction (promoted complementation / spontaneous complementation) by FRB-β10 (SmTrip10 pep86 (SEQ ID NO: 25) or SmTrip10pep289 (SEQ ID NO: 150)) in HEK293 lysates (Summary of Figures 48 and 49). (LgTrip 3546 (SEQ ID NO: 51)). [Figure 51A] FIG. 1 is a schematic diagram showing an exemplary protein-protein interaction assay or analyte detection assay using peptide-tagged binding moieties. [Figure 51B] FIG. 1 is a schematic diagram showing an exemplary protein-protein interaction assay or analyte detection assay using peptide-tagged binding moieties. [Figure 51C] FIG. 1 is a schematic diagram showing an exemplary protein-protein interaction assay or analyte detection assay using peptide-tagged binding moieties. [Figure 51D] FIG. 1 is a schematic diagram showing an exemplary protein-protein interaction assay or analyte detection assay using peptide-tagged binding moieties. [Figure 51E] FIG. 1 is a schematic diagram illustrating the following exemplary immunoassays using the components and reagents described herein: Direct immunoassay. [Figure 51F] FIG. 1 is a schematic diagram illustrating the following exemplary immunoassays using the components and reagents described herein: Indirect immunoassay. [Figure 51G] FIG. 1 is a schematic diagram showing the following exemplary immunoassays using the components and reagents described herein: competitive direct immunoassay. [Figure 51H] FIG. 1 is a schematic diagram illustrating the following exemplary immunoassays using the components and reagents described herein: competitive indirect immunoassay. [Figure 52] 1 is a schematic diagram of an exemplary multiplexed tri-molecular lateral flow assay, useful, for example, in the detection of pathogens. [Figure 53]1 is a schematic diagram of an exemplary multiplexed tri-molecular lateral flow assay, which is useful, for example, in detecting anti-viral antibodies. [Figure 54] FIG. 1 is a schematic diagram of an exemplary antibody detection assay. [Figure 55] FIG. 1 is a schematic diagram of an exemplary bead-based assay. [Figure 56] FIG. 1 is a schematic diagram of an exemplary nucleic acid detection assay. [Figure 57] 1 is a graph showing complementation promoted by FRB-FKBP in E. coli lysates in which the AI ​​(Ala-Ile) dipeptide is absent from the linker in the construct (denoted by **) (LgTrip 3546 (SEQ ID NO: 51)). [Figure 58] 1 is a graph showing complementation promoted by FRB-FKBP in HEK293 lysate (containing LgTrip 3546 (SEQ ID NO: 51)) without the AI ​​sequence dipeptide in the linker within the construct (denoted by **). [Figure 59] 1 is a graph showing promotion of luminescent complex formation by FRB-FKBP by FRB-SmTrip10 pep86 and C-terminally extended FKBP-SmTrip9 peptides in E. coli lysates (containing LgTrip 3546 (SEQ ID NO: 51)). [Figure 60] 1 is a graph showing promotion of FRB-FKBP luminescent complex formation by FRB-SmTrip10 pep289 and the C-terminally extended FKBP-SmTrip9 peptide (LgTrip 3546 (SEQ ID NO: 51)) in E. coli lysates. [Figure 61] 1 is a graph showing promotion of FRB-FKBP luminescent complex formation by FRB-SmTrip10 pep86 and the C-terminally extended FKBP-SmTrip9 peptide (LgTrip 3546 (SEQ ID NO: 51)) in HEK293 lysates. [Figure 62]1 is a graph showing the promotion of luminescent complex formation by FRB-FKBP by shortening and lengthening the SmTrip9 peptide sequence in FRB-SmTrip10 pep86 and FKBP fusions in E. coli lysates (containing LgTrip 3546 (SEQ ID NO: 51)). [Figure 63] 1 is a graph showing the promotion of luminescent complex formation by FRB-FKBP by shortening and lengthening the SmTrip9 peptide sequence in FRB-SmTrip10 pep289 and FKBP fusions in E. coli lysates (LgTrip 3546 (SEQ ID NO: 51)). [Figure 64] 1 is a graph showing the promotion of luminescent complex formation by FRB-FKBP by shortening and lengthening the SmTrip9 peptide sequence in FRB-SmTrip10 pep86 and FKBP fusions in HEK293 lysates (LgTrip 3546 (SEQ ID NO: 51)). [Figure 65] 1 is a graph showing the promotion of luminescent complex formation by FRB-FKBP by shortening and lengthening the SmTrip9 peptide sequence in FRB-SmTrip10 pep86 and FKBP fusions in HEK293 lysates (LgTrip 3546 (SEQ ID NO: 51)). [Figure 66] 1 is a graph showing promotion of luminescent complex formation by FRB-FKBP by the FKBP-SmTrip9 soluble variant and FRB-SmTrip10 pep86 in E. coli lysates (LgTrip 3546 (SEQ ID NO: 51)). [Figure 67] 1 is a graph showing the promotion of luminescent complex formation by FRB-FKBP by the FKBP-SmTrip9 soluble variant and FRB-SmTrip10 pep289 in E. coli lysates (LgTrip 3546 (SEQ ID NO: 51)). [Figure 68] 1 is a graph showing promotion of luminescent complex formation by FRB-FKBP by the FKBP-SmTrip9 soluble variant and FRB-SmTrip10 pep86 in HEK293 lysates (LgTrip 3546 (SEQ ID NO: 51)). [Figure 69]1 is a graph showing promotion of luminescent complex formation by FRB-FKBP by the FKBP-SmTrip9 soluble variant and FRB-SmTrip10 pep289 in HEK293 lysates (LgTrip 3546 (SEQ ID NO: 51)). [Figure 70] 1 is a graph showing promotion of luminescent complex formation by FRB-FKBP by the FKBP-SmTrip9 soluble variant and FRB-SmTrip10 pep86 in E. coli lysates (LgTrip 3546 (SEQ ID NO: 51)). [Figure 71] 1 is a graph showing the promotion of luminescent complex formation by FRB-FKBP by the FKBP-SmTrip9 soluble variant and FRB-SmTrip10 pep289 in E. coli lysates (LgTrip 3546 (SEQ ID NO: 51)). [Figure 72] 1 is a graph showing promotion of luminescent complex formation by FRB-FKBP by the FKBP-SmTrip9 soluble variant and FRB-SmTrip10 pep86 in HEK293 lysates (LgTrip 3546 (SEQ ID NO: 51)). [Figure 73] 1 is a graph showing promotion of luminescent complex formation by FRB-FKBP by the FKBP-SmTrip9 soluble variant and FRB-SmTrip10 pep289 in HEK293 lysates (LgTrip 3546 (SEQ ID NO: 51)). [Figure 74] 1 is a table listing the affinities and Bmax of synthetic SmTrip9 soluble variants with C-terminal extensions (LgTrip 3546 (SEQ ID NO: 51)). [Figure 75] 1 is a table listing the affinities and Bmax of synthetic SmTrip9 soluble variants with C-terminal extensions (LgTrip 3546 (SEQ ID NO: 51)). [Figure 76] 1 is a table listing the Kd and Bmax of synthetic SmTrip9 variants with different blocked ends (LgTrip 3546 (SEQ ID NO: 51)). [Figure 77A] 1 is a table listing the solubility of synthetic SmTrip9 peptides. [Figure 77B] 1 is a table listing the solubility of synthetic SmTrip9 peptides. [Figure 78A] Graph showing the affinity of SmTrip9 pep286 (SEQ ID NO: 37) to SmTrip10 pep86 (HiBiT) / LgTrip fusion (SEQ ID NOs: 210 and 212). [Figure 78B] 1 is a graph showing the affinity of SmTrip9 pep759 (SEQ ID NO: 496) for various SmTrip10 pep86 (HiBiT) / LgTrip fusions. [Figure 79] 1 is a graph showing bioluminescence after 18 hours of exposure to increasing concentrations of detergent: NanoLuc (SEQ ID NO: 3), LgBiT (SEQ ID NO: 11), LgTrip 3546 (SEQ ID NO: 51). [Figure 80] 1 is a graph showing enzyme activity in the presence of increasing detergent concentrations: NanoLuc (SEQ ID NO: 3), LgBiT (SEQ ID NO: 11), LgTrip 3546 (SEQ ID NO: 51). [Figure 81] 1 is a graph demonstrating the reversibility of FRB-FKBP-promoted bioluminescent complex formation by LgBiT (SEQ ID NO: 11) and LgTrip 3546 (SEQ ID NO: 51). [Figure 82] 1 is a table listing the results of dilutions of various SmTrip10 peptides in the presence of constant SmTrip9 pep286 (SEQ ID NO: 37) and LgTrip 3546 (SEQ ID NO: 51). [Figure 83] 1 is a table listing the results of dilutions of various SmTrip10 peptides in the presence of constant SmTrip9 pep286 (SEQ ID NO: 37) and LgTrip 3546 (SEQ ID NO: 51) dilutions. [Figure 84] 1 is a graph showing bioluminescence from an Antares-type fusion (LgTrip 3546) with SmTrip9 pep263 (SEQ ID NO: 35) and SmTrip10 pep86 (SEQ ID NO: 25) or SmTrip10 pep86+SmTrip9 pep286 (SEQ ID NO: 37). [Figure 85A]1 is a graph showing the emission spectrum from an Antares-type fusion (LgTrip 3546) (SEQ ID NO: 51) with SmTrip9 pep263 (SEQ ID NO: 35). [Figure 85B] Graph showing emission spectra from an Antares-type fusion (LgTrip 3546) (SEQ ID NO: 51) with SmTrip pep86 (HiBiT, SEQ ID NO: 25) or SmTrip10 pep86 (HiBiT, SEQ ID NO: 25) + SmTrip9 pep286 (SEQ ID NO: 37). [Figure 86] 1 is a graph showing the dilution of LgBiT (SEQ ID NO: 11) and LgTrip 3546 (SEQ ID NO: 51) by the "dark" dipeptide 272 (SEQ ID NO: 146) in the presence of dipeptide pep263 (SEQ ID NO: 35). [Figure 87] 1 is a graph comparing the inhibition of LgTrip 3546 (SEQ ID NO: 51) and LgBiT (SEQ ID NO: 11) by dark dipeptides 272 (SEQ ID NO: 146) and 273 (SEQ ID NO: 298). [Figure 88] 1 is a graph showing the inhibition of LgBiT (SEQ ID NO: 11) and LgTrip 3546 (SEQ ID NO: 51) by dark BiT167 (SEQ ID NO: 300). [Figure 89] 10 is a graph showing FRB-FKBP promotion of luminescent complex formation in E. coli lysates by complementation of the FKBP-SmTrip9 pep434 (SEQ ID NO: 230) variant with LgTrip 3546 (SEQ ID NO: 51) and FRB-HiBiT (SEQ ID NO: 25). [Figure 90] 10 is a graph showing FRB-FKBP promotion of luminescent complex formation in E. coli lysates by complementation of SmTrip9 pep434 (SEQ ID NO: 230) variant with LgTrip 3546 (SEQ ID NO: 51) and FRB-SmTrip10 pep289 (VS-HiBiT, SEQ ID NO: 150). [Figure 91A](A) Graph showing FRB-FKBP promotion of luminescent complex formation in HEK293 lysates by complementation of SmTrip9 pep435 (SEQ ID NO: 231) and SmTrip9 pep434 (SEQ ID NO: 230) variants with LgTrip 3546 (SEQ ID NO: 51) and FRB-SmTrip10 pep86 (HiBiT, SEQ ID NO: 25). [Figure 91B] (B) Graph showing FRB-FKBP promotion of luminescent complex formation in HEK293 lysates by complementation of SmTrip9 pep435 (SEQ ID NO: 231) and SmTrip9 pep434 (SEQ ID NO: 230) variants with LgTrip 3546 (SEQ ID NO: 51) and FRB-SmTrip10 pep289 (SEQ ID NO: 150). [Figure 92] Table showing results of FRB-FKBP assay using SmTrip9 823 and 840 [Figure 93] 1 is a table listing the Kd and Bmax of synthetic SmTrip9 pep435 (SEQ ID NO: 231) and SmTrip9 pep434 (SEQ ID NO: 230) variants with LgTrip 3546 (SEQ ID NO: 51). [Figure 94] 10 is a graph demonstrating wt LgTrip 2098 (SEQ ID NO: 31) and LgTrip 3546 (SEQ ID NO: 51) with pep263 (SEQ ID NO: 35) or pep331 (SEQ ID NO: 301) as bioluminescent reagents for detecting endogenously tagged (e.g., by CRISPR / Cas9) GAPDH. [Figure 95A] 1 is an exemplary SmTrip10 chemical conjugate. An example of SmTrip10 with an N-terminal cysteine ​​modification for disulfide bond formation to solvent exposed or protected cysteine ​​targets on proteins / peptides / DNA and RNA oligonucleotides / small molecules, or proteins / peptides / DNA and RNA oligonucleotides / small molecules that have been prepared using maleimides for reaction with thiols such as cysteine, or N-hydroxysuccinimide esters (NHS esters) for reaction with amines such as lysine. [Figure 95B]1 shows exemplary SmTrip10 chemical conjugates with N-terminal azido-lysine modifications for copper-catalyzed or copper-free 1,3-dipolar cycloaddition reactions ("click") with unstrained or strained alkyne targets separately introduced into protein / peptide / DNA and RNA oligonucleotides / small molecules. [Figure 95C] 1 is an exemplary SmTrip10 chemical conjugate. This is an exemplary SmTrip10 with an N-terminal N-hydroxysuccinimide ester (NHS ester) for general conjugation to nucleophilic groups (e.g., lysine, other primary amines) of proteins, peptides, DNA, and RNA oligonucleotides / small molecules. The nucleophilic group can be present on the unmodified protein / oligo / small molecule or can be chemically added for this conjugation. [Figure 95D] 1 shows exemplary SmTrip10 chemical conjugates with N-terminal propargylglycine modifications for copper-catalyzed or copper-free 1,3-dipolar cycloaddition reactions ("click") with azide, diazo, or tetrazine targets chemically or biologically introduced into proteins / peptides / DNA and RNA oligonucleotides / small molecules, respectively. [Figure 95E] Exemplary SmTrip10 Chemical Conjugates: Exemplary SmTrip10 with an N-terminal propargylglycine modification and a C-terminal fluorophore (e.g., a BODIPY dye) for copper-catalyzed or copper-free 1,3-dipolar cycloaddition reactions ("click"). [Figure 96A] Exemplary SmTrip9 pep286 chemical conjugates. (A) Example of SmTrip9-286 with a C-terminal azido-lysine modification for copper-catalyzed or copper-free 1,3-dipolar cycloaddition reactions ("click") with unstrained or strained alkyne targets, separately introduced chemically or biologically into proteins / peptides / DNA and RNA oligonucleotides / small molecules. [Figure 96B]1 is an exemplary SmTrip9 pep286 chemical conjugate. An example of SmTrip9 pep286 with a C-terminal propargylglycine modification for copper-catalyzed or copper-free 1,3-dipolar cycloaddition reactions ("click") with azide, diazo, and tetrazine targets chemically or biologically introduced into proteins / peptides / DNA and RNA oligonucleotides / small molecules, respectively. [Figure 96C] 1 is an exemplary SmTrip9 pep286 chemical conjugate, with a C-terminal cysteine ​​modification for disulfide bond formation to solvent exposed or protected cysteine ​​targets on proteins / peptides / DNA and RNA oligonucleotides / small molecules, or proteins / peptides / DNA and RNA oligonucleotides / small molecules prepared with a maleimide handle or N-hydroxysuccinimide ester. [Figure 96D] 1 is an exemplary SmTrip9 pep286 chemical conjugate. This is an example of SmTrip9 pep286 with a C-terminal cysteine ​​modification and an N-terminal BODIPY dye. The dye is not limited to BODIPY and can be any fluorophore, BRET partner, or FRET dye / quencher partner. The dye can also be incorporated via any other combination of conjugation handles provided at the C-terminus. [Figure 96E] 1 is an exemplary SmTrip9 pep286 chemical conjugate. This is an example of SmTrip9 pep286 with a C-terminal N-hydroxysuccinimide ester (NHS ester) for general conjugation to nucleophilic targets (e.g., lysine) of proteins / peptides / DNA and RNA oligonucleotides / small molecules or proteins / peptides / DNA and RNA oligonucleotides / small molecules. [Figure 96F]1 is an exemplary SmTrip9 pep286 chemical conjugate. This is an example of SmTrip9 pep286 with a C-terminal N-hydroxysuccinimide ester (NHS ester) for general conjugation to nucleophilic targets (i.e., lysines) of proteins / peptides / DNA and RNA oligonucleotides / small molecules or proteins / peptides / DNA and RNA oligonucleotides / small molecules. [Figure 97A] 1 is an exemplary SmTrip9 pep521 chemical conjugate. This is an example of SmTrip9 pep521 with a C-terminal azido-lysine modification and an N-terminal BODIPY dye. The dye is not limited to BODIPY and can be any fluorophore, BRET partner, or FRET dye / quencher partner. Dyes can also be incorporated via any other combination of conjugation handles provided at the C-terminus. [Figure 97B] 1 shows exemplary SmTrip9 pep521 chemical conjugates: Examples of SmTrip9 pep521 with C-terminal azido-lysine modifications for copper-catalyzed or copper-free 1,3-dipolar cycloaddition reactions ("click") with unstrained or strained alkyne targets chemically or biologically introduced separately into proteins / peptides / DNA and RNA oligonucleotides / small molecules. [Figure 97C] 10. Exemplary SmTrip9 pep521 chemical conjugates: Examples of SmTrip9 pep521 with a C-terminal propargylglycine modification for copper-catalyzed or copper-free 1,3-dipolar cycloaddition reactions ("click") with azide, diazo, and tetrazine targets chemically or biologically introduced into proteins / peptides / DNA and RNA oligonucleotides / small molecules, respectively. [Figure 97D]1 is an exemplary SmTrip9 pep521 chemical conjugate, with a C-terminal cysteine ​​modification for disulfide bond formation to solvent exposed or protected cysteine ​​targets on proteins / peptides / DNA and RNA oligonucleotides / small molecules, or proteins / peptides / DNA and RNA oligonucleotides / small molecules prepared using a maleimide handle or NHS ester. [Figure 97E] 1 is an exemplary SmTrip9 pep521 chemical conjugate. An example of SmTrip9 pep521 with a C-terminal N-hydroxysuccinimide ester (NHS ester) for general conjugation to nucleophilic targets (i.e., lysines) of proteins / peptides / DNA and RNA oligonucleotides / small molecules. [Figure 97F] 1 is an exemplary SmTrip9 pep521 conjugate. An example of SmTrip9 pep521 with a C-terminal N-hydroxysuccinimide ester (NHS ester) for general conjugation to nucleophilic targets (i.e., lysines) of proteins / peptides / DNA and RNA oligonucleotides / small molecules. [Figure 98A] 1 is an exemplary SmTrip9 pep524 chemical conjugate. This is an example of SmTrip9 pep524 with a C-terminal azido-lysine modification and an N-terminal BODIPY dye. The dye is not limited to BODIPY and can be any fluorophore, BRET partner, or FRET dye / quencher partner. Dyes can be incorporated by any other combination of C-terminal conjugation handles. [Figure 98B] 1 is an exemplary SmTrip9 pep524 chemical conjugate. An example of SmTrip9 pep524 with a C-terminal azido-lysine modification for copper-catalyzed or copper-free 1,3-dipolar cycloaddition reactions ("click") with unstrained or strained alkyne targets, chemically or biologically introduced separately into proteins / peptides / DNA and RNA oligonucleotides / small molecules. [Figure 98C]1. Exemplary SmTrip9 pep524 chemical conjugates: Examples of SmTrip9 pep524 with a C-terminal propargylglycine modification for copper-catalyzed or copper-free 1,3-dipolar cycloaddition reactions ("click") with azide, diazo, and tetrazine targets chemically or biologically introduced into proteins / peptides / DNA and RNA oligonucleotides / small molecules, respectively. [Figure 98D] 1 is an exemplary SmTrip9 pep524 chemical conjugate, with a C-terminal cysteine ​​modification for disulfide bond formation to solvent exposed or protected cysteine ​​targets on proteins / peptides / DNA and RNA oligonucleotides / small molecules, or proteins / peptides / DNA and RNA oligonucleotides / small molecules prepared using a maleimide handle or NHS ester. [Figure 98E] 1 is an exemplary SmTrip9 pep524 chemical conjugate. An example of SmTrip9 pep524 with a C-terminal N-hydroxysuccinimide ester (NHS ester) for general conjugation to nucleophilic targets (i.e., lysines) of proteins / peptides / DNA and RNA oligonucleotides / small molecules. [Figure 98F] 1 is an exemplary SmTrip9 pep524 chemical conjugate. An example of SmTrip9 pep524 with a C-terminal N-hydroxysuccinimide ester (NHS ester) for general conjugation to nucleophilic targets (i.e., lysines) of proteins / peptides / DNA and RNA oligonucleotides / small molecules. [Figure 99A] 1 shows an exemplary peptide-oligomer probe. A peptide displaying a reactive azide group is conjugated to an oligonucleotide displaying a reactive alkyne group to form an exemplary peptide-oligomer probe. 1 shows a peptide-oligomer conjugate of SmTrip9 pep286 (w / azide group) conjugated to a DNA oligomer containing a 5′-terminal alkyne functionality via copper-catalyzed “click” 1,3-cycloaddition. [Figure 99B] 1 shows an exemplary peptide-oligomer probe. A peptide displaying a reactive azide group is attached to an oligonucleotide displaying a reactive alkyne group to form an exemplary peptide-oligomer probe. Figure 1 shows a peptide-oligomer conjugate of SmTrip10 pep86 (HiBiT) (w / azide group) conjugated to a DNA oligomer containing a 3'-terminal alkyne functionality via copper-catalyzed "click" 1,3-cycloaddition. [Figure 100] 1 is a graph showing screening of G147 site saturation variants of SmTrip9. [Figure 101] 1 is a graph showing screening of K148 site saturation variants of SmTrip9. [Figure 102] 1 is a graph showing screening of M149 site saturation variants of SmTrip9. [Figure 103] 1 is a graph showing screening of L150 site saturation variants of SmTrip9. [Figure 104] 1 is a graph showing screening of F151 site saturation variants of SmTrip9. [Figure 105] 1 is a graph showing screening of R152 site saturation variants of SmTrip9. [Figure 106] 1 is a graph showing screening of V153 site saturation variants of SmTrip9. [Figure 107] 1 is a graph showing screening of T154 site saturation variants of SmTrip9. [Figure 108] 1 is a graph showing screening of I155 site saturation variants of SmTrip9. [Figure 109] 1 is a graph showing screening of N156 site saturation variants of SmTrip9. [Figure 110] 1 is a graph showing screening of S157 site saturation variants of SmTrip9. [Figure 111] 1 is a graph showing screening of W158 site saturation variants of SmTrip9. [Figure 112] 1 is a graph showing screening of K149 site saturation variants of SmTrip9. [Figure 113] 10 is a table of results of FRB-FKBP-promoted complementation with SmTrip9 pep435 / 434 in E. coli lysates. [Figure 114] 10 is a table of results of FRB-FKBP-promoted complementation with SmTrip9 pep435 / 434 in E. coli lysates. [Figure 115] 10 is a table of results of FRB-FKBP-promoted complementation with SmTrip9 pep435 / 434 in E. coli lysates. [Figure 116-1] 10 is a table of results of FRB-FKBP-facilitated complementation assays with combined SmTrip9 variants. [Figure 116-2] 10 is a table of results of FRB-FKBP-facilitated complementation assays with combined SmTrip9 variants. [Figure 116-3] 10 is a table of results of FRB-FKBP-facilitated complementation assays with combined SmTrip9 variants. [Figure 117] 10 is a table of results of FRB-FKBP-facilitated complementation assays with combined SmTrip9 variants. [Figure 118] 10 is a table of results of FRB-FKBP-facilitated complementation assays with combined SmTrip9 variants. [Figure 119] 10 is a table of results of FRB-FKBP-facilitated complementation assays with combined SmTrip9 variants. [Figure 120] 10 is a table of results of FRB-FKBP-facilitated complementation assays with combined SmTrip9 variants. [Figure 121] 10 is a table of results of FRB-FKBP-facilitated complementation assays with combined SmTrip9 variants. [Figure 122A] 10 is a table of results of FRB-FKBP-facilitated complementation assays with combined SmTrip9 variants. [Figure 122B]10 is a table of results of FRB-FKBP-facilitated complementation assays with combined SmTrip9 variants. [Figure 123] 1 is a table of Kd and Bmax of SmTrip9 synthetic peptides. [Figure 124] 1 is a table of Kd and Bmax of SmTrip9 synthetic peptides. [Figure 125] 1 is a table of Kd and Bmax of SmTrip9 synthetic peptides. [Figure 126] 1 is a table of Kd and Bmax of SmTrip9 synthetic peptides. [Figure 127] 1 is a table of Kd and Bmax of SmTrip9 synthetic peptides. [Figure 128] 1 is a table of Kd and Bmax of SmTrip9 synthetic peptides. [Figure 129] 1 is a table of Kd and Bmax of SmTrip9 synthetic peptides. [Figure 130] 1 is a table of Kd and Bmax of SmTrip9 synthetic peptides. [Figure 131A] 1 is a table of the solubility of synthetic SmTrip9 peptides. [Figure 131B] 1 is a table of the solubility of synthetic SmTrip9 peptides. [Figure 131C] 1 is a table of the solubility of synthetic SmTrip9 peptides. [Figure 132] 1 is a graph of biochemical co-titration of SmTrip9 synthetic peptide and pep289. [Figure 133] 16 is a graph of biochemical co-dilution of SmTrip9 synthetic peptide and pep289. [Figure 134] Graph of biochemical co-dilution of SmTrip9 and SmTrip10 synthetic peptides. [Figure 135] Graph of biochemical co-dilution of pep521 and alternative SmTrip10 synthetic peptides. [Figure 136] SDS PAGE gel of strand removal (purification) from LgTrip 3546 template. [Figure 137A] 1 is a graph of strand removal proteins with various combinations of peptides. [Figure 137B] 1 is a graph of strand removal proteins with various combinations of peptides. [Figure 137C] 1 is a graph of strand removal proteins with various combinations of peptides. [Figure 137D] 1 is a graph of strand removal proteins with various combinations of peptides. [Figure 138A] Graph of 6, 7, 8, 9, or 10 strand removal (purification) from the LgTrip 3546 template. [Figure 138B] Graph of 6, 7, 8, 9, or 10 strand removal (purification) from the LgTrip 3546 template. [Figure 139A] 1 is a graph of Kd and Bmax values ​​of dipeptide dilutions. [Figure 139B] 1 is a graph of Kd and Bmax values ​​of dipeptide dilutions. [Figure 139C] 1 is a graph of Kd and Bmax values ​​of dipeptide dilutions. [Figure 139D] 1 is a graph of Kd and Bmax values ​​of dipeptide dilutions. [Figure 139E] 1 is a graph of Kd and Bmax values ​​of dipeptide dilutions. [Figure 140] FIG. 1 is a schematic diagram showing the approach taken to develop a solution-based homogeneous quantitative assay for the anti-TNFα biologics Remicade, Humira, and Enbrel using a trimolecular Protein G and TNFα fusion protein. [Figure 141] 1 is a graph showing quantitative analysis of TNFα inhibitor dose response by accelerated complementation using SmTrip9 pep521-Protein G (SEQ ID NO: 268) and TNFα-SmTrip10 pep289 (VS-HiBiT, SEQ ID NO: 150) fusion proteins with purified LgTrip 3546 (SEQ ID NO: 51) in a solution-based homogeneous assay. [Figure 142]1 is a graph showing quantitative analysis of 10 nM infliximab by facilitated complementation using SmTrip9 pep521-Protein G (SEQ ID NO: 268) and TNFα-SmTrip10 pep289 (VS-HiBiT, SEQ ID NO: 150) fusion proteins with purified LgTrip 3546 (SEQ ID NO: 51) in the presence of complex sample matrices including human serum and urine using a solution-based homogeneous assay. [Figure 143] 1 is a graph showing the binding kinetics of signal generation indicative of 100 pM infliximab by accelerated complementation using SmTrip9 pep521-Protein G (SEQ ID NO: 268) and TNFα-SmTrip10 pep289 (VS-HiBiT, SEQ ID NO: 150) fusion proteins with purified LgTrip 3546 (SEQ ID NO: 51) in a solution-based homogeneous assay. [Figure 144] 1 is a graph showing signal generation indicative of 10 nM infliximab by accelerated complementation using different SmTrip9 pep(X)-Protein G variants and TNFα-SmTrip10 pep289 (VS-HiBiT, SEQ ID NO: 150) fusion protein with purified LgTrip 3546 (SEQ ID NO: 51) in a solution-based homogeneous assay. [Figure 145] FIG. 1 is a schematic diagram showing the approach taken to develop a homogeneous cell-based quantitative assay for the anti-EGFR biologics panitumumab and cetuximab using HEK293 cells expressing SmTrip9-Protein G fusion protein and SmTrip10 pep289-EGFR (SEQ ID NO: 150). [Figure 146] 1 is a graph showing quantification of panitumumab by accelerated complementation using cells expressing SmTrip9 pep521-Protein G (SEQ ID NO: 268) fusion protein and SmTrip10 pep289-EGFR (VS-HiBiT, SEQ ID NO: 150) with purified LgTrip 3546 (SEQ ID NO: 51) in a cell-based homogeneous assay. [Figure 147]1 is a graph showing real-time binding kinetics of signal generation measuring cetuximab by accelerated complementation with cells expressing SmTrip9 pep521-Protein G (SEQ ID NO: 268) fusion protein and SmTrip10 pep289-EGFR (VS-HiBiT, SEQ ID NO: 150) in a cell-based homogeneous assay. [Figure 148] 1 is a graph showing signal generation indicative of 1 nM panitumumab by complementation promoted by cells expressing different SmTrip9 pep(X)-Protein G variants and SmTrip10 pep289-EGFR (VS-HiBiT, SEQ ID NO: 150) in combination with purified LgTrip 3546 (SEQ ID NO: 51) in a cell-based homogeneous assay. [Figure 149] 1 is a graph showing quantification of panitumumab dose response by complementation promoted by cells expressing different SmTrip9 pep(X)-Protein G variants and SmTrip10 pep289-EGFR (VS-HiBiT, SEQ ID NO: 150) in combination with purified LgTrip 3546 (SEQ ID NO: 51) in a cell-based homogeneous assay. [Figure 150] Figure 1 shows the quantification of human IL-1 beta using paired antibodies chemically labeled with HaloTag-SmTrip9 pep521 (SEQ ID NO: 268) and HaloTag-SmTrip10 pep289 (SEQ ID NO: 150) in a solution-based homogeneous assay. Figure 2 shows the real-time binding kinetics of human troponin using paired antibodies chemically labeled with NanoTrip. [Figure 151] 1 is a graph showing real-time binding kinetics for quantification of human troponin using paired antibodies chemically labeled with HaloTag-SmTrip9 pep521 (SEQ ID NO: 268) and HaloTag-SmTrip10 pep289 (SEQ ID NO: 150) in a solution-based homogeneous assay. [Figure 152]A specific peptide responsible for targeting proteins to specific subcellular compartments was fused to the LgBiT-HaloTag. (A) LgBiT-Membrane Sensor: LgBiT is green, and the nucleus is blue. (B) LgBiT-Nuclear Sensor: LgBiT is green, and the nucleus is blue. (C) LgBiT-Mitochondria Sensor: LgBiT is green, MitoTracker is red, and the nucleus is blue. (D) LgBiT-ER Sensor: LgBiT is green, the ER marker is red, and the nucleus is blue. [Figure 153] Translocation assay. POIs are endogenously tagged with HiBiT. Upon stimulation, the POIs translocate to different intracellular compartments, such as the nucleus. The LgBiT-nucleus sensor can be used to detect this translocation event, as HiBiT contacts LgBiT, resulting in a luminescent signal. [Fig. 154] Membrane translocation assay using wild-type LgBiT sensor. PKCα-HiBiT cell lines were transfected with wild-type LgBiT-membrane sensor. The strong interaction between LgBiT and HiBiT resulted in spontaneous complementation, which resulted in no response to PMA stimulation. [Figure 155] Membrane translocation assay using the LgBiT* sensor (SEQ ID NO: 979). PKCα-HiBiT cell lines were transfected with different amounts of DNA encoding the LgBiT*-membrane sensor. Upon PMA treatment, PKCα-HiBiT translocates to the plasma membrane, where the LgBiT*-membrane sensor remains. Assembly between HiBiT and LgBiT* generates a luminescent signal that is proportional to the amount of PKCα-HiBiT on the membrane. The assay is sensitive and robust. Dilutions of PMA yielded similar EC50 values ​​(EC50 = 2.0 nM) regardless of the sensor input. The fold response varied between 12- and 19-fold, depending on the sensor input. [Figure 156]Nuclear translocation assay using the LgBiT* sensor. The p65-HiBiT cell line was transfected with DNA encoding the LgBiT* nuclear sensor. Addition of TNFα recruits p65 to the nucleus, where the LgBiT* nuclear sensor is localized. Complementation occurs between HiBiT and LgBiT*, resulting in the production of light. The signal intensity reflects the concentration of p65 in the nucleus. Dilution of TNFα resulted in an EC50 of 0.7 ng / mL with a response fold of 4. Real-time measurements showed that 30 minutes was required for p65 to reach maximum accumulation in the nucleus upon stimulation. [Figure 157A] (A) Graph showing the affinity and Bmax of LgBiT variants for HiBiT. [Figure 157B] (B) Table showing the affinity and Bmax of LgBiT variants for HiBiT. [Figure 158] Graph showing the affinity of LgBiT variant lysates for HiBiT. [Figure 159] 1 is a graph and table showing the relative bioluminescence produced in the presence of substrate for various combinations of truncated luciferase polypeptides and peptide components. [Figure 160] LgTrip was diluted to 0.2 nM in TBS + 0.01% BSA + 0.01% Tergitol and pep289 was added to 25 μM. This solution was used as the diluent for a 5-fold serial dilution series of the SmTrip9 peptide. Samples were equilibrated at room temperature for 10 minutes and then dispensed in triplicate onto the assay plate. 20 μM furimazine in TBS + 0.01% BSA + 0.01% Tergitol was added to the samples at a 1:1 (volume:volume) ratio. The plate was incubated for 10 minutes and luminescence was read. To measure VS-HiBiT Kd, the same protocol was followed, except that saturating dilutions of SmTrip9 (20× Kd) and VS-HiBiT were used. [Figure 161]LgTrip was diluted to 0.2 nM in TBS + 0.01% BSA + 0.01% Tergitol and pep289 was added to 25 μM. This solution was used as the diluent for a 5-fold serial dilution series of the SmTrip9 peptide. Samples were equilibrated at room temperature for 10 minutes and then dispensed in triplicate onto the assay plate. 20 μM furimazine in TBS + 0.01% BSA + 0.01% Tergitol was added to the samples at a 1:1 (volume:volume) ratio. The plate was incubated for 10 minutes and luminescence was read. To measure VS-HiBiT Kd, the same protocol was followed, except that saturating dilutions of SmTrip9 (20× Kd) and VS-HiBiT were used. [Figure 162] Dipeptides were diluted to 5 μM and serially diluted 5-fold using TBS + 0.01% BSA + 0.01% Tergitol containing 0.2 nM LgTrip as diluent. Samples were incubated at room temperature for 10 minutes and added to the assay plate in triplicate. A 20-fold dilution of live cell furimazine substrate was added to the samples, 1:1 (volume:volume) in TBS + 0.01% BSA + 0.01% Tergitol, and the plate was read after 10 minutes on a GloMax® luminometer. [Figure 163] LgTrip variants were grown overnight at 37°C in LB containing 100 μg / ml ampicillin. Cells were diluted 20-fold in induction medium (LB containing 100 μg / ml ampicillin and 0.1% rhamnose (w / v)) and induced for 4 hours at 37°C with shaking. 10 μl of each induced sample was diluted with 250 μl of lysis buffer (0.3×PLB + 25 mM HEPES pH 7.5). 80 μl of the lysate was further diluted with 2 ml of lysis buffer. A 10-fold dilution series of pep788 (SEQ ID NO: 414) was performed starting with 10 μM peptide using Nano-Glo with 50 μM furimazine as diluent. Peptide dilutions and lysate were mixed 1:1 (volume:volume), incubated at room temperature for 10 minutes, and luminescence was read. [Fig. 164]LgTrip variants were grown overnight at 37°C in LB containing 100 μg / ml ampicillin. Cells were diluted 20-fold in induction medium (LB containing 100 μg / ml ampicillin and 0.1% (w / v) rhamnose) and induced for 4 hours at 37°C with shaking. 10 μl of each induced sample was diluted with 250 μl of lysis buffer (0.3×PLB + 25 mM HEPES pH 7.5). 80 μl of lysate was further diluted with 2 ml of lysis buffer. A 5-fold dilution series of pep759 (SEQ ID NO: 496) was performed starting with 50 μM peptide using Nano-Glo with 50 μM furimazine and 50 μM pep289 (SEQ ID NO: 826) as diluents. Peptide dilutions and lysate were mixed 1:1 (volume:volume), incubated at room temperature for 10 minutes, and luminescence was read. [Figure 165] LgTrip variants were grown overnight at 37°C in LB containing 100 μg / ml ampicillin. Cells were diluted 20-fold into induction medium (LB containing 100 μg / ml ampicillin and 0.1% (w / v) rhamnose) and induced for 20 hours at 25°C with shaking. 20 μl of each induction solution was diluted with 40 μl of lysis buffer (0.3× PLB + 25 mM HEPES pH 7.5) and lysed for 15 minutes at room temperature. The lysates were diluted 1,000-fold in 1× TBS + 0.01% BSA. 50 μl of each sample was transferred to a PCR plate and incubated at 80°C for 1.5 hours in a thermocycler. Controls were incubated on ice. Samples were equilibrated to room temperature and diluted 1:100 in 1× TBS + 0.01% BSA. 25 μl of each sample was transferred to an assay plate and mixed with 25 μl of 400 nM pep788 (SEQ ID NO: 414) / (TBS + 0.01% BSA + 20x diluted live cell furimazine substrate). Samples were incubated at room temperature for 10 minutes and luminescence was read. [Figure 166]LgTrip variants were purified using the Promega MagneHis™ Protein Purification System according to the manufacturer's protocol and diluted to 0.2 nM in TBS + 0.01% BSA + 0.01% Tergitol + 25 μM pep788. This solution was used as the diluent for a 5-fold serial dilution series of SmTrip9 peptide. Samples were equilibrated at room temperature for 10 minutes and dispensed in triplicate into assay plates. 20 μM furimazine in TBS + 0.01% BSA + 0.01% Tergitol was added to the samples in a 1:1 (volume:volume) ratio. The plate was incubated for 10 minutes and luminescence was read. To measure VS-HiBiT Kd, the same protocol was followed, except that saturating dilutions of SmTrip9 (25 μM) and VS-HiBiT were used. [Figure 167] The LgTrip variants were purified using the Promega MagneHis™ Protein Purification System according to the manufacturer's protocol and diluted to 0.2 nM in TBS + 0.01% BSA + 0.01% Tergitol + 25 μM pep840. This solution was used as the diluent for a 5-fold serial dilution series of the SmTrip9 peptide. Samples were equilibrated at room temperature for 10 minutes and dispensed in triplicate into assay plates. 20 μM furimazine in TBS + 0.01% BSA + 0.01% Tergitol was added to the samples in a 1:1 (volume:volume) ratio. The plate was incubated for 10 minutes, and luminescence was read. To measure VS-HiBiT Kd, the same protocol was followed, except that saturating dilutions of SmTrip9 (25 μM) and VS-HiBiT were used. [Figure 168]The LgTrip variants were purified using the Promega MagneHis™ Protein Purification System according to the manufacturer's protocol and diluted to 0.2 nM in TBS + 0.01% BSA + 0.01% Tergitol + 25 μM pep289. This solution was used as the diluent for a 5-fold serial dilution series of the SmTrip9 peptide. Samples were equilibrated at room temperature for 10 minutes and dispensed in triplicate into assay plates. 20 μM furimazine in TBS + 0.01% BSA + 0.01% Tergitol was added to the samples in a 1:1 (volume:volume) ratio. The plate was incubated for 10 minutes, and luminescence was read. To measure VS-HiBiT Kd, the same protocol was followed, except that saturating dilutions of SmTrip9 (25 μM) and VS-HiBiT were used. [Figure 169] MagneHis-purified LgTrip variants were diluted to 20 nM in TBS + 0.01% BSA. Triplicate 100 μl aliquots of each sample were loaded into 200 μl thin-walled PCR tubes. Samples were incubated at 70°C in a thermal cycler. At various time points, samples were withdrawn and equilibrated to room temperature. Samples were diluted to 0.2 nM (5 μl in 495 μl) in TBS + 0.01% BSA. 25 μl of each diluted sample was mixed with 25 μl of TBS + 0.01% BSA + 20-fold diluted live cell substrate furimazine + 400 nM pep788 (SEQ ID NO: 414). Samples were incubated for 10 minutes and then read in GMM+. Half-lives were calculated by nonlinear regression. [Figure 170]Cultures of FKBP_SmTrip9 variants and FRB-SmTrip10 were grown overnight at 37°C in LB + 100µg / ml ampicillin. Cells were diluted 1:20 into LB containing 0.15% glucose, 0.1% rhamnose, and 100µg / ml ampicillin. Cultures were induced at 25°C with shaking for approximately 20 hours. PLB assay reagent was prepared using 444nM MagneHis-purified LgTrip variants, 90-fold diluted FRB-SmTrip10 cultures, and + / -35nM rapamycin. 90µl of assay reagent was added to each well of a 96-well assay plate. FKBP_SmTrip9 cultures were diluted 1:10 in PLB, and 10µl was added to the assay plate. Samples were incubated at room temperature for 30 minutes. 100 μl of Nano-Glo containing 50 uM furimazine was added to the assay plate wells and luminescence was read after 5 minutes on a GloMax® luminometer. [Figure 171] Cultures of FKBP_SmTrip9 variants and FRB-SmTrip10 were grown overnight at 37°C in LB + 100µg / ml ampicillin. Cells were diluted 1:20 into LB containing 0.15% glucose, 0.1% rhamnose, and 100µg / ml ampicillin. Cultures were induced at 25°C with shaking for approximately 20 hours. PLB assay reagent was prepared using 444nM MagneHis-purified LgTrip variants, 90-fold diluted FRB-SmTrip10 cultures, and + / -35nM rapamycin. 90µl of assay reagent was added to each well of a 96-well assay plate. FKBP_SmTrip9 cultures were diluted 1:10 in PLB, and 10µl was added to the assay plate. Samples were incubated at room temperature for 30 minutes. 100 μl of Nano-Glo containing 50 uM furimazine was added to the assay plate wells and luminescence was read after 5 minutes on a GloMax® luminometer. [Fig. 172]LgTrip variants were grown overnight at 37°C in LB containing 100 μg / ml ampicillin. Cells were diluted 20-fold into induction medium (LB containing 100 μg / ml ampicillin and 0.1% (w / v) rhamnose) and induced for 20 hours at 25°C with shaking. 20 μl of each induction solution was diluted with 40 μl of lysis buffer (0.3× PLB + 25 mM HEPES pH 7.5) and lysed for 15 minutes at room temperature. The lysates were diluted 1,000-fold in 1× TBS + 0.01% BSA. 50 μl of each sample was transferred to a PCR plate and incubated at 70°C for 1.5 hours in a thermocycler. Controls were incubated on ice. Samples were equilibrated to room temperature and diluted 1:100 in 1× TBS + 0.01% BSA. 25 μl of each sample was transferred to an assay plate and mixed with 25 μl of 400 nM pep788 (SEQ ID NO: 414) / (TBS + 0.01% BSA + 20x diluted live cell furimazine substrate). Samples were incubated at room temperature for 10 minutes and luminescence was read. [Figure 173] LgTrip variants were grown overnight at 37°C in LB containing 100 μg / ml ampicillin. Cells were diluted 20-fold into induction medium (LB containing 100 μg / ml ampicillin and 0.1% (w / v) rhamnose) and induced for 20 hours at 25°C with shaking. 20 μl of each induction solution was diluted with 40 μl of lysis buffer (0.3x PLB + 25 mM HEPES pH 7.5) and lysed for 15 minutes at room temperature. Lysates were diluted 1,000-fold in 1x TBS + 0.01% BSA. 50 μl of each sample was transferred to a PCR plate and incubated in a Veritas thermocycler at two temperature gradients: 75°C to 100°C over 10 minutes, or 50°C to 75°C over 1.5 hours. Controls were incubated on ice. Samples were equilibrated to room temperature and diluted 1:100 in 1x TBS + 0.01% BSA. 25 μl of each sample was transferred to an assay plate and mixed with 25 μl of 400 nM pep788 (SEQ ID NO: 414) / (TBS + 0.01% BSA + 20x diluted live cell furimazine substrate). Samples were incubated at room temperature for 10 minutes and luminescence was read. [Fig. 174]MagneHis-purified LgTrip variants were diluted to 20 nM in TBS + 0.01% BSA. Triplicate 100 μl aliquots of each sample were loaded into 200 μl thin-walled PCR tubes. Samples were incubated at 70°C in a thermal cycler. At various time points, samples were withdrawn and equilibrated to room temperature. Samples were diluted to 0.2 nM (5 μl in 495 μl) in TBS + 0.01% BSA. 25 μl of each diluted sample was mixed with 25 μl of TBS + 0.01% BSA + 20-fold diluted live cell substrate furimazine + 400 nM pep788 (SEQ ID NO: 414). Samples were incubated for 10 minutes and then read in GMM+. [Figure 175] The LgTrip variants were purified using the Promega MagneHis™ Protein Purification System according to the manufacturer's protocol and diluted to 0.2 nM in TBS + 0.01% BSA + 0.01% Tergitol + 25 μM pep521. This solution was used as the diluent for a 5-fold serial dilution series of the SmTrip9 peptide. Samples were equilibrated at room temperature for 10 minutes and dispensed in triplicate into assay plates. 20 μM furimazine in TBS + 0.01% BSA + 0.01% Tergitol was added to the samples in a 1:1 (volume:volume) ratio. The plate was incubated for 10 minutes, and luminescence was read. To measure VS-HiBiT Kd, the same protocol was followed, except that saturating dilutions of SmTrip9 (12.5 μM) and VS-HiBiT were used. [Figure 176]The LgTrip variants were purified using the Promega MagneHis™ Protein Purification System according to the manufacturer's protocol and diluted to 0.2 nM in TBS + 0.01% BSA + 0.01% Tergitol + 25 μM pep840. This solution was used as the diluent for a 5-fold serial dilution series of the SmTrip9 peptide. Samples were equilibrated at room temperature for 10 minutes and dispensed in triplicate into assay plates. 20 μM furimazine in TBS + 0.01% BSA + 0.01% Tergitol was added to the samples in a 1:1 (volume:volume) ratio. The plate was incubated for 10 minutes and luminescence was read. To measure VS-HiBiT Kd, the same protocol was followed, except that saturating dilutions of SmTrip9 (12.5 μM) and VS-HiBiT were used. [Figure 177] The LgTrip variants were purified using the Promega MagneHis™ Protein Purification System according to the manufacturer's protocol and diluted to 0.2 nM in TBS + 0.01% BSA + 0.01% Tergitol + 25 μM pep289. This solution was used as the diluent for a 5-fold serial dilution series of the SmTrip9 peptide. Samples were equilibrated at room temperature for 10 minutes and dispensed in triplicate into assay plates. 20 μM furimazine in TBS + 0.01% BSA + 0.01% Tergitol was added to the samples in a 1:1 (volume:volume) ratio. The plate was incubated for 10 minutes, and luminescence was read. To measure VS-HiBiT Kd, the same protocol was followed, except that saturating dilutions of SmTrip9 (25 μM) and VS-HiBiT were used. [Figure 178]The LgTrip variants were purified using the Promega MagneHis™ Protein Purification System according to the manufacturer's protocol and diluted to 0.2 nM in TBS + 0.01% BSA + 0.01% Tergitol + 25 μM pep289. This solution was used as the diluent for a 5-fold serial dilution series of the SmTrip9 peptide. Samples were equilibrated at room temperature for 10 minutes and dispensed in triplicate into assay plates. 20 μM furimazine in TBS + 0.01% BSA + 0.01% Tergitol was added to the samples in a 1:1 (volume:volume) ratio. The plate was incubated for 10 minutes, and luminescence was read. To measure VS-HiBiT Kd, the same protocol was followed, except that saturating dilutions of SmTrip9 (25 μM) and VS-HiBiT were used. [Figure 179] The LgTrip variants were purified using the Promega MagneHis™ Protein Purification System according to the manufacturer's protocol and diluted to 0.2 nM in TBS + 0.01% BSA + 0.01% Tergitol + 25 μM pep289. This solution was used as the diluent for a 5-fold serial dilution series of the SmTrip9 peptide. Samples were equilibrated at room temperature for 10 minutes and dispensed in triplicate into assay plates. 20 μM furimazine in TBS + 0.01% BSA + 0.01% Tergitol was added to the samples in a 1:1 (volume:volume) ratio. The plate was incubated for 10 minutes and luminescence was read. To measure VS-HiBiT Kd, the same protocol was followed, except that saturating dilutions of SmTrip9 (20× Kd) and VS-HiBiT were used. [Figure 180]The LgTrip variants were purified using the Promega MagneHis™ Protein Purification System according to the manufacturer's protocol and diluted to 0.2 nM in TBS + 0.01% BSA + 0.01% Tergitol + 25 μM pep289. This solution was used as the diluent for a 5-fold serial dilution series of the SmTrip9 peptide. Samples were equilibrated at room temperature for 10 minutes and dispensed in triplicate into assay plates. 20 μM furimazine in TBS + 0.01% BSA + 0.01% Tergitol was added to the samples in a 1:1 (volume:volume) ratio. The plate was incubated for 10 minutes and luminescence was read. To measure VS-HiBiT Kd, the same protocol was followed, except that saturating dilutions of SmTrip9 (20× Kd) and VS-HiBiT were used. [Figure 181] LgTrip variant cultures were grown overnight at 37°C in LB + 100µg / ml ampicillin. Cells were diluted 1:20 in LB containing 0.15% glucose, 0.1% rhamnose, and 100µg / ml ampicillin and induced for approximately 20 hours at 25°C with shaking. Cells were diluted 1000-fold in PLB assay reagent (0.3x PLB + 25mM HEPES pH 7.5) and lysed for 20 minutes. Five-fold serial dilutions of SmTrip9 pep840 were made in NanoGlo + 50µM furimazine + 25µM pep289 and mixed 1:1 (volume:volume) with LgTrip lysis solution. Samples were incubated for 10 minutes at room temperature and read on a GloMax® luminometer. [Figure 182]The LgTrip variants were purified using the Promega MagneHis™ Protein Purification System according to the manufacturer's protocol and diluted to 0.2 nM in TBS + 0.01% BSA + 0.01% Tergitol + 25 μM pep289. This solution was used as the diluent for a 5-fold serial dilution series of the SmTrip9 peptide. Samples were equilibrated at room temperature for 10 minutes and dispensed in triplicate into assay plates. 20 μM furimazine in TBS + 0.01% BSA + 0.01% Tergitol was added to the samples in a 1:1 (volume:volume) ratio. The plate was incubated for 10 minutes, and luminescence was read. To measure VS-HiBiT Kd, the same protocol was followed, except that saturating dilutions of SmTrip9 (25 μM) and VS-HiBiT were used. [Figure 183] MagneHis-purified LgTrip variants were diluted to 20 nM in TBS + 0.01% BSA. Triplicate 100 μl aliquots of each sample were loaded into 200 μl thin-walled PCR tubes. Samples were incubated at 70°C in a thermal cycler. At various time points, samples were withdrawn and equilibrated to room temperature. Samples were diluted to 0.2 nM (5 μl in 495 μl) in TBS + 0.01% BSA. 25 μl of each diluted sample was mixed with 25 μl of TBS + 0.01% BSA + 20-fold diluted live cell substrate furimazine + 200 nM pep900 (SEQ ID NO: 907). Samples were incubated for 10 minutes and then read in GMM+. Half-lives were calculated by nonlinear regression. [Figure 184]Cultures of FKBP_SmTrip9 variants and FRB-SmTrip10 were grown overnight at 37°C in LB + 100µg / ml ampicillin. Cells were diluted 1:20 into LB containing 0.15% glucose, 0.1% rhamnose, and 100µg / ml ampicillin. Cultures were induced for approximately 20 hours at 25°C with shaking. PLB assay reagent was prepared using 444nM MagneHis-purified LgTrip variants, 90x diluted FRB-SmTrip10 cultures, and + / - 35nM rapamycin. 90µl of assay reagent was added to each well of a 96-well assay plate. FKBP_SmTrip9 cultures were diluted 1:10 in PLB, and 10µl was added to the assay plate. Samples were incubated at room temperature for 30 minutes. 100 μl of Nano-Glo containing 50 uM furimazine was added to the assay plate wells and luminescence was read after 5 minutes on a GloMax® luminometer. [Figure 185] LgTrip variant cultures were grown overnight at 37°C in LB + 100µg / ml ampicillin. Cells were diluted 1:20 in LB containing 0.15% glucose, 0.1% rhamnose, and 100µg / ml ampicillin and induced for approximately 20 hours at 25°C with shaking. Cells were diluted 5000-fold in 0.3x PLB assay reagent and lysed for 20 minutes. Five-fold serial dilutions of the dipeptides pep788, pep900, or SmTrip9 pep840, containing the saturating pep289, were made in Nano-Glo + 50µM furimazine and mixed 1:1 (volume:volume) with the LgTrip variant lysis solution. Samples were incubated for 10 minutes at room temperature and read on a GloMax® luminometer. Bmax was calculated by nonlinear regression. [Figure 186]LgTrip variants were diluted to 20 nM in 2 ml TBS + 0.01% BSA. 100 μl of each sample was dispensed into a 96-well PCR plate in duplicate, with two rows per plate (two plates were prepared). The plates were incubated in a Veritas thermocycler for 3 hours at a high (75-100°C) or low (50-75°C) temperature gradient. Samples were placed at 70°C, and then aliquots were transferred to room temperature at various time points. At each time point, the samples were mixed with a pipette and then diluted 1:100 (5 μl in 495 μl) with TBS + 0.01% BSA. 25 μl of each sample was dispensed into a white assay plate, and 25 μl of 200 nM pep788 or pep900 in TBS + 0.01% BSA + 20x diluted LCS was added. The plates were incubated for 10 minutes and then read in GMM+. [Figure 187] Cultures of FKBP_SmTrip9 variants and FRB-SmTrip10 were grown overnight at 37°C in LB + 100µg / ml ampicillin. Cells were diluted 1:20 into LB containing 0.15% glucose, 0.1% rhamnose, and 100µg / ml ampicillin. Cultures were induced for approximately 20 hours at 25°C with shaking. PLB assay reagent was prepared using 444nM MagneHis-purified LgTrip variants, 90-fold diluted FRB-SmTrip10 cultures, and + / -35nM rapamycin. 90µl of assay reagent was added to each well of a 96-well assay plate. FKBP_SmTrip9 cultures were diluted 1:10 in PLB, and 10µl was added to the assay plate. Samples were incubated at room temperature for 30 minutes. 100 μl of Nano-Glo containing 50 uM furimazine was added to the assay plate wells and luminescence was read after 5 minutes on a GloMax® luminometer. [Figure 188]LgTrip3546 was diluted to 1 nM in OptiMEM + 10% FBS. 12 μM solutions of chain 9 peptides 521 and 693 were prepared in OptiMem + 10% FBS. Using each chain 9 dilution, a 3-fold dilution series of each chain 10 peptide was prepared, starting at 20 μM. 90 μl of each dilution series was transferred to a white assay plate, followed by the addition of 10 μl of 1 μM LgTrip3546 stock solution. The plate was placed on an orbital shaker set at 600 RPM for 30 minutes. A detection reagent consisting of 10 mM DTT and 50 μM furimazine in OptiMEM + 10% FBS was prepared, and 11 μl was added to the sample. The plate was placed on an orbital shaker and mixed at room temperature for 5 minutes. The plate was read on a GloMax® Multi+ luminometer. Kd and Bmax were calculated using single-site specific binding in GraphPad Prism. [Figure 189] Protein purification. Cultures of each sample were started from isolated colonies in LB + 30 μg / ml KAN and grown for 20 hours at 37°C. Cultures were diluted 1:100 (500 μl in 50 ml) into LB supplemented with 30 μg / ml KAN, 0.1% rhamnose, and 0.15% glucose. Cultures were grown for 20 hours at 25°C. Cells were centrifuged and resuspended in 9 ml 100 mM HEPES pH 7.5 + 1 ml FastBreak Lysis Buffer + 200 μl RQ DNAse I and placed on an orbital mixer at 4°C for 30 minutes (an aliquot of the total lysate was saved). Each sample was centrifuged (7000 RPM, 15 minutes) to clarify the lysate, and the protein was purified using the MagneHis™ Purification System (Promega V8550). One ml of MagneHis™ magnetic resin was added to each cleared lysate and placed on an orbital mixer for 10 minutes. Each sample was washed three times with wash buffer, then collected twice with 500 μl of elution buffer. Samples were dialyzed into 1×TBS for 2 hours using a 10,000 MWCO dialysis device (Thermo 88404). [Figure 190]Comparison of the luminescence of NanoLuc (ATG-462) with the monomeric LgBiT-SmBiT protein. Purified protein was diluted to 0.2 nM in TBS + 0.01% BSA. 50 μl of each sample was mixed with 50 μl of furimazine / NanoGlo® buffer (N113, N112), and luminescence was measured in a GMM+ luminometer 3 minutes after adding the substrate. NanoLuc was approximately 2-fold brighter than the monomeric construct. [Figure 191] Km measurement. Purified protein was diluted to 0.2 nM in TBS + 0.01% BSA. A two-fold dilution series of furimazine was prepared in Nano-Glo® buffer starting at 50 uM (40 ul in 2 ml), then diluted 1 ml to 1 ml. 50 ul of each sample was mixed in duplicate with 50 ul of the dilution series and incubated at room temperature for 3 minutes, after which luminescence was read in GMM+. [Figure 192] Stability of monomeric LgBiT-SmBiT protein compared to NanoLuc (ATG-462). Each sample was diluted to 2 nM in TBS + 0.01% BSA, dispensed into multiple wells of a 96-well PCR plate, and placed in a thermocycler with a 30-minute temperature gradient. After 30 minutes of incubation, 5 μl of each sample was mixed with 45 μl of TBS + 0.01% BSA, and 50 μl of furimazine diluted in NanoGlo® buffer was added. The plate was incubated for 3 minutes, and luminescence was measured using GMM+. Temperature gradient A: 54°C, 57°C, 60°C, 63°C, 66°C, and 70°C. Temperature gradient B: 55°C, 60°C, 65°C, 70°C, 75°C, and 80°C. Temperature gradient C: 65°C, 70°C, 75°C, 80°C, 85°C, and 90°C. [Figure 193]Comparison of thermal stability. Each sample was diluted to 2 nM in TBS + 0.01% BSA, dispensed into multiple wells of a 96-well PCR plate, and placed in a thermocycler set to a 30-minute temperature gradient (75°C, 80°C, 85°C, 90°C, 95°C, and 100°C). After 30 minutes of incubation, 5 ul of each sample was mixed with 45 ul of TBS + 0.01% BSA, and 50 ul of furimazine diluted in NanoGlo® buffer was added. The mixture was incubated for 3 minutes, and luminescence was read in GMM+. G at position 159 improves stability compared to S at position 159. [Figure 194] Comparison of the stability of LgBiT-SmBiT variants with NanoLuc. Samples were diluted to 2 nM, dispensed into wells of a 96-well PCR plate, and placed in a thermocycler set at 60°C. Aliquots were removed at various time points and placed on ice. After all samples were incubated, they were equilibrated to room temperature and 5 ul of each sample was mixed with 45 ul of TBS + 0.01% BSA. 50 ul of NanoGlo® buffer + 50 uM furimazine was added, the plate was incubated for 3 minutes, and then luminescence was measured in a GMM+ luminometer. The stability curves at 60°C were consistent with the temperature gradient curves, with LgBiT-SmBiT being slightly more stable than NanoLuc and LgBiT-HiBiT being significantly more stable than NanoLuc. [Figure 195] Stability of monomeric LgBiT-SmBiT at elevated temperatures. Samples were diluted to 200 nM in TBS + 0.01% BSA and then further diluted to 0.2 nM (4 ul in 4 ml). 50 ul of ATG-462 or ATG-3564 was mixed with 50 ul of NanoGlo™ buffer + 50 uM furimazine or 50 ul of TBS + 0.01% BSA + 20 uM furimazine in the wells of a thin-walled PCR tray, and the tray was placed in a Veritas thermocycler with a temperature gradient of 55°C, 60°C, 65°C, 70°C, 75°C, and 80°C. Samples were withdrawn at various time points, and luminescence was measured in a GMM+ luminometer. [Figure 196]Stability of monomeric LgBiT-SmBiT at elevated temperature for 30 minutes. was diluted to 200 nM in TBS + 0.01% BSA, then further diluted to 0.2 nM (4 ul in 4 ml). 50 ul of ATG-462 or ATG-3564 was mixed with 50 ul of NanoGlo™ buffer + 50 uM furimazine or 50 ul of TBS + 0.01% BSA + 20 uM furimazine in the wells of a thin-walled PCR tray, and the tray was placed in a Veritas thermocycler with a temperature gradient of 55°C, 60°C, 65°C, 70°C, 75°C, and 80°C. Luminescence was measured at 30 minutes. [Figure 197] Measurement of Km and Vmax of purified NanoLuc® variants. Purified protein was diluted to 0.2 nM in TBS + 0.01% BSA. A two-fold dilution series of furimazine in Nano-Glo® buffer was performed starting at 50 uM (40 ul in 2 ml), then diluted 1 ml to 1 ml. 50 ul of each sample was mixed with 50 ul of the dilution series in duplicate, incubated at room temperature for 3 minutes, and luminescence was read on a GMM+ luminometer. [Figure 198] heat load. [Figure 199] Heat challenge with stable variants. Each sample was diluted to 2 nM in TBS + 0.01% BSA, dispensed into multiple wells of two 96-well PCR plates, and placed in a thermocycler set to a temperature gradient (75°C, 80°C, 85°C, 90°C, and 95°C) over 30 minutes. After 30 minutes of incubation, 5 ul of each sample was mixed with 45 ul of TBS + 0.01% BSA, and 50 ul of furimazine diluted in NanoGlo® buffer was added. The mixture was incubated for 3 minutes, and luminescence was measured in a GMM+ luminometer. [Figure 200]Comparison of pep521 and pep40 with detector proteins. Each protein was diluted to 20 nM in TBS + 0.01% BSA. 3-fold dilution series of pep521 and pep840, starting at 20 μM, were prepared in TBS + 0.01% BSA. 50 μl of each enzyme dilution was mixed with 50 μl of each peptide dilution in duplicate and pre-equilibrated by incubation on a shaker for 10 minutes. Assay buffer was prepared by diluting Nano-Glo Live Cell Substrate (LCS; Promega; N205) 30-fold in TBS + 0.01% BSA. 100 μl was added to each well, incubated for 5 minutes, and luminescence was measured using a GMM+ luminometer. Background readings were obtained from samples without peptide. [Figure 201] Comparison of the strand 9 detector protein pep840. Each protein was diluted to 200 nM in TBS + 0.01% BSA. A 3-fold dilution series of pep840, starting at 0.5 μM, was prepared in TBS + 0.01% BSA. 50 μl of each enzyme dilution was mixed with 50 μl of each peptide dilution in quadruplicate and pre-equilibrated by incubation on a shaker for 10 minutes. Assay buffer was prepared by diluting Nano-Glo LCS (Promega N205) 30-fold in TBS + 0.01% BSA, adding 100 μl to each well, incubating for 5 minutes, and measuring luminescence in a GMM+ luminometer. Background readings were obtained from samples without peptide. [Figure 202]Testing a series of linkers. Overnight cultures of each sample were prepared in LB + 100 μg / ml ampicillin. The next day, the cultures were diluted 1:20 (150 μl in 3 ml) with LB + 0.1% rhamnose + 100 μg / ml ampicillin, grown at 37°C for 4 hours, and then lysed in PLB lysis buffer (0.3x passive lysis buffer (Promega) + 25 mM HEPES pH 7.5) (4.5 ml of PLB lysis buffer for 500 μl of lysate). To perform the assay, the lysate was diluted 1:100 in TBS + 0.01% BSA. A 3-fold dilution series of chain 9 (pep840) was prepared, starting at 10 nM, and 50 μl was mixed with 50 μl of the diluted lysate from each sample and incubated for 20 minutes at room temperature on an orbital shaker set at 600 rpm. 100 ul Nano-Glo® buffer + 50 uM furimazine (N113) was added and luminescence was measured in a GMM+ luminometer. Each clone except ATG-5485, which has a 5AA linker, gave RLU values ​​similar to ATG-4992. [Figure 203] Linker comparison: 8GS (ATG-4992) vs. 11GS (ATG-5490). ATG-4992 and ATG-5490 were purified using the MagneHis™ purification system and diluted to 100 nM in CO2-independent medium + 10% FBS. 3-fold serial dilutions of pept840 were prepared in Nano-Glo® buffer + 50 μM furimazine, starting at 10 nM. 50 μl of each enzyme dilution was mixed with 50 μl of the above peptide dilutions in quadruplicate. Luminescence was measured over time on a GMM+ luminometer. Plotted data are from 50-minute kinetic readings. ATG-5490 showed approximately 2-fold higher signal compared to ATG-4992. [Figure 204]Kd of ATG-4992 and ATG-5490 mutants. Overnight cultures of each sample were prepared in LB + 100 μg / ml ampicillin. The next day, the cultures were diluted 1:20 (3 ml per 150 μl) with LB + 0.1% rhamnose + 100 μg / ml ampicillin, grown at 37°C for 4 hours, and lysed in PLB lysis buffer (4.5 ml of PLB lysis buffer per 500 μl of lysate). To perform the assay, the lysates were diluted 1:100 in TBS + 0.01% BSA. A 3-fold dilution series of chain 9 (pep840) was prepared, starting at 40 μM, and 50 μl was mixed with 50 μl of the diluted lysate of each sample and incubated for 20 minutes at room temperature on an orbital shaker set at 600 rpm. 100 ul of Nano-Glo® buffer + 50 uM furimazine (N113) was added and luminescence was measured in a GMM+ luminometer. [Figure 205] Comparison of ATG-4992 and ATG-5490 variants. Protein was purified (MagneHis™ Purification System, Promega) and diluted to 100 nM in CO2-independent medium + 10% FBS. 3-fold serial dilutions of pep840, starting at 2 nM, were prepared in Nano-Glo® buffer + 50 μM furimazine. 50 μl of each enzyme dilution was mixed, in triplicate, with 50 μl of each peptide diluted in furimazine / NanoGlo solution. Luminescence was measured every 5 minutes. Data from the 60-minute time point were plotted. When combined with pep840, clones ATG-5515 and ATG-5517 had lower calculated Kd values, higher RLU values, and a slightly higher signal over background. [Figure 206]Biochemical analysis. Samples were purified using the MagneHis™ Protein Purification System (Promega V8500) and diluted to 0.2 nM in TBS + 0.01% BSA. A 3-fold dilution series of VS-HiBiT (pep289) was prepared in TBS + 0.01% Tergitol. One series started at 400 nM, and the other at 20 μM. 50 μl of each diluted enzyme (0.2 nM) was mixed with 50 μl of each peptide dilution and incubated on a shaker for 10 minutes. 100 μl of furimazine diluted 250-fold in TBS + 0.01% BSA was added, the plate was placed on a shaker for 5 minutes, and luminescence was measured using a GMM+ luminometer. Kd and Bmax were calculated using single-site specific binding regression in GraphPad Prism. [Figure 207] Activity at various pH levels. Each purified enzyme sample was diluted to 0.2 nM in TBS + 0.01% BSA, and 20 μM VS-HiBiT (pep289) was added to each solution and incubated at room temperature for 20 minutes. 990 μl of each pH buffer was added to a well of a deep-well plate, 10 μl of furimazine (Promega, N113) was added, and 50 μl of each enzyme / peptide dilution was added along with 50 μl of the pH buffer / furimazine solution. Samples were incubated at room temperature for 12 minutes, and luminescence was measured in a GMM+ luminometer. Data were normalized to the pH 8.49 sample. [Figure 208]Biochemical comparison of ATG-5823, ATG-5824, and ATG-5825 with ATG-5146 (pep263). Proteins (ATG-5823, ATG-5824, and ATG-5825) were purified using the MagneHis™ Purification System (Promega). ATG-5146 was purified using an AKTA equipped with a nickel-Sepharose column. The purified protein was first diluted to 200 nM in TBS + 0.01% BSA and then further diluted to 0.2 nM in TBS + 0.01% BSA. A 3-fold dilution series of pep263, starting at 100 nM, was prepared in TBS + 0.01% BSA + 0.02% Tergitol. 50 μl of each enzyme was mixed with 50 μl of the peptide dilution series. Samples were incubated for 10 minutes on an orbital shaker (600 RPM). After incubation, 100 μl of LCS (N205 Promega) was diluted 1:30 in TBS + 0.01% BSA and added to each sample. Samples were incubated for 3 minutes at room temperature, and then luminescence was measured in the GloMaxMulti+. [Figure 209] Biochemical comparison of ATG-5826 and ATG-5827 with pep86. Proteins were purified using the MagneHis™ Purification System (Promega V8500). Purified proteins were first diluted to 200 nM in TBS + 0.01% BSA and then further diluted to 0.2 nM in TBS + 0.01% BSA. Two 2-fold dilution series of pep86 were prepared in TBS + 0.01% BSA + 0.02% Tergitol, starting at 1 uM and 100 nM. 50 ul of ATG-5826 and ATG-5827 were mixed with 50 ul of the above peptide dilution series starting at 1 uM. 50 ul of LgBiT protein (Promega N401C) was mixed with the above peptide dilution series starting at 100 nM. Samples were incubated for 10 minutes on an orbital shaker (600 RPM). After incubation, 100 μl of LCS (N205 Promega) was diluted 1:30 in TBS + 0.01% BSA and added to each sample. Samples were incubated for 3 minutes at room temperature, and then luminescence was measured in the GloMaxMulti+. [Figure 210] Biochemical comparison of ATG-5826 and ATG-5827 with pep114. Proteins were purified using the MagneHis™ Purification System (Promega V8500). Purified proteins were first diluted to 200 nM in TBS + 0.01% BSA and then further diluted to 0.2 nM in TBS + 0.01% BSA. Two 2-fold dilution series of pep114, starting at 1 mM, were prepared in TBS + 0.01% BSA + 0.02% Tergitol. 50 μl of ATG-5826, ATG-5827, and LgBiT proteins (Promega N401C) were mixed with the peptide dilution series. Samples were incubated on an orbital shaker (600 RPM) for 30 minutes. After incubation, 100ul of LCS (N205 Promega) was diluted 1:30 in TBS + 0.01% BSA and added to each sample. Samples were incubated at room temperature for 3 minutes and then luminescence was measured in the GloMaxMulti+. [Figure 211] SDS PAGE analysis. Each protein was diluted to 0.1 ug / ml in TBS + 1x SDS loading dye. Samples were heated at 70°C for 5 minutes, then 3 ul was loaded onto an SDS PAGE gel. [Figure 212] Exemplary assay formats for detecting SARS-COV-2. [Figure 213] Exemplary reagents used to screen for reactive peptides: SulfoSE reacts with lysines on the target protein, a PEG6 linker, and pyridinium sulfonic acid (PSA) confer solubility; PSA has a UV signature that can be used to measure labeling density. [Figure 214] Spike-in antibody dilution series assay for alternative labeling strategies to detect SARS-COV-2. [Figure 215] Detection of purified SARS-CoV-2 nucleocapsid antigen. [Figure 216]Samples tested with the lyophilized antigen assay in the portable assay. A patient's nasal swab was placed into a reagent tube and the foil seal at the bottom of the tube was broken. A buffer solution containing reagent cap was placed on top of the reagent tube containing the sample and secured in place. The buffer capsule was broken and released, then shaken. The reagent tube was inserted into a portable immunometer and the sample was analyzed. [Figure 217] Monomeric NanoBiT containing fluorofurimazine. Each test enzyme was diluted in TBS + 0.01% BSA. A dilution series containing either furimazine (N205) or JRW-1677 was prepared in either TBS (starting at 20 μM) or NanoGlo buffer (starting at 25 μM). Each substrate was serially diluted in either TBS + 0.01% BSA or NanoGlo® buffer (Promega N112). 50 μl of each enzyme dilution was mixed with 50 μl of each substrate dilution and incubated for 3 minutes, after which luminescence was measured in a GMM+ luminometer. Kinetic parameters for furimazine and JRW-1677 were calculated. When TBS was used to dilute the substrate, NanoLuc (ATG-462) produced higher RLU values ​​(approximately 10-20 fold) compared to the monomeric NanoBiT constructs in both furimazine and JRW-1667. NanoLuc and the monomeric constructs exhibited similar RLU values ​​in the furimazine / NanoGlo buffer, but only NanoLuc exhibited enhanced luminescence when JRW-1667 was used. The monomeric NanoBiT constructs exhibited lower RLU values ​​in both furimazine and JRW-1667, but the RLU values ​​were similar for the two buffers and the two substrates. Km values ​​were lower in TBS + 0.01% BSA compared to NanoGlo; however, in general, Km values ​​were similar for each condition, except for ATG-3562 (monomeric LgBiT-SmBiT), which had a lower calculated Km for all conditions tested. [Figure 218]HaloTag-LgBiT* - Expression and fluorescence imaging in mammalian cells. Immunofluorescence images of HaloTag-LgBiT* in HeLa cells show that HaloTag-LgBiT* is ubiquitously distributed throughout the cells. Left: red channel only, center: blue channel only, right: overlap. [Figure 219] Nuclear sensor - mammalian cell expression and fluorescence imaging. Immunofluorescence images of the nuclear sensor in HeLa cells show that the nuclear sensor is localized to the nucleus. Left: red channel only; center: blue channel only. Right: overlap. [Figure 220] Mitochondrial sensor - mammalian cell expression and fluorescence imaging. Immunofluorescence image of the mitochondrial sensor in HeLa cells shows that the mitochondrial sensor is localized in the mitochondrial matrix. DNA staining is blue. Left: red channel only; center: green channel only; right: overlap. [Figure 221] ER sensor - mammalian cell expression and fluorescence imaging. Immunofluorescence image of the endoplasmic reticulum (ER) sensor in HeLa cells shows that the ER sensor is localized to the ER. DNA staining is blue. Left: green channel only; center: red channel only; right: overlap. [Figure 222] Golgi Sensor - Expression and Fluorescence Imaging in Mammalian Cells. Fluorescent antibody imaging of the Golgi sensor in HeLa cells showed that the Golgi sensor was localized to the Golgi. The Golgi-GFP marker stained only the cis-Golgi. Some of our Golgi sensors did not colocalize with the Golgi GFP marker, suggesting that our Golgi sensors may be present in both the cis-Golgi and trans-Golgi. DNA staining is blue. Left: red channel only; center: green channel only; right: overlap. [Figure 223] Lysosomal sensor - mammalian cell expression and fluorescence imaging. Immunofluorescence image of the lysosomal sensor in HeLa cells shows that the sensor is localized to lysosomes. DNA staining is blue. Left: red channel only; center: green channel only; right: overlap. [Figure 224] Membrane sensor - mammalian cell expression and fluorescence imaging. Immunofluorescence image of the membrane sensor in HeLa cells shows that the membrane sensor is localized to the plasma membrane. DNA staining is blue. Left: red channel only; center: green channel only; right: overlap. [Figure 225] Membrane sensors - mammalian cell expression and luminescence assay. PKCα translocation under PMA stimulation. PKCα-HiBiT clone (HeLa) was transiently transfected with the membrane sensors. Transfected cells were treated with phorbol 12-myristate 13-acetate (PMA) and kinetic measurements were performed. The EC50 values ​​of all three membrane sensors were similar, suggesting that the affinity of the LgBiT variants for HiBiT does not affect the PKCα translocation event. [Figure 226] Membrane / HaloTag-LgBiT sensor - mammalian cell expression and luminescence assay. Reversibility of the HiBiT-LgBiT* complex. As PMA concentration increases, more PKCα is recruited to the plasma membrane, resulting in a stronger luminescence signal as more PKCα-HiBiT complements the LgBiT* membrane sensor. Conversely, less PKCα accumulates in the cytosol, thus decreasing the luminescence signal as it associates with HaloTag-LgBiT (the cytosolic sensor). [Figure 227] PKCα translocation events. Kinetic measurement of PKCα translocation events. The accumulation of PKCα at the plasma membrane can be kinetically measured upon association with the membrane sensor. Under PMA stimulation, PKCα reaches maximum accumulation at the plasma membrane at t = 16 min. DETAILED DESCRIPTION OF THE INVENTION

[0066] definition Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the embodiments described herein, certain preferred methods, compositions, devices, and materials are described herein. However, in describing the materials and methods of the present invention, it should be understood that the invention is not limited to the specific molecules, compositions, methodologies, or protocols described herein, as these may vary through routine experimentation and optimization. It should also be understood that the terminology used herein is for the purpose of describing particular versions or embodiments only, and is not intended to limit the scope of the embodiments described herein.

[0067] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, will prevail. Therefore, the following definitions apply in connection with the embodiments described herein:

[0068] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "peptide" is a reference to one or more peptides and equivalents thereof known to those skilled in the art, and so forth.

[0069] As used herein, the term "and / or" includes any and all combinations of the listed items, including any one of the listed items. For example, "A, B, and / or C" includes A, B, C, AB, AC, BC, and ABC, each of which is considered to be individually described by the recitation "A, B, and / or C."

[0070] As used herein, the term "comprise" and its linguistic variants mean the presence of the recited feature(s), component(s), method step(s), etc., and do not exclude the presence of additional feature(s), component(s), method step(s), etc. Conversely, the term "consisting of" and its linguistic variants mean the presence of the recited feature(s), component(s), method step(s), etc., and excludes any unrecited feature(s), component(s), method step(s), etc., except for impurities ordinarily associated therewith. The phrase "consisting essentially of" means the recited feature(s), component(s), method step(s), etc., plus any additional feature(s), component(s), method step(s), etc. that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using the open language "comprising." Such embodiments encompass multiple closed "consisting of" and / or "consisting essentially of" embodiments, which may alternatively be claimed or described using such language.

[0071] As used herein, the term "substantially" means that the recited property, parameter, and / or value need not be achieved exactly, but rather that deviations or variations, including, for example, tolerances, measurement errors, measurement precision limits, and other factors known to those of skill in the art, may occur in amounts that do not eliminate the effect the property is intended to provide. A substantially absent property or characteristic (e.g., substantially non-luminescent) can be a property or characteristic that is within the noise range, below background, below the detection capability of the assay being used, or that is only a fraction (e.g., less than 1%, less than 0.1%, less than 0.01%, less than 0.001%, less than 0.00001%, less than 0.000001%, less than 0.0000001%) of a significant property (e.g., the luminescence intensity of a bioluminescent protein or bioluminescent complex).

[0072] As used herein, the term "bioluminescence" refers to the production and emission of light by a chemical reaction catalyzed or enabled by an enzyme, protein, protein complex, or other biological molecule (e.g., a bioluminescent complex). In typical embodiments, a substrate of a bioluminescent entity (e.g., a bioluminescent protein or bioluminescent complex) is converted by the bioluminescent entity to an unstable form, after which the substrate emits light.

[0073] As used herein, the term "complementary" refers to the characteristic of two or more structural elements (e.g., peptides, polypeptides, nucleic acids, small molecules, etc.) that they can hybridize, dimerize, or otherwise form a complex with one another. For example, "complementary peptides and polypeptides" can assemble to form a complex. Complementary components may require, for example, assistance (facilitation) to place the components in a conformation suitable for complementation, assistance (facilitation) to position the components in proximity suitable for complementation, assistance (facilitation) to colocalize complementary components, assistance (facilitation) to lower the interaction energy for complementarity, assistance (facilitation) to overcome insufficient affinity for each other, etc. to form a complex (e.g., from interacting elements).

[0074] As used herein, the term "complex" refers to an assembly or aggregation of molecules (e.g., peptides, polypeptides, etc.) that are in direct and / or indirect contact with one another. In one aspect, "contact" or more specifically "direct contact" means that two or more molecules are in sufficient proximity such that non-covalent attractive interactions, such as van der Waals forces, hydrogen bonding, ionic interactions, and hydrophobic interactions, dominate their interaction. In such aspects, a complex of molecules (e.g., peptides and polypeptides) forms under assay conditions such that the complex is thermodynamically favored (e.g., compared to the unaggregated or uncomplexed states of its constituent molecules). As used herein, unless otherwise specified, the term "complex" refers to an assembly of two or more molecules (e.g., peptides, polypeptides, or combinations thereof).

[0075] As used herein, the term "non-luminescent" refers to an entity (e.g., a peptide, polypeptide, complex, protein, etc.) that exhibits the characteristic of not emitting a detectable amount of light in the visible spectrum (e.g., in the presence of a substrate). For example, an entity may be referred to as non-luminescent if it exhibits no detectable luminescence in a given assay. As used herein, the term "non-luminescent" is synonymous with the term "substantially non-luminescent." In some embodiments, an entity is considered to be "non-luminescent" if any luminescence is small enough so as not to produce background that interferes with a particular assay.

[0076] As used herein, the terms "non-luminescent peptide" and "non-luminescent polypeptide" refer to a peptide or polypeptide that exhibits substantially no luminescence (e.g., in the presence of a substrate) or an amount below noise (e.g., 100-fold, 200-fold, 500-fold, 1 x 10) when compared to a significant signal (e.g., a luminescent complex) using a typical instrument (e.g., a luminometer) under standard conditions (e.g., physiological conditions, assay conditions, etc.). 3 1 / 10, 1 x 10 4 1 / 10, 1 x 10 5 1 / 10, 1 x 106 1 / 10, 1 x 10 7 In some embodiments, such non-luminescent peptides and polypeptides assemble under the reference conditions described herein to form a bioluminescent complex.

[0077] As used herein, the term "interacting element" refers to a moiety that assists or facilitates the assembly of non-luminescent elements to form a bioluminescent complex. In some embodiments, a pair of interacting elements (also referred to as an "interacting pair") is attached to a pair of non-luminescent elements (e.g., non-luminescent peptides), and the formation of a bioluminescent complex is facilitated by an attractive interaction between the two interacting elements. However, the present invention is not limited to such a mechanism, and an understanding of the mechanism is not required to practice the present invention. The interacting element may facilitate the formation of a bioluminescent complex by any suitable mechanism (e.g., bringing the non-luminescent elements into close proximity, positioning the non-luminescent elements in a conformation suitable for stable interaction, reducing the activation energy for complex formation, a combination thereof, etc.). The interacting element may be a protein, polypeptide, peptide, small molecule, cofactor, nucleic acid, lipid, carbohydrate, antibody, etc. An interacting pair may consist of two identical interacting elements (i.e., a homogeneous pair) or two different interacting elements (i.e., a heterogeneous pair). In the case of heterologous pairs, the interacting members may be the same type of moiety (e.g., a polypeptide) or two different types of moieties (e.g., a polypeptide and a small molecule). In some embodiments in which complex formation by an interacting pair is investigated, the interacting pair may be referred to as a "target pair" or "pair of interest," and the individual interacting members are referred to as "target elements" (e.g., "target peptide," "target polypeptide," etc.) or "elements of interest" (e.g., "peptide of interest," "polypeptide of interest," etc.).

[0078] As used herein, the term "low affinity" describes a molecular interaction between two or more (e.g., three) entities that is too weak to result in significant complex formation between the entities unless at concentrations significantly higher (e.g., 2x, 5x, 10x, 100x, 1000x or more) than physiological or assay conditions, or facilitated by the formation of a secondary complex of the attached elements (e.g., interacting elements).

[0079] As used herein, the term "high affinity" describes an intermolecular interaction between two or more (e.g., three) entities that is strong enough to cause detectable complex formation under physiological or assay conditions without facilitation by the formation of a secondary complex of the attached elements (e.g., interacting elements).

[0080] As used herein, the term "colocalization element" refers to a moiety that promotes colocalization of non-luminescent elements. In some embodiments, a pair of non-luminescent elements has sufficient affinity to form a complex when the non-luminescent elements are colocalized at sufficient concentrations. In such embodiments, a pair (e.g., a pair) of colocalization elements (also referred to as a "colocalization pair") is attached to a pair of non-luminescent elements (e.g., non-luminescent peptides), and colocalization of the two colocalization elements (e.g., within an intracellular compartment, tissue, in solution, on a solid matrix support, etc.) promotes colocalization of the non-luminescent elements, thereby promoting the formation of a bioluminescent complex. However, the present invention is not limited to such mechanisms, and an understanding of such mechanisms is not required to practice the present invention. In some embodiments, due to the ability of the non-luminescent elements to self-assemble into a luminescent complex, the colocalization elements do not need to interact directly to promote complex formation. The colocalization elements can be proteins, polypeptides, peptides, small molecules, cofactors, nucleic acids, lipids, carbohydrates, antibodies, etc. A colocalized pair may consist of two identical colocalized elements (i.e., a homogeneous pair) or two different colocalized elements (i.e., a heterogeneous pair). In the case of a heterogeneous pair, the colocalized elements may be the same type of moiety (e.g., a polypeptide) or two different types of moieties (e.g., a polypeptide and a small molecule). In some embodiments in which the localization of a colocalized pair is investigated, the colocalized pair may be referred to as a "target pair" or "pair of interest," and the individual colocalized elements are referred to as "target elements" (e.g., "target peptide," "target polypeptide," etc.) or "elements of interest" (e.g., "peptide of interest," "polypeptide of interest," etc.).

[0081] As used herein, the term "coelenterazine" refers to naturally occurring ("native") coelenterazine. As used herein, the terms "coelenterazine analogs" or "coelenterazine derivatives" include those disclosed in WO2003 / 040100; U.S. Patent Application No. 12 / 056073 (paragraph

[0086] ); U.S. Patent No. 8,669,103; WO2012 / 061529; U.S. Patent Publication No. 2017 / 0233789; and U.S. Patent Publication No. 2018 / 0030059 (the disclosures of which are incorporated herein by reference in their entireties), as well as free coelenterazine analogs. Coelenterazine refers to synthetic analogs (e.g., derivatives or variants) and natural analogs of coelenterazine, including coelenterazine-N, coelenterazine-F, coelenterazine-H, coelenterazine-HCP, coelenterazine-CP, coelenterazine-C, coelenterazine-E, coelenterazine-FCP, bisdeoxycoelenterazine ("coelenterazine-HH"), coelenterazine-I, coelenterazine-ICP, coelenterazine-V, and 2-methylcoelenterazine. In some embodiments, coelenterazine analogs include pro-substrates such as those described in U.S. Patent Application No. 12 / 056073; U.S. Publication No. 2012 / 0707849; and U.S. Publication No. 2014 / 0099654 (incorporated herein by reference in their entireties).

[0082] As used herein, the term "pre-existing protein" refers to an amino acid sequence that physically existed before a particular event or date. A "peptide that is not a fragment of a pre-existing protein" is a short chain of amino acids that is not a fragment or subsequence of a protein (e.g., synthetic or naturally occurring) that physically existed before the design and / or synthesis of the peptide.

[0083] As used herein, the term "fragment" refers to a peptide or polypeptide that results from cleavage or "fragmentation" of a larger whole entity (e.g., protein, polypeptide, enzyme, etc.), or that has been prepared to have the same sequence. Thus, a fragment is a subsequence of a whole entity (e.g., protein, polypeptide, enzyme, etc.) and is made and / or engineered from that whole entity. A peptide or polypeptide that is not a subsequence of an existing whole protein is not a fragment (e.g., not a fragment of an existing protein). A peptide or polypeptide that is "not a fragment of an existing bioluminescent protein" is an amino acid chain that (1) physically existed prior to the design and / or synthesis of the peptide or polypeptide, and (2) is not a subsequence of a protein (e.g., natural or synthetic) that exhibits high bioluminescent activity.

[0084] As used herein, the term "subsequence" refers to a peptide or polypeptide that has 100% sequence identity with a portion of another, larger peptide or polypeptide. A subsequence is an exact sequence match for a portion of a longer chain of amino acids.

[0085] The term "amino acid" refers to natural amino acids, unnatural amino acids, and amino acid analogs, all of which, unless otherwise specified, are D and L stereoisomers (if their structures allow for such stereoisomeric forms).

[0086] Naturally occurring amino acids include alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0087] Unnatural amino acids include pentafluorophenylalanine ("Z"), azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, naphthylalanine ("naph"), aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, tertiary butylglycine ("tBuG"), 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoproline ("hPro" or "homoP"), hydroxylysine, allo-hydroxylysine, 3-hydroxyproline ("3Hyp"), 4-hydroxyisobutyric ... These include, but are not limited to, proline ("4Hyp"), isodesmosine, allo-isoleucine, N-methylalanine ("MeAla" or "Nime"), N-alkylglycines ("NAG") including N-methylglycine, N-methylisoleucine, N-alkylpentylglycines ("NAPG") including N-methylpentylglycine, N-methylvaline, naphthylalanine, norvaline ("Norval"), norleucine ("Norleu"), octylglycine ("OctG"), ornithine ("Orn"), pentylglycine ("pG" or "PGly"), pipecolic acid, thioproline ("ThioP" or "tPro"), homolysine ("hLys"), and homoarginine ("hArg"). Non-naturally occurring reactive amino acids are described, for example, in Boutureira, O. and GJ Bernardes (2015) "Advances in chemical protein modification." Chem Rev 115(5):2174-2195, which is incorporated herein by reference in its entirety.

[0088] The term "amino acid analog" refers to a natural or unnatural amino acid in which one or more of the C-terminal carboxy group, the N-terminal amino group, and the side chain bioactive group are reversibly or irreversibly chemically blocked or otherwise modified with another bioactive group. For example, aspartic acid-(β-methyl ester) is an amino acid analog of aspartic acid, N-ethylglycine is an amino acid analog of glycine, and alanine carboxamide is an amino acid analog of alanine. Other amino acid analogs include methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine ​​sulfoxide, and S-(carboxymethyl)-cysteine ​​sulfone. Amino acid analogs can include amino acids with various protecting groups (Isidro-Llobet, A., et al. (2009). "Amino Acid-Protecting Groups." Chemical Reviews 109(6):2455-2504, incorporated herein by reference in its entirety).

[0089] As used herein, unless otherwise specified, the terms "peptide" and "polypeptide" refer to a polymeric compound of two or more amino acids linked through a backbone by peptide amide bonds (--C(O)NH--). The term "peptide" typically refers to short amino acid polymers (e.g., chains having fewer than 30 amino acids), while the term "polypeptide" typically refers to longer amino acid polymers (e.g., chains having more than 30 amino acids).

[0090] As used herein, unless otherwise specified, the term "dipeptide" refers to a peptide or small polypeptide (e.g., less than 70 amino acids, less than 60 amino acids, less than 50 amino acids, etc.) comprising two peptide segments (e.g., directly or indirectly fused / attached (e.g., via a linker (e.g., a peptide linker (e.g., 1-10 amino acids (e.g., a single glycine))) corresponding to the two β-strands of luciferase (e.g., a "β9 / β10 dipeptide" corresponding to the β9 and β10 strands of the OgLuc luciferase polypeptide)).

[0091] As used herein, unless otherwise specified, the term "tripeptide" refers to a peptide or small polypeptide (e.g., less than 100 amino acids, less than 90 amino acids, less than 80 amino acids, etc.) comprising three peptide segments (e.g., directly or indirectly fused / attached (e.g., via a linker (e.g., a peptide linker (e.g., 1-10 amino acids (e.g., a single glycine)))) corresponding to the three β-strands of luciferase (e.g., a "β8-10 tripeptide" corresponding to the β-strand of the OgLuc luciferase polypeptide).

[0092] As used herein, the terms "peptidomimetic" and "peptide analog" refer to peptide- or polypeptide-like molecules that mimic sequences derived from proteins or peptides. Peptidomimetics can contain amino acid analogs, peptoid amino acids, and / or non-amino acid components, either alone or in combination with amino acids (e.g., natural or unnatural amino acids). Examples of peptidomimetics include chemically modified peptides / polypeptides, peptoids (in which side chains are attached to the nitrogen atom of the peptide backbone instead of the alpha carbon), beta peptides (in which the amino group is attached to the beta carbon instead of the alpha carbon), and the like.

[0093] As used herein, the term "peptoid" refers to a class of peptidomimetics in which the nitrogen atoms of the peptide backbone, rather than the alpha carbons, are functionalized with side chains.

[0094] As used herein, the term "artificial" refers to a composition or system that is designed or prepared by man and does not exist in nature, for example, an artificial peptide, artificial peptoid, or artificial nucleic acid that contains a non-naturally occurring sequence (e.g., a peptide that does not have 100% identity to a naturally occurring protein or fragment thereof).

[0095] As used herein, a "conservative" amino acid substitution refers to the replacement of an amino acid in a peptide or polypeptide with another amino acid having similar chemical properties, e.g., size or charge. For purposes of this disclosure, each of the following eight groups contains amino acids that are conservative substitutions for one another: 1) alanine (A) and glycine (G), 2) aspartic acid (D) and glutamic acid (E), 3) asparagine (N) and glutamine (Q), 4) arginine (R) and lysine (K), 5) isoleucine (I), leucine (L), methionine (M), and valine (V); 6) phenylalanine (F), tyrosine (Y) and tryptophan (W), 7) serine (S) and threonine (T), and 8) Cysteine ​​(C) and methionine (M).

[0096] Naturally occurring residues can be divided into classes based on the properties of their common side chains, for example, polar positively charged (or basic) (histidine (H), lysine (K), and arginine (R)), polar negatively charged (or acidic) (aspartic acid (D), glutamic acid (E)), polar neutral (serine (S), threonine (T), asparagine (N), glutamine (Q)), nonpolar aliphatic (alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M)), nonpolar aromatic (phenylalanine (F), tyrosine (Y), tryptophan (W)), proline and glycine, and cysteine. As used herein, a "semi-conservative" amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid within the same class.

[0097] In some embodiments, unless otherwise specified, conservative or semi-conservative amino acid substitutions may also include non-naturally occurring amino acid residues that have similar chemical properties to the natural residues. These non-natural residues are typically incorporated by chemical peptide synthesis rather than synthesis in biological systems. These include, but are not limited to, peptidomimetics and other inverted or reversed amino acid moieties. Embodiments herein may, in some embodiments, be limited to natural amino acids, non-natural amino acids, and / or amino acid analogs.

[0098] Non-conservative substitutions may involve the exchange of a member of one class for a member of another class.

[0099] As used herein, the term "sequence identity" refers to the degree to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have the same sequence composition of monomeric subunits. The term "sequence similarity" refers to the degree to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have similar polymer sequences. For example, similar amino acids are those that share the same biophysical properties and can be classified into, for example, acidic (e.g., aspartic acid, glutamic acid), basic (e.g., lysine, arginine, histidine), nonpolar (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) families. "Percent sequence identity" (or "percent sequence similarity") is calculated by: (1) comparing two optimally aligned sequences over a comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window); (2) determining the number of positions containing identical (similar) monomers (e.g., positions where the same amino acid is found in both sequences, positions where a similar amino acid is found in both sequences) to obtain the number of matched positions; (3) dividing the number of matched positions by the total number of positions within the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window); and (4) multiplying the result by 100 to obtain the percent sequence identity or percent sequence similarity. For example, if peptide A and peptide B are both 20 amino acids long and have identical amino acids at all but one position, peptide A and peptide B have 95% sequence identity. If the amino acids at non-identical positions share the same biophysical properties (e.g., both are acidic), peptide A and peptide B will have 100% sequence similarity. As another example, if peptide C is 20 amino acids long and peptide D is 15 amino acids long, and 14 of the 15 amino acids in peptide D are identical to some amino acids in peptide C, then peptides C and D have 70% sequence identity, but peptide D has 93.3% sequence identity over the optimal comparison window of peptide C.For purposes of calculating "percent sequence identity" (or "percent sequence similarity") herein, any gap in the aligned sequences is treated as a mismatch at that position.

[0100] Any peptide / polypeptide described herein as having a particular percent sequence identity or similarity (e.g., at least 70%) with a reference SEQ ID NO: can be expressed as having a maximum number of substitutions (or terminal deletions) relative to that reference sequence. For example, a sequence having at least Y% sequence identity (e.g., 90%) with SEQ ID NO: Z (e.g., 100 amino acids) can have up to X substitutions (e.g., 10) compared to SEQ ID NO: Z, and thus can also be expressed as having no more than X (e.g., 10) substitutions compared to SEQ ID NO: Z.

[0101] As used herein, the term "wild-type" refers to a gene or gene product (e.g., a protein, polypeptide, peptide, etc.) that has the characteristics (e.g., sequence) of that gene or gene product isolated from a naturally occurring source and that is most frequently observed in a population. In contrast, the term "variant" or "variant" refers to a gene or gene product that exhibits an alteration in sequence when compared to the wild-type gene or gene product. Note that a "naturally occurring variant" is a gene or gene product that occurs in nature but has an altered sequence when compared to the wild-type gene or gene product, and is not the most frequently occurring sequence. An "artificial variant" is a gene or gene product that has an altered sequence when compared to the wild-type gene or gene product and does not occur in nature. A variant gene or gene product can be a naturally occurring sequence that occurs in nature but is not the most commonly occurring variant of the gene or gene product, or a "synthetic" sequence created by human or experimental intervention.

[0102] As used herein, the term "physiological conditions" encompasses any conditions compatible with living cells, such as primarily aqueous conditions of temperature, pH, salinity, chemical composition, etc., compatible with living cells.

[0103] As used herein, the term "sample" is used in the broadest sense. In one sense, this term is meant to include specimens or culture media obtained from any source, as well as biological and environmental samples. Biological samples can be obtained from animals (including humans) and encompass liquids, solids, tissues, and gases. Biological samples include blood products such as plasma and serum. Samples can also refer to cell lysates or purified forms of the enzymes, peptides, and / or polypeptides described herein. Cell lysates can include cells lysed with a lysing agent or lysates such as rabbit reticulocyte or wheat germ lysates. Samples can also include cell-free expression systems. Environmental samples include environmental materials such as surface materials, soil, water, crystals, and industrial samples. However, such examples should not be construed as limiting the types of samples applicable to the present invention.

[0104] As used herein, the terms "fusion," "fusion polypeptide," and "fusion protein" refer to a chimeric protein containing a first protein or polypeptide of interest (e.g., a substantially non-luminescent peptide) linked to a second, different peptide, polypeptide, or protein (e.g., an interacting element).

[0105] As used herein, the terms "conjugated" and "conjugation" refer to the covalent joining of two molecular entities (e.g., post-synthesis and / or during synthetic preparation). The chemical (e.g., "chemically" conjugated) or enzymatic attachment of a peptide or small molecule tag to a protein or small molecule is an example of conjugation.

[0106] The term "binding moiety" refers to a domain that specifically binds to an antigen or epitope independently of a distinct epitope or antigen-binding domain. A binding moiety can be an antibody, an antibody fragment, a receptor domain that binds to a target ligand, a protein that binds to an immunoglobulin (e.g., Protein A, Protein G, Protein A / G, Protein L, Protein M), a binding domain of a protein that binds to an immunoglobulin (e.g., Protein A, Protein G, Protein A / G, Protein L, Protein M), an oligonucleotide probe, a peptide nucleic acid, a DARPin, an aptamer, an affimer, a purified protein (either the analyte itself or a protein that binds to the analyte), and an analyte-binding domain(s) of a protein, etc. Table A provides a list of exemplary binding moieties that can be used singly or in various combinations in the methods, systems, and assays (e.g., immunoassays) herein. [Table 1]

[0107] As used herein, the term "antibody" refers to a whole antibody molecule or a fragment thereof (e.g., fragments such as Fab, Fab', and F(ab')2, variable light chain, variable heavy chain, Fv, etc.), which may be a polyclonal or monoclonal antibody, or a recombinant antibody, chimeric antibody, humanized antibody, human antibody, etc. As used herein, when an antibody or other entity "specifically binds to" or "specifically recognizes" an antigen or epitope, the antibody or other entity preferentially recognizes the antigen in a complex mixture of proteins and / or macromolecules and binds to the antigen or epitope with substantially higher affinity than other entities that do not display the antigen or epitope. In this regard, "substantially higher affinity" means an affinity high enough to allow detection of the antigen or epitope distinguished from multiple entities using a desired assay or measurement device. Typically, it is at least 10 7 M -1 (e.g., 10 7 M-1 Less than 10 8 M -1 Less than 10 9 M -1 Less than 10 10 M -1 Less than 10 11 M -1 Less than 10 12 M -1 Less than 10 13 M -1 less than the binding constant (K a ) refers to a binding affinity having a specific epitope. In certain such embodiments, the antibody can bind to different antigens as long as the different antigens contain that particular epitope. In certain instances, for example, homologous proteins from different species may contain the same epitope.

[0108] As used herein, the term "antibody fragment" refers to a portion of a full-length antibody that contains at least a portion of the antigen-binding or variable region. Antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, Fd, variable light chain, variable heavy chain, diabody, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. See, e.g., Hudson et al. (2003) Nat. Med. 9:129-134, incorporated herein by reference in its entirety. In certain embodiments, antibody fragments are produced by enzymatic or chemical cleavage (e.g., papain and pepsin digestion of antibodies) of intact antibodies produced by recombinant DNA technology or chemical polypeptide synthesis. For example, a "Fab" fragment contains one light chain and one heavy chain C. H1 and variable regions. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. An "Fab" fragment contains one light chain and one C H1 Domain and C H2The Fab′ fragment contains one heavy chain with an additional constant region extending between the heavy and light chain domains. Interchain disulfide bonds can form between the two heavy chains of the Fab′ fragment to form a “F(ab′)2” molecule. The “Fv” fragment contains the variable regions from both the heavy and light chains but lacks the constant regions. The single-chain Fv (scFv) fragment contains the heavy and light chain variable regions linked by a flexible linker to form a single polypeptide chain with an antigen-binding region. Exemplary single-chain antibodies are described in detail in WO 88 / 01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203 (incorporated herein by reference in their entireties). In certain instances, a single variable region (e.g., heavy or light chain variable region) can have the ability to recognize and bind to an antigen. Other antibody fragments will be understood by those skilled in the art.

[0109] As used herein, the term "peptide tag" refers to a peptide that can be attached (e.g., post-synthesis or during synthesis) or fused to another entity (e.g., a protein of interest, a molecule of interest, an interacting element, a co-localization element, etc.). A peptide tag may or may not be attached to another entity. As used herein, a peptide tag is typically capable of forming a bioluminescent complex with another peptide tag and a polypeptide under appropriate conditions. In embodiments in which a peptide tag is attached to another entity, the peptide tag is chemically conjugated to another molecule (e.g., a peptide, polypeptide, nucleic acid, other small molecule, or other macromolecule), chemically synthesized to be part of another molecule, genetically fused to another peptide or polypeptide molecule, etc.

[0110] As used herein, the term "polypeptide component" is used synonymously with the term "polypeptide component of a bioluminescent complex." As used herein, a polypeptide component is typically capable of forming a bioluminescent complex with a pair of peptide tags under appropriate conditions.

[0111] As used herein, the term "Oplophorus luciferase" ("OgLuc") refers to a light-emitting polypeptide having significant sequence identity, structural conservation, and / or functional activity of the luciferase produced by or derived from the deep-sea shrimp, Oplophorus gracilirostris. In particular, OgLuc polypeptide refers to a light-emitting polypeptide having significant sequence identity, structural conservation, and / or functional activity of the mature 19 kDa subunit (e.g., without a signal sequence) of the Oplophorus luciferase protein complex, e.g., SEQ ID NOS: 1 (WT OgLuc) and 3 (NanoLuc), which comprises 10 β-strands (β1, β2, β3, β4, β5, β6, β7, β8, β9, β10), and which utilizes a substrate, such as coelenterazine or a coelenterazine derivative, to generate luminescence.

[0112] As used herein, the term "β9-like peptide" refers to a peptide (or peptide tag) that contains significant sequence identity, structural conservation, and / or functional activity with the β (beta)9 chain of the OgLuc polypeptide. In particular, a β9-like peptide is a peptide that can structurally complement an OgLuc polypeptide lacking the β9 chain, resulting in enhanced luminescence of the complex compared to the OgLuc polypeptide in the absence of the β9-like peptide. Other "βX-like peptides" may be similarly named (e.g., β1-like, β2-like, β3-like, β4-like, β5-like, β6-like, β7-like, β8-like, β9-like).

[0113] As used herein, the term "β10-like peptide" refers to a peptide (or peptide tag) that contains significant sequence identity, structural conservation, and / or functional activity with the β (beta)10 chain of the OgLuc polypeptide. In particular, a β10-like peptide is a peptide that can structurally complement an OgLuc polypeptide lacking the β10 chain, resulting in enhanced luminescence of the complex compared to the OgLuc polypeptide in the absence of the β10-like peptide. Other "βX-like peptides" may be similarly named (e.g., β1-like, β2-like, β3-like, β4-like, β5-like, β6-like, β7-like, β8-like, β9-like).

[0114] As used herein, "β 1~8 The term "β-like polypeptide" refers to a polypeptide that has sequence and structural similarity to the β (beta) 1-8 chains of the OgLuc polypeptide, but lacks the β (beta) 9 and 10 chains. Y~Z "β-like polypeptides" may be named similarly (e.g., β- 1~4 -like polypeptide, β 2~8 -like polypeptide, β 5~10 -like polypeptides).

[0115] As used herein, the term "NANOLUC" refers to an artificial luciferase or bioluminescent polypeptide commercially produced by Promega Corporation and corresponding to SEQ ID NO:3.

[0116] As used herein, the term "LgBiT" refers to a β-LgBiT that is useful, for example, in bimolecular complementation to form a bioluminescent complex and corresponds to SEQ ID NO: 11. 1~9 It refers to a polypeptide that corresponds to a polypeptide-like polypeptide.

[0117] As used herein, the term "SmBiT" refers to a β-SmBiT that is useful, for example, in bimolecular complementation to form a bioluminescent complex, but has low affinity for LgBiT (e.g., requires facilitation for complex formation) and corresponds to SEQ ID NO: 13. 10 It refers to a peptide that corresponds to a peptide-like peptide.

[0118] As used herein, the term "HiBiT" refers to a β-HiBiT that is useful, for example, in bimolecular complementation to form a bioluminescent complex, but has low affinity for LgBiT (e.g., requires facilitation for complex formation) and corresponds to SEQ ID NO: 15. 10 HiBiT refers to a peptide corresponding to a HiBiT-like peptide. HiBiT has the same sequence as "SmHiTrip10" (SEQ ID NO: 25) and "pep86" (also referred to as SmTrip10, pep86, etc.), terms which may be used interchangeably.

[0119] As used herein, the term "LgTrip" corresponds to SEQ ID NO: 17 and refers to a β9-like and β9-like complex to form a bioluminescent complex. 10 β to form trimolecular complementation or bioluminescent complexes with β-like peptides 9~10 Useful in bimolecular complementation with β-like dipeptides 1~8 LgTrip variants include LgTrip 2098 (with His tag: SEQ ID NO: 31, without His tag: SEQ ID NO: 304) and LgTrip 3546 (with His tag: SEQ ID NO: 51, without His tag: SEQ ID NO: 302).

[0120] As used herein, "SmTrip10" refers to a β-antibody that is useful, for example, in trimolecular complementation to form a bioluminescent complex. 10 It refers to a peptide that corresponds to a peptide-like peptide.

[0121] As used herein, "SmTrip9" refers to a peptide corresponding to a β9-like peptide that is useful, for example, in trimolecular complementation to form a bioluminescent complex.

[0122] Detailed Description Provided herein are bioluminescent polypeptides and compositions and methods for the assembly of tri- or multi-molecular bioluminescent complexes. In certain embodiments, bioluminescent complexes are formed when three or more peptide and / or polypeptide components (e.g., separate or fused as dipeptides or tripeptides) interact.

[0123] Experiments conducted during the development of embodiments herein demonstrated the ability of two small peptide components (e.g., a β10-like peptide and a β9-like peptide) and one polypeptide component (e.g., a β 1~8The present disclosure demonstrates that a trimolecular luciferase comprising a luciferase-like polypeptide assembles to form a light-emitting complex. Experiments conducted during the development of embodiments herein further demonstrate the formation of bioluminescent complexes from up to five fragments of luciferase (or variants of such fragments), for example, a polypeptide fragment (or variants thereof) and one or more peptide, dipeptide, or tripeptide fragments (or variants of such fragments).

[0124] Commercially available NANOLUC luciferase (Promega Corporation) contains 10 β (beta) chains (β1, β2, β3, β4, β5, β6, β7, β8, β9, β10). U.S. Patent No. 9,797,889 (incorporated herein by reference in its entirety) describes the β 1~9 -like polypeptides and β 10 describes the development and use of a complementation system containing a peptide-like peptide (the actual polypeptide and peptide sequences in US Pat. No. 9,797,889 differ from the corresponding sequences in NANOLUC and wild-type native OgLuc).

[0125] In experiments conducted during the development of embodiments herein, β 1~9 The β-like polypeptide was further split by removal of the β9 chain. The remaining portion (β 1~8The SmTrip-like polypeptide is referred to herein as LgTrip 2098 (SEQ ID NO: 17, or SEQ ID NO: 31 (with His tag)). Experiments attempted to reconstitute a luminescent complex from LgTrip and two peptides corresponding to the β9 chain (SmTrip9 pep245, SEQ ID NO: 23) and the β10 chain (SmTrip10 pep86; HiBiT, SEQ ID NO: 15, a β10 sequence optimized for use in high-affinity bimolecular systems). Experiments demonstrated that poorly expressed LgTrip 2098 (SEQ ID NO: 17, or SEQ ID NO: 31 (with His tag)) in E. coli was unstable and susceptible to surface deactivation. During development of embodiments herein, experiments were conducted to develop artificial variants that exhibit one or more (e.g., all) of enhanced stability, enhanced expression, enhanced activity, enhanced molecular interaction, etc., and that can be used in systems for reconstituting bioluminescent complexes with peptides corresponding to the β9 chain (e.g., β9-like peptide (e.g., SmTrip9 pep245; SEQ ID NO: 23)) and the β10 chain (e.g., β10-like peptide (e.g., SmTrip10 pep86, HiBiT; SEQ ID NO: 25)). Experiments conducted during development of embodiments herein demonstrate, for example, that LgTrip 3092 (SEQ ID NO: 19) or LgTrip 3546 (SEQ ID NO: 51) can form luminescent complexes with suitable β9-like peptides (e.g., SmTrip9 pep245, SEQ ID NO: 23) and β10-like peptides (e.g., SmTrip10 pep86, HiBiT; SEQ ID NO: 25).During the development of the embodiments herein, experiments were conducted to develop artificial polypeptide components (e.g., SEQ ID NOs: 19, 21, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, and additional variants thereof) and peptide tags (e.g., the peptides listed in Table 1 and additional variants thereof) that have enhanced properties for reconstitution of light-emitting complexes.

[0126] Further experiments conducted during the development of embodiments herein have demonstrated the ability to synthesize other polypeptide components (e.g., β-glucan-1-phosphate dehydrogenase) derived from the NANOLUC-based, NanoBiT-based, and NanoTrip-based systems, polypeptides, and peptides described herein. 1~5 Dear, β 1~6 Dear, β 1~7 β-like, ... 10 like), dipeptides (e.g., β 6~7 Dear, β 7~8 Dear, β 8~9 Dear, β 9~10 like), tripeptides (e.g., β 6~8 Dear, β 7~9 Dear, β 8~10 like), polypeptides (e.g., β 6~10 Dear, β 6~9 Dear, β 7~10Experiments performed during the development of embodiments herein demonstrate the formation of bioluminescent complexes from two or more (e.g., two, three, four, five, etc.) peptide and polypeptide components that collectively comprise the entire length of a luciferase construct (e.g., a full-length luciferase polypeptide comprising 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or ranges therebetween) sequence identity to SEQ ID NO: 788 or 789).

[0127] In some embodiments, two peptide tags (e.g., a β9-like peptide (e.g., SmTrip9) and a β10-like peptide (e.g., SmTrip10)) and a polypeptide component (e.g., β 1~8 Provided herein are compositions and methods for the assembly of bioluminescent complexes from IgG1- and IgG2-like polypeptides (eg, LgTrip).

[0128] In some embodiments, the polypeptide component (e.g., β 1~5 Dear, β 1~6 Dear, β 1~7 Like, or β 1~8 -like polypeptides) as well as complementary peptide(s) (e.g., β6-like, β7-like, β8-like, β9-like, β 10 like), dipeptide(s) (e.g., β 6~7 Dear, β 7~8 Dear, β 8~9 Dear, β 9~10 like), tripeptides (e.g., β 6~8 Dear, β 7~9 Dear, β 8~10 β-like), and / or polypeptide(s) (e.g., 6~10 Dear, β 6~9 Dear, β 7~10 Provided herein are compositions and methods for the assembly of bioluminescent complexes from nucleophilic saccharides (e.g., saccharides), such as saccharides containing ... of interest.

[0129] In some embodiments, the peptide tag and one or more (e.g., two, three, four, five, etc.) of the polypeptide components are not fragments of an existing protein (e.g., not structurally complementary subsequences of a known polypeptide sequence). However, in other embodiments, the peptide tag and one or more of the polypeptide components can be fragments of a known or existing protein, polypeptide, or peptide. In certain embodiments, the bioluminescent activity of a polypeptide component (of a bioluminescent complex) is enhanced (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 10 ... 2 double, 10 3 double, 10 4 double, 10 5 double, 10 6fold, or even more). In some embodiments, provided herein are peptide (peptide tag) / polypeptide elements that can assemble into bioluminescent complexes for, for example, detecting and monitoring molecular interactions (e.g., protein-protein, protein-DNA, protein-RNA interactions, RNA-DNA, protein-small molecule, RNA-small molecule, DNA-DNA, RNA-RNA, PNA-DNA, PNA-RNA, etc.). In some embodiments, peptide / dipeptide / tripeptide tags (e.g., β6-like, β7-like, β8-like, β9-like (e.g., SmTrip9), and / or β10-like peptides (e.g., SmTrip10), and / or dipeptides and tripeptides thereof) are fused or otherwise linked to interacting elements. In certain embodiments, the peptide / dipeptide / tripeptide tag and polypeptide components do not form a complete bioluminescent complex without the promotion of interaction between the interacting elements for the purpose of detecting / monitoring molecular interactions. However, interaction (e.g., binding) of the interacting elements with each other (or to a target molecule or complex) promotes the formation of a bioluminescent complex. In some embodiments, the bioluminescent signal from the bioluminescent complex (or the ability to generate such a signal in the presence of a substrate) serves as a reporter of the formation of a complex by the interacting elements. If an interacting complex is formed, a bioluminescent complex is formed and a bioluminescent signal is detected / measured / monitored (e.g., in the presence of a substrate). If an interacting complex cannot be formed (e.g., due to unfavorable conditions, due to unstable interactions between interacting elements, due to mismatched interacting elements), a bioluminescent complex is not formed and no bioluminescent signal is generated (e.g., in the presence of a substrate). In some embodiments, the bioluminescent signal from the bioluminescent complex (or the ability to generate such a signal in the presence of a substrate) serves as a reporter of the binding of the interacting elements to a target. If binding to the target occurs, a bioluminescent complex is formed and a bioluminescent signal is detected / measured / monitored (e.g., in the presence of a substrate).If binding to the target is not possible (e.g., due to unfavorable conditions, due to an unstable interaction between the interacting element and the target, due to the absence of the target), no bioluminescent complex will form and no bioluminescent signal will be produced.

[0130] In certain embodiments, the interacting elements are two molecules of interest (e.g., protein(s) of interest, small molecule(s) of interest, etc.). For example, an assay can be performed to detect the interaction of two molecules of interest by tethering each to a separate peptide / dipeptide / tripeptide tag (e.g., β6-like, β7-like, β8-like, β9-like (e.g., SmTrip9), and / or β10-like (e.g., SmTrip10) peptide, and / or dipeptide and tripeptide thereof). When the molecules of interest interact (e.g., transiently interact, stably interact, etc.), the peptide / dipeptide / tripeptide tags are brought into close proximity in a suitable conformation, and a bioluminescent complex is formed between the peptide / dipeptide / tripeptide tag and the polypeptide component of the bioluminescent complex (and a bioluminescent signal is generated / detected (e.g., in the presence of a substrate)). If there is no interaction between the molecules of interest, the peptide / dipeptide / tripeptide tags are not in close proximity and / or are not oriented in a manner that promotes complex formation with the polypeptide component of the bioluminescent complex, a bioluminescent complex will not form, and no bioluminescent signal will be produced (in the presence of substrate). Such embodiments can be used to study the effect of inhibitors on complex formation, the effect of mutations on complex formation, the effect of conditions (e.g., temperature, pH, etc.) on complex formation, the interaction of small molecules (e.g., therapeutic drug candidates) with target molecules, etc.

[0131] In some embodiments, peptide / dipeptide / tripeptide tags (e.g., β6-like, β7-like, β8-like, β9-like (e.g., SmTrip9), and / or β10-like peptides (e.g., SmTrip10), and / or dipeptides and tripeptides thereof) and polypeptide components (e.g., β1-5-like, β1-6-like, β1-7-like, β1-8-like (e.g., LgTrip) polypeptides) are provided that can assemble into a bioluminescent complex without the promotion of interacting elements. In such embodiments, a bioluminescent complex is formed when the peptide / dipeptide / tripeptide tag and polypeptide components are simultaneously present (e.g., co-localized) within the same sample, subcellular compartment, cell, tissue, etc. In some embodiments, provided herein are peptide / dipeptide / tripeptide (tag) / polypeptide elements that can assemble into bioluminescent complexes for use in detecting and monitoring (e.g., without molecular interaction) the co-localization of molecular elements (e.g., proteins, nucleic acids, small molecules, lipids, carbohydrates, cellular structures, etc.). In some embodiments, the bioluminescent complexes are formed from peptide / dipeptide / tripeptide tags and polypeptide components that collectively span the entire β1-β10-like sequence. In some embodiments, the peptide / dipeptide / tripeptide tags are fused or otherwise linked to a co-localization element. In certain embodiments, the peptide / dipeptide / tripeptide tags and polypeptide components can form a bioluminescent complex without facilitation (e.g., without interacting elements), particularly for detecting / monitoring co-localization (e.g., without molecular interaction). Upon co-localization (e.g., within the same cell, on the same surface, in the same subcellular compartment, in the same tissue, etc.) of the co-localization elements (e.g., fused to the peptide / dipeptide / tripeptide tag), the formation of a bioluminescent complex (from the peptide / dipeptide / tripeptide tag and the polypeptide component) is promoted, regardless of whether the co-localization elements interact. In some embodiments, the bioluminescent signal from the bioluminescent complex (or the ability to generate such a signal in the presence of substrate) serves as a reporter of the co-localization of the co-localization elements.If the co-localization element co-localizes, a bioluminescent complex of the polypeptide component and the peptide / dipeptide / tripeptide tag fused to the co-localization element is formed and a bioluminescent signal is detected / measured / monitored (e.g., in the presence of a substrate). If the co-localization element does not co-localize, a bioluminescent complex is not formed and no bioluminescent signal is generated (e.g., in the presence of a substrate).

[0132] In certain embodiments, a colocalization pair comprises two molecules of interest (e.g., protein(s) of interest, small molecule(s) of interest, etc.). For example, an assay can be performed to detect colocalization of two molecules of interest (e.g., within a cell, subcellular compartment, tissue, etc.) by tethering each to a distinct dipeptide / tripeptide tag (e.g., β6-like, β7-like, β8-like, β9-like (e.g., SmTrip9), and / or β10-like (e.g., SmTrip10) peptide, and / or dipeptides and tripeptides thereof). When the molecules of interest colocalize, the peptide tags are in close proximity in a suitable conformation, a bioluminescent complex is formed with the polypeptide components (e.g., β1-5-like, β1-6-like, β1-7-like, β1-8-like (e.g., LgTrip) polypeptide), and a bioluminescent signal is generated / detected (e.g., in the presence of a substrate). If the molecules of interest are not co-localized, the polypeptide component and the peptide / dipeptide / tripeptide tag will not interact to form a complex and no bioluminescent signal will be generated (e.g., in the presence of a substrate). Such embodiments can be used to study the co-localization of molecules of interest under a variety of conditions.

[0133] In some embodiments, systems, assays, and devices are provided that include dipeptide / tripeptide tags (e.g., β6-like, β7-like, β8-like, β9-like (e.g., SmTrip9), and / or β10-like (e.g., SmTrip10) peptides and / or dipeptides and tripeptides thereof) and polypeptide components (e.g., β1-5-like, β1-6-like, β1-7-like, β1-8-like (e.g., LgTrip) polypeptides) for detecting analytes (e.g., small molecules, peptides, proteins, antibodies, nucleic acids, etc.) in a sample. In some embodiments, the peptide / dipeptide / tripeptide tags are tethered or fused to a target analyte, multiple target analytes, detection / binding agents (e.g., binding moieties, binding sequences, etc.) that recognize secondary analytes bound by the target analytes, secondary binding agents that bind to primary binding agents, etc. In some embodiments, various combinations of peptide / dipeptide / tripeptide tags tethered / fused to the above-mentioned detection / binding agents are used in assays and devices for detecting / quantifying / identifying analytes in a sample. Exemplary systems useful in the assays and devices are shown, for example, in Figures 51-56 and described herein.

[0134] In some embodiments, a dipeptide (e.g., a β9 / β10-like dipeptide) and a polypeptide component (e.g., a β 1~8 Provided herein are compositions and methods for the assembly of bioluminescent complexes from dipeptide-like (e.g., LgTrip) polypeptides. In some embodiments, the dipeptide and polypeptide components are not fragments of existing proteins (e.g., are not structurally complementary subsequences of known polypeptide sequences). However, in other embodiments, the dipeptide and / or polypeptide components can be fragments of known or existing proteins, polypeptides, or peptides. In certain embodiments, the bioluminescent activity of the polypeptide components (of the bioluminescent complex) is enhanced (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 10 ... 2 double, 10 3 double, 10 4 double, 10 5double, 10 6 times, or more). In some embodiments, β 1~8 The β-like polypeptide 1~9 In some embodiments, the β-like polypeptide exhibits weaker background luminescence than the β-like polypeptide. 1~8 The β-like polypeptide 1~9 These polypeptides exhibit increased thermal and chemical stability compared to similar polypeptides.

[0135] In some embodiments, provided herein are bioluminescent conjugates, including but not limited to, those that include any of the following combinations of peptide, dipeptide, tripeptide, and polypeptide components: β1-5-like polypeptide + β6-like peptide + β7-like peptide + β8-like peptide + β9-like peptide + β10-like peptide, β1-5-like polypeptide + β6-like peptide + β7-like peptide + β8-like peptide + β9 / 10-like dipeptide, β1-5-like polypeptide + β6-like peptide + β7 / 8-like dipeptide + β9 / 10-like dipeptide, β1-5-like polypeptide + β6 / 7 / 8-like tripeptide + β9 / 10-like dipeptide, β1-5-like polypeptide + β6-like peptide + β7 / 8 / 9-like tripeptide + β10-like peptide, β1-6-like polypeptide + β7-like peptide + β8-like peptide + β9-like peptide + β10-like peptide, β1-6-like polypeptide + β7-like peptide + β8-like peptide + β9 / 10-like dipeptide, β1-6-like polypeptide + β7 / 8-like dipeptide + β9 / 10-like dipeptide, β1-6-like polypeptide + β6 / 7 / 8-like dipeptide + β9-like peptide + β10-like peptide, β1-6-like polypeptide + β7 / 8 / 9-like tripeptide + β10-like peptide, β1-7-like polypeptide + β8-like peptide + β9-like peptide + β10-like peptide, β1-7-like polypeptide + β8-like peptide + β9 / 10-like dipeptide, β1-7-like polypeptide + β8 / 9-like dipeptide + β10-like peptide, β1-7-like polypeptide + β8 / 9 / 10-like tripeptide, β1-8-like polypeptide + β9-like peptide + β10-like peptide, β1-8-like polypeptide + β9 / 10-like dipeptide, β1-5-like polypeptide + β6-10-like polypeptide, β1-5-like polypeptide + β6-9-like polypeptide + β10-like peptide, and • β1-5-like polypeptide + β7-10-like polypeptide + β6-like peptide. The above combinations are not limiting and other combinations of peptide, dipeptide, tripeptide, and polypeptide components are within the scope of this specification.

[0136] In some embodiments, a β1-5-like polypeptide comprises positions 1-102 of SEQ ID NO: 788. In some embodiments, a β1-6-like polypeptide comprises positions 1-124 of SEQ ID NO: 788. In some embodiments, a β1-7-like polypeptide comprises positions 1-133 of SEQ ID NO: 788. In some embodiments, a β1-8-like polypeptide comprises positions 1-148 of SEQ ID NO: 788.

[0137] In some embodiments, a set of β5-10-like peptides / dipeptides / tripeptides / polypeptides collectively comprises positions 103-170 of SEQ ID NO: 788 or 789. In some embodiments, a set of β6-10-like peptides / dipeptides / tripeptides / polypeptides collectively comprises positions 125-170 of SEQ ID NO: 788 or 789. In some embodiments, a set of β7-10-like peptides / dipeptides / tripeptides / polypeptides collectively comprises positions 134-170 of SEQ ID NO: 788 or 789. In some embodiments, a set of β8-10-like peptides / dipeptides / tripeptides / polypeptides collectively comprises positions 149-170 of SEQ ID NO: 788 or 789.

[0138] In some embodiments, one or more components of a bioluminescent complex span a partial beta strand of a base luciferase described herein (e.g., OgLuc, NANOLUC, SEQ ID NO:788, SEQ ID NO:789, etc.). The division between peptide, dipeptide, tripeptide, and polypeptide components can be found at the division point between the beta strands or can occur at positions −1, −2, −3, 4, −5, +1, +2, +3, +4, +5, or more from the division point specified by the sequences herein. In some embodiments, peptide, dipeptide, tripeptide, and polypeptide components spanning the entire sequence of a base luciferase described herein (e.g., OgLuc, NANOLUC, SEQ ID NO:788, SEQ ID NO:789, etc.) can form a bioluminescent complex even if the division point of the component is not between beta strands.

[0139] For example, the split position between β5 and β6 can be between positions 102 and 103 of SEQ ID NO:788, or in some embodiments, such a split position can be located up to five residues before or after that position (e.g., after positions 96, 97, 98, 99, 100, 101, 103, 104, 105, 106, 107). In some embodiments, the split position between β6 and β7 can be located between positions 124 and 125 of SEQ ID NO:788, or in some embodiments, such a split position can be located up to five residues before or after that position (e.g., after positions 118, 119, 120, 121, 122, 123, 125, 126, 127, 128, 129). In some embodiments, the split position between β7 and β8 can be between positions 133 and 134 of SEQ ID NO:788, or in some embodiments, such a split position can be located up to five residues before or after that position (e.g., after positions 127, 128, 129, 130, 131, 132, 134, 135, 136, 137, 138). In some embodiments, the split position between β8 and β9 can be between positions 148 and 149 of SEQ ID NO:788, or in some embodiments, such a split position can be located up to five residues before or after that position (e.g., after positions 142, 143, 144, 145, 146, 147, 149, 150, 151, 152, 153).

[0140] In some embodiments, two consecutively adjacent peptide, dipeptide, tripeptide, and polypeptide components within the sequence of a base luciferase (e.g., OgLuc, NANOLUC, SEQ ID NO:788, SEQ ID NO:789, etc.) comprise all amino acids of the corresponding portion of the base sequence. In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, or more) amino acids adjacent to the division point in the base sequence are absent from the corresponding peptide, dipeptide, tripeptide, and / or polypeptide component.

[0141] In some embodiments, provided herein are peptides that include 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) sequence identity with one of the following: β6-like - GVTPNKLNYFGRPYEGIAVFDG (SEQ ID NO: 802), β7-like - KKITTTGTL (SEQ ID NO: 803), ● β8-like - WNGNKIIDERLITPD (sequence number 804), β9-like - GSMLFRVTINS (SEQ ID NO: 805), β10-like (high affinity)—VSGWRLFKKIS (SEQ ID NO: 806), and • β10-like (low affinity) - VTGYRLFEEIL (SEQ ID NO: 807).

[0142] In some embodiments, provided herein are peptides that include 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) sequence identity with one of the following: β6 / 7-like - GVTPNKLNYFGRPYEGIAVFDGKKITTTGTL (SEQ ID NO: 808), β7 / 8-like - KKITTTGTLWNGNKIIDERLITPD (sequence number 809), β8 / 9-like - WNGNKIIDERLITPDGSMLFRVTINS (SEQ ID NO: 810), β9 / 10-like (high affinity)—GSMLFRVTINSVSGWRLFKKIS (SEQ ID NO: 811), and • β9 / 10-like (low affinity)—GSMLFRVTINSVTGYRLFEEIL (SEQ ID NO: 812).

[0143] In some embodiments, provided herein are tripeptides that contain 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) sequence identity to one of the following: β6 / 7 / 8-like - GVTPNKLNYFGRPYEGIAVFDGKKITTTGTLWNGNKIIDERLITPD (SEQ ID NO: 813), β7 / 8 / 9-like - KKITTTGTLWNGNKIIDERLITPDGSMLFRVTINS (SEQ ID NO: 814), B8 / 9 / 10-like (high affinity) - WNGNKIIDERLITPDGSMLFRVTINSVSGWRLFKKIS (SEQ ID NO: 815), and ●B8 / 9 / 10-like (low affinity)- WNGNKIIDERLITPDGSMLFRVTINSVTGYRLFEEIL (SEQ ID NO: 816).

[0144] In some embodiments, provided herein are polypeptides that comprise 40% or greater (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) sequence identity to one of the following: β1-5-like - MVFTLDDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIMRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVID (SEQ ID NO: 790), β6-10-like (high affinity) - GVTPNKLNYFGRPYEGIAVFDGKKITTTGTLWNGNKIIDERLITPDGSMLFRVTINSVSGWRLFKKIS (SEQ ID NO: 794), β6-10-like (low affinity) - GVTPNKLNYFGRPYEGIAVFDGKKITTTGTLWNGNKIIDERLITPDGSMLFRVTINSVTGYRLFEEIL (SEQ ID NO: 798), β6-9-like - GVTPNKLNYFGRPYEGIAVFDGKKITTTGTLWNGNKIIDERLITPDGSMLFRVTINS (SEQ ID NO: 829), β7-10-like (high affinity)—KKITTTGTLWNGNKIIDERLITPDGSMLFRVTINSVSGWRLFKKIS (SEQ ID NO: 795), and • β7-10-like (low affinity)—KKITTTTGTLWNGNKIIDERLITPDGSMLFRVTINSVTGYRLFEEIL (SEQ ID NO: 799).

[0145] In some embodiments, a polypeptide component (e.g., of a pair of peptides / polypeptides or a bioluminescent complex) comprises 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) sequence identity to one of SEQ ID NOs: 788, 789, 790, 791, 792, and 793.

[0146] In some embodiments, the peptide / dipeptide / tripeptide components (e.g., tags) (e.g., of a pair of peptides / polypeptides or a bioluminescent complex) collectively comprise 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) sequence identity with one of SEQ ID NOs: 794, 795, 796, 797, 798, 799, 800, and 801.

[0147] In some embodiments, provided herein are sets of building blocks and complexes of the peptides, dipeptides, tripeptides, and polypeptides listed above. In certain embodiments, sets of building blocks are selected that span all 10 beta strands of the underlying luciferase sequence.

[0148] In some embodiments, the interaction, co-localization, detection, and other methods, assays, and techniques described herein for use with two peptide tag systems (e.g., β9-like (e.g., SmTrip9) peptides, β10-like (e.g., SmTrip10) peptides, and polypeptide components (e.g., β1-8-like (e.g., LgTrip) polypeptides)) are also useful with the dipeptide systems described herein (e.g., β9 / 10-like dipeptides and polypeptide components). In some embodiments, the dipeptide has a high affinity for the polypeptide component, and in such embodiments, a bioluminescent complex is formed when the dipeptide and polypeptide component come into contact (e.g., co-localized, added to a sample, etc.) without facilitating interaction. In some embodiments, the dipeptide has a low affinity for the polypeptide component. However, in such embodiments, bioluminescent complexes are not formed when the dipeptide and polypeptide components come into contact (e.g., co-localized, added to a sample, etc.) without facilitating interaction. As with the two-peptide tag systems described herein (e.g., a β9-like (e.g., SmTrip9) peptide, a β10-like (e.g., SmTrip10) peptide, and a polypeptide component (e.g., a β1-8-like (e.g., LgTrip) polypeptide)), dipeptide / polypeptide pairs of varying affinity can be selected for different applications. In some embodiments, systems, methods, and assays for two-component complementation systems are described in U.S. Pat. No. 9,797,890 (incorporated herein by reference in its entirety), and all such systems, methods, and assays are useful for use with the dipeptide / polypeptide systems described herein.

[0149] In some embodiments, the interaction, co-localization, detection, and other methods, assays, and techniques described herein for use with two peptide tag systems (e.g., β9-like (e.g., SmTrip9) peptides, β10-like (e.g., SmTrip10) peptides, and polypeptide components (e.g., β1-8-like (e.g., LgTrip) polypeptides)) are also useful with systems including any suitable combination of peptides, dipeptides, tripeptides, and polypeptides described herein. In some embodiments, the components have high affinity for each other, and in such embodiments, a bioluminescent complex is formed when the components contact (e.g., co-localize, are added to a sample, etc.) without facilitating interaction. In some embodiments, one or more of the components have low affinity for one or more of the other components, and in such embodiments, a bioluminescent complex is formed. In this configuration, bioluminescent complexes do not form when the components come into contact (e.g., co-localized, added to a sample, etc.) without facilitating interaction. Other systems with varying affinities, such as the two-peptide tag systems described herein (e.g., β9-like (e.g., SmTrip9) peptide, β10-like (e.g., SmTrip10) peptide, and polypeptide components (e.g., β1-8-like (e.g., LgTrip) polypeptide)), may be provided for different applications. In some embodiments, systems, methods, and assays for two-component complementation systems are described in U.S. Pat. No. 9,797,890 (incorporated herein by reference in its entirety), and all such systems, methods, and assays are useful for use with the various peptide, dipeptide, tripeptide, and polypeptide systems described herein.

[0150] In some embodiments, provided herein are complementary panels of interchangeable peptide / dipeptide / tripeptide tags (e.g., β6-like, β7-like, β8-like, β9-like (e.g., SmTrip9), and / or β10-like (e.g., SmTrip10) peptides and / or dipeptides and tripeptides thereof) and polypeptide components (e.g., β1-5-like, β1-6-like, β1-7-like, β1-8-like (e.g., LgTrip) polypeptides) with variable affinities and light emission upon formation of bioluminescent complexes therefrom (e.g., high affinity / high light emission, medium affinity / high light emission, low affinity / moderate light emission, etc.). Utilizing different combinations of peptide / dipeptide / tripeptide tags and polypeptide components provides a flexible system with a variety of sets ranging from low affinity, light emission, expression levels, stability, solubility, and other variable properties to higher affinity, light emission, expression levels, stability, solubility, and other variable properties. This adaptability allows the detection / monitoring / identification / quantification of analytes, molecular interactions, co-localization, and / or other properties to be fine-tuned to the particular molecule(s) of interest and / or condition being studied, expanding the range of molecular interactions and / or co-localization that can be detected / monitored / identified / quantified to include interactions with very high or low affinity. Further provided herein are methods for developing and testing non-luminescent elements and panels of non-luminescent elements.

[0151] In some embodiments, due to the small size of the tags (e.g., peptide tags) herein (e.g., compared to larger polypeptides and proteins), they are resistant to denaturation (they do not have the tertiary structure required for function).

[0152] In some embodiments, peptide / dipeptide / tripeptide tags and polypeptide components can be selected based on the molecule being studied or the protein of interest. In some embodiments, different peptide / dipeptide / tripeptide tags (e.g., β6-like, β7-like, β8-like, β9-like (e.g., SmTrip9), and / or β10-like (e.g., SmTrip10) peptides and / or their dipeptides and tripeptides) and polypeptide components (e.g., β1-5-like, β1-6-like, β1-7-like, β1-8-like (e.g., LgTrip) polypeptides) may require interaction complexes (e.g., complexes of interacting elements) of different strength, duration, and / or stability to result in the formation of a bioluminescent complex. In some embodiments, a highly stable interaction complex is required to generate a detectable bioluminescent signal (e.g., in the presence of a substrate). In other embodiments, even weak or transient interaction complexes result in the formation of a bioluminescent complex. In yet other embodiments, a bioluminescent complex forms in the absence of an interaction complex, so long as the peptide / dipeptide / tripeptide tag and polypeptide component co-localize. In some embodiments, the strength or extent of the interaction complex is directly proportional to the strength of the resulting bioluminescent signal. Some peptide / dipeptide / tripeptide tag / polypeptide component pairs have high millimolar dissociation constants (e.g., K d Other peptide / dipeptide / tripeptide tag / polypeptide component pairs produce a detectable signal when combined with an interaction complex at low millimolar (e.g., K > 100 mM) to produce a bioluminescent complex with a detectable signal. d <100 mM), micromolar (e.g., K d <1 mM), nanomolar (e.g., K d <1 μM), or even picomolar (e.g., K d The interaction requires an interaction pair with a dissociation constant of <1 nM.

[0153] In some embodiments, the peptide / dipeptide / tripeptide tags and / or polypeptide components herein are not fragments of existing proteins (e.g., existing bioluminescent proteins). In some embodiments, neither the peptide / dipeptide / tripeptide tags nor the polypeptide components used to form the complex are fragments of existing proteins (e.g., the same existing protein, an existing bioluminescent protein, etc.). In some embodiments, neither the peptide tags (e.g., β9-like (e.g., SmTrip9) and β10-like (e.g., SmTrip10) peptides; β9 / β10-like dipeptides, etc.) that assemble together to form the bioluminescent complex nor the polypeptide components (e.g., β 1~8 In some embodiments, the peptide / dipeptide / tripeptide tag or polypeptide component of a bioluminescent complex for use in embodiments of the present invention is not a subsequence of an existing protein. In some embodiments, a non-luminescent element for use in embodiments described herein does not comprise a structurally complementary subsequence of an existing protein.

[0154] In some embodiments, the peptide / dipeptide / tripeptide tags herein (e.g., β6-like, β7-like, β8-like, β9-like (e.g., SmTrip9) and / or β10-like (e.g., SmTrip10) peptides, and / or dipeptides and tripeptides thereof) are non-luminescent or substantially non-luminescent when associated with each other in the absence of the polypeptide components (e.g., β1-5-like, β1-6-like, β1-7-like, β1-8-like (e.g., LgTrip) polypeptides). In some embodiments, the polypeptide components ... In some embodiments, a single peptide / dipeptide / tripeptide tag (e.g., a β6-like, β7-like, β8-like, β9-like (e.g., SmTrip9), and / or β10-like (e.g., SmTrip10) peptide, and / or dipeptide and tripeptide thereof) and polypeptide component is non-luminescent or substantially non-luminescent in the absence of a second, third, or fourth peptide / dipeptide / tripeptide tag (e.g., in the presence or absence of a substrate). In certain embodiments, when placed under suitable conditions (e.g., physiological conditions), multiple peptide / dipeptide / tripeptide tags and polypeptide components interact to form a bioluminescent complex that generates a bioluminescent signal in the presence of a substrate.

[0155] In certain embodiments, the interacting element and / or co-localizing element and the peptide / dipeptide / tripeptide tag are attached, fused, linked, conjugated, etc. In typical embodiments, a first peptide / dipeptide / tripeptide tag and a first interacting element (or a first co-localizing element) are attached to each other, and a second peptide / dipeptide / tripeptide tag and a second interacting element (or a second co-localizing element) are attached to each other. Attachment of the peptide / dipeptide / tripeptide tag to the interacting element (or co-localizing element) can be achieved by any suitable mechanism, chemistry, linker, etc. The interacting element (or co-localizing element) and the peptide / dipeptide / tripeptide tag are typically attached by a covalent bond, although non-covalent linkage of the two elements is also provided. In some embodiments, the peptide / dipeptide / tripeptide tag and the interacting element (or co-localizing element) are directly conjugated, while in other embodiments, they are connected by a linker. In some embodiments, the peptide / dipeptide / tripeptide tag and the interacting element (or co-localizing element) are provided as a genetic / recombinant fusion. In some embodiments, endogenous tagging with the peptide / dipeptide / tripeptide tag herein (e.g., under endogenous regulatory control) allows for monitoring of normal cellular function using the tools described herein. For example, a protein of interest can be endogenously tagged with a high-affinity β9 / β10-like dipeptide (e.g., using CRISPR / Cas9), and then spontaneous complementation by LgTrip (or its variants) is monitored in cells, animals, lysates, etc.In other embodiments, the peptide tag and the interacting element (or co-localization element) are linked by chemical modification / chemical conjugation, such as native chemical ligation, Staudinger ligation, "traceless" Staudinger ligation, amide coupling, activated ester methods, methods targeting lysine, tyrosine, and cysteine ​​residues, imine bond formation (with or without orthoboronic acids), boronic acid / diol interactions, disulfide bond formation, copper-based and copper-free azide, diazo, and tetrazine "click" chemistry, UV-promoted thiol-ene conjugation, diazirine photolabeling, Diels-Alder cycloaddition, metathesis reactions, Suzuki cross-coupling, thiazolidine coupling (step-4), streptavidin / biotin complementation, HaloTag / chloroalkane substrate complementation, etc. In some embodiments, the peptide / dipeptide / tripeptide tag and interacting element (or co-localizing element) are synthetically produced (e.g., solid phase synthesis, solution phase synthesis, etc.). In some embodiments, the interacting element (or co-localizing element) is produced (e.g., synthetically or recombinantly) or obtained (e.g., from crude lysates, extracted proteins, purified proteins, etc.) by any suitable means.

[0156] In some embodiments, where the interacting element (or co-localization element) is a peptide or polypeptide, the peptide / dipeptide / tripeptide tag (e.g., β6-like, β7-like, β8-like, β9-like (e.g., SmTrip9), and / or β10-like (e.g., SmTrip10) peptides, and / or their dipeptides and tripeptides) and the interacting element (or co-localization element) are contained within a single amino acid chain. In some embodiments, the single amino acid chain comprises, consists of, or consists essentially of the peptide / dipeptide / tripeptide tag and the interacting element (or co-localization element). In some embodiments, the single amino acid chain comprises, consists of, or consists essentially of the peptide / dipeptide / tripeptide tag, the interacting element (or co-localization element), optionally one or more N-terminal sequences, C-terminal sequences, regulatory elements (e.g., promoters, translation start sites, etc.), and linker sequences. In some embodiments, the peptide / dipeptide / tripeptide tag and the interacting element (or co-localization element) are contained within a fusion polypeptide. In some embodiments, a first fusion of a peptide / dipeptide / tripeptide tag and an interacting element (or co-localizing element) and a second fusion of a peptide / dipeptide / tripeptide tag and an interacting element (or co-localizing element) are expressed separately, while in other embodiments a fusion protein is expressed that comprises or consists of both an interacting (or co-localizing) and a peptide / dipeptide / tripeptide tag.

[0157] In some embodiments, a first fusion protein comprising a first peptide / dipeptide / tripeptide tag (e.g., a β6-like, β7-like, β8-like, β9-like (e.g., SmTrip9), and / or β10-like (e.g., SmTrip10) peptide, and / or dipeptides and tripeptides thereof) and a first interacting element and a second fusion protein comprising a second peptide / dipeptide / tripeptide tag (e.g., a β6-like, β7-like, β8-like, β9-like (e.g., SmTrip9), and / or β10-like (e.g., SmTrip10) peptide, and / or dipeptides and tripeptides thereof) and a second interacting element are expressed in the same cell. In some embodiments, a first fusion protein comprising a first peptide / dipeptide / tripeptide tag and a first co-localization element and a second fusion protein comprising a second peptide / dipeptide / tripeptide tag and a second co-localization element are expressed in the same cell. In some embodiments, the first and second fusion proteins are purified and / or isolated from cells. In some embodiments, the interaction and / or co-localization of the fusion proteins is analyzed intracellularly. In some embodiments, the interaction and / or co-localization of the fusion proteins is analyzed in a cell lysate. In other embodiments, the first and second fusion proteins are expressed in separate cells and combined for signal detection (e.g., after purification and / or isolation, after fusion of cells or portions of cells, by transfer of the fusion proteins from one cell to the other, or by secretion of one or more fusion proteins into the extracellular medium). In some embodiments, one or more fusion proteins are expressed in a cell lysate (e.g., rabbit reticulocyte lysate) or in a cell-free system. In some embodiments, one or more fusion proteins are expressed from the genome of a virus or other cellular pathogen.In some embodiments, the polypeptide components for complex formation (with the first and second fusion proteins) (e.g., β1-5-like, β1-6-like, β1-7-like, β1-8-like (e.g., LgTrip) polypeptides) and any other peptide / dipeptide / tripeptide components (e.g., β6-like, β7-like, β8-like, β9-like (e.g., SmTrip9), and / or β10-like (e.g., SmTrip10) peptides, and / or dipeptides and tripeptides thereof) are expressed in the same cell or cell lysate as one or both of the tag-containing fusion proteins. In some embodiments, the peptide / dipeptide / tripeptide / polypeptide components for complex formation with the peptide / dipeptide / tripeptide tags (in the first and second fusion proteins) are expressed in a different cell or cell lysate than one or both of the peptide tag-containing fusion proteins. In some embodiments, the peptide / dipeptide / tripeptide / polypeptide components for complex formation with the peptide / dipeptide / tripeptide tag (in the first and second fusion proteins) are added to cells, cell lysates, or other samples containing the fusion proteins containing the peptide tag.

[0158] In some embodiments, the systems (e.g., peptide / dipeptide / tripeptide tags, peptide / dipeptide / tripeptide / polypeptide components, substrates, vectors, etc.) and methods herein are useful in analyzing samples (e.g., colocalization, molecular interactions, target detection / quantification / identification / monitoring, etc.). In some embodiments, one or more of the components of the systems herein are added to and / or provided in or expressed within a sample. Suitable samples that may be useful in embodiments herein include, but are not limited to, blood, plasma, serum, urine, saliva, cells, cell lysates, tissues, tissue homogenates, purified nucleic acids, stool, vaginal secretions, cerebrospinal fluid, allantoic fluid, water, biofilms, soil, dust, food, beverages, agricultural products, plants, etc.

[0159] In certain embodiments, nucleic acids, DNA, RNA, vectors, etc. are provided that encode the peptide / dipeptide / tripeptide tags described herein (e.g., β6-like, β7-like, β8-like, β9-like (e.g., SmTrip9), and / or β10-like (e.g., SmTrip10) peptides, and / or dipeptides and tripeptides thereof) and polypeptide components (e.g., β1-5-like, β1-6-like, β1-7-like, β1-8-like (e.g., LgTrip) polypeptides), fusion polypeptides, fusion proteins, etc. Such nucleic acids and vectors can be used for expression, transformation, gene transfer, injection, etc.

[0160] In some embodiments, the peptide / dipeptide / tripeptide tag (e.g., β6-like, β7-like, β8-like, β9-like (e.g., SmTrip9), and / or β10-like (e.g., SmTrip10) peptides, and / or their dipeptides and tripeptides) and the interacting element, co-localization element, or binding agent are connected by a linker. In some embodiments, the linker connects the signaling element and the interacting element or co-localization element while providing a desired amount of space / distance between the elements. In some embodiments, the linker allows both the signaling element and the interacting element to simultaneously form their respective complexes (e.g., luminescent complex and interacting complex). In some embodiments, the linker helps the interacting element promote the formation of the luminescent complex. In some embodiments, once the interacting complex is formed, the linker connecting each peptide / dipeptide / tripeptide tag to its respective interacting element positions the peptide tags at the appropriate distance and conformation to form a bioluminescent complex. In some embodiments, the interacting or co-localizing element and the peptide / dipeptide / tripeptide tag are held in close proximity (e.g., less than 4 monomer units) by a linker. In some embodiments, the linker provides a desired amount of distance (e.g., 1, 2, 3, 4, 5, 6, 10, 20, or more monomer units) between the peptide tag and the interacting element (e.g., to prevent undesired interactions between the peptide / dipeptide / tripeptide tag and the interacting or co-localizing element due to steric issues, to allow proper orientation of the non-emissive element upon formation of the interacting complex, to allow propagation of complex formation from the interacting complex to the emissive complex, etc.). In certain embodiments, the linker provides appropriate attachment chemistry between the peptide / dipeptide / tripeptide tag and the interacting element. The linker may improve the synthetic process for producing the peptide / dipeptide / tripeptide tag and the interacting or co-localizing element (e.g., by allowing them to be synthesized as a single unit, by allowing post-synthetic conjugation of the two elements, etc.).

[0161] In some embodiments, the linker is any suitable chemical moiety capable of linking, binding, or tethering a peptide / dipeptide / tripeptide tag (e.g., β6-like, β7-like, β8-like, β9-like (e.g., SmTrip9), and / or β10-like (e.g., SmTrip10) peptides, and / or dipeptides and tripeptides thereof) or polypeptide component (e.g., β1-5-like, β1-6-like, β1-7-like, β1-8-like (e.g., LgTrip) polypeptides) to an interacting element or co-localization element. In some embodiments, the linker is a polymer of one or more repeating or non-repeating monomeric units (e.g., nucleic acid chain, amino acid chain, carbon-containing polymer chain, carbon chain, etc.). When the peptide / dipeptide / tripeptide tag and interacting element, co-localization element, or binding agent are part of a fusion protein, the linker (if present) is typically an amino acid chain. In cases where the peptide / dipeptide / tripeptide tag and the interacting element, co-localization element, or binding agent are tethered to one another after expression of the individual elements, the linker may comprise any chemical moiety having a functional group (or reactive group) at either end that is reactive with the functional groups of the peptide tag and the interacting element or co-localization element, respectively. Any suitable moiety capable of tethering the signal element and the interacting element, co-localization element, and / or binding agent may be useful as a linker.

[0162] A wide variety of linkers can be used. In some embodiments, the linker is a single covalent bond. In some embodiments, the linker comprises a linear or branched, cyclic or heterocyclic, saturated or unsaturated structure having 1 to 20 non-hydrogen atoms (e.g., C, N, P, O, and S), and is composed of any combination of alkyl, ether, thioether, imine, carboxylic acid, amine, ester, carboxamide, sulfonamide, hydrazide linkages, and aromatic or heteroaromatic linkages. In some embodiments, the linker is longer than 20 non-hydrogen atoms (e.g., 21 non-hydrogen atoms, 25 non-hydrogen atoms, 30 non-hydrogen atoms, 40 non-hydrogen atoms, 50 non-hydrogen atoms, 100 non-hydrogen atoms, etc.). In some embodiments, the linker comprises 1 to 50 non-hydrogen atoms (in addition to hydrogen atoms) selected from the group of C, N, P, O, and S (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 non-hydrogen atoms).

[0163] The scope of the embodiments herein is not limited by the type of linker available. The peptide / dipeptide / tripeptide tag, polypeptide component, and interacting element, co-localizing element, or binding agent may be linked either directly (e.g., a linker consisting of a single covalent bond) or via a suitable linker. The embodiments are not limited to any particular linker group. A variety of linker groups are contemplated, and suitable linkers include, but are not limited to, alkyl groups, methylene carbon chains, ethers, polyethers, alkylamide linkers, peptide linkers, modified peptide linkers, poly(ethylene glycol) (PEG) linkers, streptavidin-biotin or avidin-biotin linkers, polyamino acids (e.g., polylysine), functionalized PEGs, polysaccharides, glycosaminoglycans, dendritic polymers (see WO 93 / 06868 and To The linker may comprise a cleavable polymer, such as those described in WO 94 / 08629, WO 94 / 09056, and WO 96 / 26754 (incorporated herein by reference in their entireties), an oligonucleotide linker, a phospholipid derivative, an alkenyl chain, an alkynyl chain, a disulfide, or a combination thereof. In some embodiments, the linker is cleavable (e.g., enzymatically (e.g., TEV protease site), chemically, photoinducibly, etc.).

[0164] In some embodiments, peptide / dipeptide / tripeptide tags (e.g., β6-like, β7-like, β8-like, β9-like (e.g., SmTrip9), and / or β10-like (e.g., SmTrip10) peptides, and / or dipeptides and tripeptides thereof) and polypeptide components (e.g., β1-5-like, β1-6-like, β1-7-like, β1-8-like (e.g., LgTrip) polypeptides), recognition elements, interaction elements, co-localization elements, binding agents, analytes, substrates, etc. are attached (e.g., by any suitable chemistry) to or contained within a solid surface or matrix. In some embodiments, one or more system components are attached (e.g., by any suitable chemistry) to or contained within a solid surface or matrix, while other components are added to the solid surface or matrix (e.g., in solution (e.g., in a sample)). Suitable solid surfaces include, but are not limited to, beads (e.g., magnetic beads), chips, tubes, plates, particles, membranes, paper, etc. In some embodiments, the solid surface / matrix may be any suitable material, for example, Ahlstrom CytoSep, nitrocellulose, cellulose acetate, cellulose (e.g., Whatman FTA-DMPK-A, B, and C cards; Whatman ET 3 / Chr; Whatman Protein Saver 903 cards; Whatman Grade 1 filter paper; Whatman FTA Elute;Ahlstrom 226 specimen collection form), Noviplex Plasma Prep Cards, polypropylene membranes, PVDF, nitrocellulose membranes (Millipore Nitrocellular Hi Flow Plus), polytetrafluoroethylene membranes, mixed cellulose esters, glass fiber media (e.g., Whatman Unifilter Plates, glass fiber filter membranes, Agilent dry matrix spotting cards, Ahlstrom Grade 8950, etc.), plastics (e.g., polyester, polypropylene, polyethersulfone, poly(methacrylate), acrylic polymers, polytetrafluoroethylene, etc.), natural and synthetic polymers (e.g., polymer blends, block copolymers, etc.), sugars (e.g., pullulan, trehalose, maltose, sucrose, cellulose, etc.), polyamides (e.g., natural (e.g., wool, silk, etc.) and synthetic (e.g., aramid, nylon, etc.)), metals (e.g., aluminum, cadmium, chromium, cobalt, copper, iron, manganese, nickel, platinum, palladium, rhodium, silver, gold, tin, titanium, tungsten, vanadium, zinc, etc.), alloys (e.g., aluminum alloys (e.g., Al-Li, alumel, duralumin, Magnox, Zama, etc.), Iron alloys (e.g., steel, stainless steel, surgical stainless steel, silicon steel, tool steel, cast iron, mirror iron, etc.), cobalt alloys (e.g., Stellite, Talonite, etc.), nickel alloys (e.g., nickel silver, chromel, mumetal, Monel metal, nichrome, Nicrosil, Nisil, Nitinol, etc.), copper alloys (e.g., beryllium copper, Billon, brass, bronze, phosphor bronze, Constantan, cupronickel, Bellmetal, Devarda's alloy, plated alloys, nickel silver, Nordic Gold, Royal Gold, Tumbaga, etc.), silver alloys (e.g., sterling silver, etc.), tin alloys (e.g., Britannium, pewter, solder, etc.), gold alloys (e.g., electrum, white gold, etc.), amalgam, ELISPot plates, immunoassay plates, tissue culture plates, etc.;

[0165] In some embodiments, peptide / dipeptide / tripeptide tags (e.g., β6-like, β7-like, β8-like, β9-like (e.g., SmTrip9), and / or β10-like (e.g., SmTrip10) peptides, and / or dipeptides and tripeptides thereof) and polypeptide components of light-emitting complexes (e.g., β1-5-like, β1-6-like, β1-7-like, β1-8-like (e.g., LgTrip) polypeptides) are provided that have less than 100% sequence identity and / or similarity to any portion of an existing luciferase (e.g., firefly luciferase, Renilla luciferase, Oplophorus luciferase, or the highly sensitive Oplophorus luciferases described in U.S. Patent Application Nos. 2010 / 0281552 and 2012 / 0174242, which are incorporated by reference in their entireties). Certain embodiments involve the formation of bioluminescent complexes of peptide / dipeptide / tripeptide tags and polypeptide components that have less than 100% sequence identity to all or a portion (e.g., 8 amino acids or more, less than about 25 amino acids for the peptide) of SEQ ID NO:1 (e.g., the entire sequence of wild-type Oplophorus luciferase) and / or SEQ ID NO:3 (e.g., the entire sequence of NANOLUC). Certain embodiments involve the formation of bioluminescent complexes from peptide / dipeptide / tripeptide tag and polypeptide components that have less than 100% but more than 40% (e.g., more than 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%) sequence identity to all or a portion (e.g., 8 amino acids or more, less than about 25 amino acids for peptides) of SEQ ID NO:1 (e.g., the entire sequence of wild-type Oplophorus luciferase) and / or SEQ ID NO:3 (e.g., the entire sequence of NANOLUC). In some embodiments, peptide / dipeptide / tripeptide tag and polypeptide components that have less than 100% sequence similarity to a portion (e.g., 8 amino acids or more, less than about 25 amino acids for peptides) of SEQ ID NO:1 (e.g., the entire sequence of wild-type Oplophorus luciferase) and / or SEQ ID NO:3 (e.g., the entire sequence of NANOLUC) are provided.In some embodiments, peptide / dipeptide / tripeptide tag and polypeptide components are provided that have less than 100% but more than 40% (e.g., more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, more than 99%) sequence similarity to a portion (e.g., 8 amino acids or more, less than about 25 amino acids for peptides) of SEQ ID NO:1 (e.g., the entire sequence of wild-type Oplophorus luciferase) and / or SEQ ID NO:3 (e.g., the entire sequence of NANOLUC). In some embodiments, peptide / dipeptide / tripeptide tags are provided that have less than 100% sequence identity and / or similarity to about a 25 amino acid or less portion of SEQ ID NO:1 (e.g., the entire sequence of wild-type Oplophorus luciferase) and / or SEQ ID NO:3 (e.g., the entire sequence of NANOLUC), where two of such peptides, when combined under appropriate conditions (e.g., stabilized by an interacting pair, brought into proximity by a co-localizing element, etc.), form a bioluminescent complex with a polypeptide component that has less than 100% but more than 40% (e.g., more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, more than 99%) sequence identity and / or similarity to another portion of SEQ ID NO:1 (e.g., the entire sequence of wild-type Oplophorus luciferase) and / or SEQ ID NO:3 (e.g., the entire sequence of NANOLUC).In some embodiments, peptide / dipeptide / tripeptide tags are provided that have less than 100% sequence identity and / or similarity to about a 25 amino acid or less portion of SEQ ID NO:1 (e.g., the entire sequence of wild-type Oplophorus luciferase) and / or SEQ ID NO:3 (e.g., the entire sequence of NANOLUC), where a pair of such peptide tags, when combined under appropriate conditions (e.g., stabilized by an interacting pair, brought into proximity by a co-localizing element, etc.), form a bioluminescent complex with a polypeptide component that has less than 100% but more than 40% (e.g., more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, more than 99%) sequence identity and / or similarity to another portion of SEQ ID NO:1 (e.g., the entire sequence of wild-type Oplophorus luciferase) and / or SEQ ID NO:3 (e.g., the entire sequence of NANOLUC). In some embodiments, peptide / dipeptide / tripeptide tags are provided that have less than 100% but more than 40% (e.g., more t...

Claims

1. A peptide comprising an amino acid sequence having more than 40% but less than 100% sequence identity with SEQ ID NO:23 and less than 100% sequence identity with SEQ ID NO:6, SEQ ID NO:9, and / or SEQ ID NO:29, wherein when the peptide is contacted with a second peptide consisting of SEQ ID NO:25 and a polypeptide complement consisting of SEQ ID NO:17, the bioluminescent signal generated in the presence of coelenterazine or a coelenterazine analog substrate is substantially increased compared to the bioluminescent signal generated by the peptide and the coelenterazine substrate alone.

2. The peptide of claim 1, wherein the bioluminescent signal is substantially increased when the peptide associates with the second peptide and the polypeptide complement.

3. The peptide of claim 1, which exhibits enhanced one or more traits compared to the peptides of SEQ ID NO: 6 and / or SEQ ID NO: 9, wherein the traits are selected from affinity for the second peptide and the polypeptide complement, expression, solubility, stability, and bioluminescent activity when combined with the second peptide and the polypeptide complement.

4. The peptide of claim 1 , wherein the amino acid sequence is not a naturally occurring protein or a fragment thereof.

5. The peptide of claim 4 , wherein the amino acid sequence contains unnatural amino acids, amino acid analogs, and / or peptoid amino acids.

6. A nucleic acid comprising a sequence encoding the peptide of claim 1.

7. A fusion polypeptide comprising the peptide of claim 1 and an additional amino acid sequence.

8. The fusion polypeptide of claim 7 , wherein the additional amino acid sequence is selected from the group consisting of a protein of interest, an interacting element, a co-localization element, and a binding moiety.

9. 9. The fusion polypeptide of claim 8, wherein the additional amino acid sequence is a binding moiety selected from the group consisting of an antibody (polyclonal, monoclonal, and / or recombinant antibody), an antibody fragment, Protein A, the Ig-binding domain of Protein A, Protein G, the Ig-binding domain of Protein G, Protein A / G, the Ig-binding domain of Protein A / G, Protein L, the Ig-binding domain of Protein L, Protein M, the Ig-binding domain of Protein M, an oligonucleotide probe, a peptide nucleic acid, a DARPin, an aptamer, an affimer, a purified protein (either the analyte itself or a protein that binds to the analyte), and an analyte-binding domain(s) of a protein.

10. 9. The fusion polypeptide of claim 8, wherein the additional amino acid sequence is a first interacting polypeptide configured to form a complex with a second interacting polypeptide upon contact of the first interacting polypeptide and the second interacting polypeptide.

11. 9. The fusion polypeptide of claim 8, wherein the additional amino acid sequence is a first co-localization polypeptide configured to co-localize with a second co-localization polypeptide within a subcellular compartment, cell, tissue, or organism.

12. The fusion polypeptide of claim 8, wherein the additional amino acid sequence is a protein of interest and a potential drug target.

13. A nucleic acid comprising a sequence encoding the fusion polypeptide of claim 7.

14. A peptide comprising an amino acid sequence having more than 40% but less than 100% sequence identity with SEQ ID NO: 25 and less than 100% sequence identity with SEQ ID NO: 7 and / or SEQ ID NO: 10, wherein when the peptide is contacted with a second peptide consisting of SEQ ID NO: 23 and a polypeptide complement consisting of SEQ ID NO: 17, the bioluminescent signal generated in the presence of coelenterazine or a coelenterazine analog substrate is substantially increased compared to the bioluminescent signal generated by the peptide and the coelenterazine substrate alone.

15. 15. The peptide of claim 14, wherein the bioluminescent signal is substantially increased when the peptide associates with the second peptide and the polypeptide complement.

16. 15. The peptide of claim 14, which exhibits enhanced one or more traits compared to the peptides of SEQ ID NO: 7 and / or SEQ ID NO: 10, wherein the traits are selected from affinity for the second peptide and the polypeptide complement, expression, solubility, stability, and bioluminescent activity when combined with the second peptide and the polypeptide complement.

17. 15. The peptide of claim 14, wherein the amino acid sequence is not a naturally occurring protein or a fragment thereof.

18. 18. The peptide of claim 17, wherein the amino acid sequence contains unnatural amino acids, amino acid analogs, and / or peptoid amino acids.

19. A nucleic acid comprising a sequence encoding the peptide of claim 14.

20. A fusion polypeptide comprising the peptide of claim 14 and an additional amino acid sequence.

21. 21. The fusion polypeptide of claim 20, wherein the additional amino acid sequence is selected from the group consisting of a protein of interest, an interacting element, a co-localization element, and a binding moiety.

22. 22. The fusion polypeptide of claim 21, wherein the additional amino acid sequence is a binding moiety selected from the group consisting of an antibody (polyclonal, monoclonal, and / or recombinant antibody), an antibody fragment, Protein A, the Ig-binding domain of Protein A, Protein G, the Ig-binding domain of Protein G, Protein A / G, the Ig-binding domain of Protein A / G, Protein L, the Ig-binding domain of Protein L, Protein M, the Ig-binding domain of Protein M, an oligonucleotide probe, a peptide nucleic acid, a DARPin, an aptamer, an affimer, a purified protein (either the analyte itself or a protein that binds to the analyte), and an analyte-binding domain(s) of a protein.

23. 22. The fusion polypeptide of claim 21, wherein the additional amino acid sequence is a first interacting polypeptide configured to form a complex with a second interacting polypeptide upon contact of the first interacting polypeptide and the second interacting polypeptide.

24. 22. The fusion polypeptide of claim 21, wherein the additional amino acid sequence is a first co-localization polypeptide configured to co-localize with a second co-localization polypeptide within a subcellular compartment, cell, tissue, or organism.

25. 22. The fusion polypeptide of claim 21, wherein the additional amino acid sequence is a protein of interest and a potential drug target.

26. A nucleic acid comprising a sequence encoding the fusion polypeptide of claim 20.

27. (a) a first peptide comprising an amino acid sequence having more than 40% but less than 100% sequence identity with SEQ ID NO:25 and less than 100% sequence identity with SEQ ID NO:7 and / or SEQ ID NO:10; (b) a second peptide comprising an amino acid sequence having more than 40% but less than 100% sequence identity with SEQ ID NO:23 and less than 100% sequence identity with SEQ ID NO:6, SEQ ID NO:9, and SEQ ID NO:29, The composition, wherein when the first peptide is contacted with the second peptide and a polypeptide complement consisting of SEQ ID NO: 17, the bioluminescent signal generated in the presence of coelenterazine or a coelenterazine analog substrate is substantially increased compared to the bioluminescent signal generated by the first peptide and / or the second peptide and the coelenterazine substrate alone.

28. 28. The composition of claim 27, wherein the bioluminescent signal is substantially increased when the first peptide associates with the second peptide and the polypeptide complement.

29. 28. The composition of claim 27, wherein the first peptide exhibits one or more enhanced traits compared to the peptides of SEQ ID NO: 7 and / or SEQ ID NO: 10, and the second peptide exhibits one or more enhanced traits compared to the peptides of SEQ ID NO: 6, SEQ ID NO: 9 and SEQ ID NO: 29, the traits being selected from affinity for the second peptide and the polypeptide complement, expression, solubility, stability, and bioluminescent activity when combined with the second peptide and the polypeptide complement.

30. 28. The composition of claim 27, wherein the amino acid sequence of the first and / or second peptide is not a naturally occurring protein or a fragment thereof.

31. 31. The composition of claim 30, wherein the amino acid sequences of the first and / or second peptides contain unnatural amino acids, amino acid analogs, and / or peptoid amino acids.

32. 28. A composition comprising a nucleic acid comprising a sequence encoding the first and second peptides of claim 27.

33. 28. A composition comprising a fusion polypeptide comprising the first and second peptides of claim 27 and an additional amino acid sequence.

34. 34. The composition of claim 33, wherein the additional amino acid sequence is selected from the group consisting of a protein of interest, an interacting element, a co-localization element, and a binding moiety.

35. 35. The composition of claim 34, wherein the additional amino acid sequence is a binding moiety selected from the group consisting of an antibody (polyclonal, monoclonal, and / or recombinant antibody), an antibody fragment, Protein A, the Ig-binding domain of Protein A, Protein G, the Ig-binding domain of Protein G, Protein A / G, the Ig-binding domain of Protein A / G, Protein L, the Ig-binding domain of Protein L, Protein M, the Ig-binding domain of Protein M, an oligonucleotide probe, a peptide nucleic acid, a DARPin, an aptamer, an affimer, a purified protein (either the analyte itself or a protein that binds to the analyte), and an analyte-binding domain(s) of a protein.

36. 35. The composition of claim 34, wherein the additional amino acid sequence is a first interacting polypeptide that is configured to form a complex with a second interacting polypeptide upon contact of the first interacting polypeptide and the second interacting polypeptide.

37. 35. The composition of claim 34, wherein the additional amino acid sequence is a first co-localization polypeptide configured to co-localize with a second co-localization polypeptide within a subcellular compartment, cell, tissue, or organism.

38. 35. The composition of claim 34, wherein the additional amino acid sequence is a protein of interest and a potential drug target.

39. 34. A composition comprising a nucleic acid comprising a sequence encoding the composition of claim 33.

40. A polypeptide comprising an amino acid sequence having more than 40% but less than 100% sequence identity with SEQ ID NO: 17 and less than 100% sequence identity with SEQ ID NO: 5, SEQ ID NO: 8, and / or SEQ ID NO: 27, wherein when the polypeptide is contacted with a first peptide consisting of SEQ ID NO: 23 and a second peptide consisting of SEQ ID NO: 25, the bioluminescent signal generated in the presence of coelenterazine or a coelenterazine analog substrate is substantially increased compared to the bioluminescent signal generated by the peptides and the coelenterazine substrate alone.

41. 41. The polypeptide of claim 40, wherein the bioluminescent signal is substantially increased when the polypeptide associates with the first and second peptides.

42. 41. The polypeptide of claim 40, wherein the polypeptide exhibits enhanced one or more traits compared to the polypeptides of SEQ ID NO: 5 and / or SEQ ID NO: 8, the traits being selected from affinity for the first and / or second peptides, expression, solubility, stability, and bioluminescent activity when combined with the first and second peptides.

43. 41. The polypeptide of claim 40, wherein the amino acid sequence is not a naturally occurring protein or fragment thereof.

44. 44. The polypeptide of claim 43, wherein the amino acid sequence contains unnatural amino acids, amino acid analogs, and / or peptoid amino acids.

45. 41. A nucleic acid comprising a sequence encoding the polypeptide of claim 40.

46. 41. A fusion polypeptide comprising the polypeptide of claim 40 and an additional amino acid sequence.

47. 47. The fusion polypeptide of claim 46, wherein the additional amino acid sequence is selected from the group consisting of a protein of interest, an interacting element, a co-localization element, and a binding moiety.

48. 48. The fusion polypeptide of claim 47, wherein the additional amino acid sequence is a binding moiety selected from the group consisting of an antibody (polyclonal, monoclonal, and / or recombinant antibody), an antibody fragment, Protein A, the Ig-binding domain of Protein A, Protein G, the Ig-binding domain of Protein G, Protein A / G, the Ig-binding domain of Protein A / G, Protein L, the Ig-binding domain of Protein L, Protein M, the Ig-binding domain of Protein M, an oligonucleotide probe, a peptide nucleic acid, a DARPin, an aptamer, an affimer, a purified protein (either the analyte itself or a protein that binds to the analyte), and an analyte-binding domain(s) of a protein.

49. 48. The fusion polypeptide of claim 47, wherein the additional amino acid sequence is a first interacting polypeptide configured to form a complex with a second interacting polypeptide upon contact of the first interacting polypeptide and the second interacting polypeptide.

50. 48. The fusion polypeptide of claim 47, wherein the additional amino acid sequence is a first co-localization polypeptide configured to co-localize with a second co-localization polypeptide within a subcellular compartment, cell, tissue, or organism.

51. 48. The fusion polypeptide of claim 47, wherein the additional amino acid sequence is a protein of interest and a potential drug target.

52. 47. A nucleic acid comprising a sequence encoding the fusion polypeptide of claim 46.

53. A polypeptide comprising an amino acid sequence having 40% or more sequence identity with SEQ ID NO:51 and / or 302 and less than 100% sequence identity with SEQ ID NO:5, SEQ ID NO:8, and SEQ ID NO:27, wherein when the polypeptide is contacted with a first peptide consisting of SEQ ID NO:23 and a second peptide consisting of SEQ ID NO:25, the bioluminescent signal generated in the presence of coelenterazine or a coelenterazine analog substrate is substantially increased compared to the bioluminescent signal generated by the peptides and the coelenterazine substrate alone.

54. 54. The polypeptide of claim 53, wherein the bioluminescent signal is substantially increased when the polypeptide associates with the first and second peptides.

55. 54. The polypeptide of claim 53, wherein the polypeptide exhibits enhanced one or more traits compared to the polypeptides of SEQ ID NO: 5 and / or SEQ ID NO: 8, the traits being selected from affinity for the first and / or second peptides, expression, solubility, stability, and bioluminescent activity when combined with the first and second peptides.

56. 54. The polypeptide of claim 53, wherein the amino acid sequence is not a naturally occurring protein or fragment thereof.

57. 57. The polypeptide of claim 56, wherein the amino acid sequence contains unnatural amino acids, amino acid analogs, and / or peptoid amino acids.

58. 54. A nucleic acid comprising a sequence encoding the polypeptide of claim 53.

59. 54. A fusion polypeptide comprising the polypeptide of claim 53 and an additional amino acid sequence.

60. 60. The fusion polypeptide of claim 59, wherein the additional amino acid sequence is selected from the group consisting of a protein of interest, an interacting element, a co-localization element, and a binding moiety.

61. 61. The fusion polypeptide of claim 60, wherein the additional amino acid sequence is a binding moiety selected from the group consisting of an antibody (polyclonal, monoclonal, and / or recombinant antibody), an antibody fragment, Protein A, the Ig-binding domain of Protein A, Protein G, the Ig-binding domain of Protein G, Protein A / G, the Ig-binding domain of Protein A / G, Protein L, the Ig-binding domain of Protein L, Protein M, the Ig-binding domain of Protein M, an oligonucleotide probe, a peptide nucleic acid, a DARPin, an aptamer, an affimer, a purified protein (either the analyte itself or a protein that binds to the analyte), and an analyte-binding domain(s) of a protein.

62. 61. The fusion polypeptide of claim 60, wherein the additional amino acid sequence is a first interacting polypeptide configured to form a complex with a second interacting polypeptide upon contact of the first interacting polypeptide and the second interacting polypeptide.

63. 61. The fusion polypeptide of claim 60, wherein the additional amino acid sequence is a first co-localization polypeptide configured to co-localize with a second co-localization polypeptide within a subcellular compartment, cell, tissue, or organism.

64. 61. The fusion polypeptide of claim 60, wherein the additional amino acid sequence is a protein of interest and a potential drug target.

65. 60. A nucleic acid comprising a sequence encoding the fusion polypeptide of claim 59.

66. (a) a polypeptide comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO: 17, 21, or 302 and less than 100% sequence identity to SEQ ID NO: 5 and / or SEQ ID NO: 8; (b) a first peptide comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO:23 and less than 100% sequence identity to SEQ ID NO:6 and / or SEQ ID NO:9; (c) a second peptide comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO:25 and less than 100% sequence identity to SEQ ID NO:7 and / or SEQ ID NO:10, The bioluminescent conjugate produces substantially increased bioluminescence in the presence of a coelenterazine or coelenterazine analog substrate compared to the coelenterazine or coelenterazine analog substrate in the presence of the polypeptide alone, the first peptide alone, the second peptide alone, and any two of the polypeptide, the first peptide, and the second peptide.

67. 67. The bioluminescent complex of claim 66, wherein the first peptide is a first peptide tag and the second peptide is a second peptide tag, and the first and second peptide tags are each linked to a moiety independently selected from the group consisting of a molecule of interest, a peptide of interest, a protein of interest, an interacting element, a co-localization element, or a binding moiety.

68. The bioluminescent complex of claim 67, wherein the first peptide tag or the second peptide tag is linked to a drug or drug candidate, and the other peptide tag is linked to a drug target or drug target candidate, and the intensity of the bioluminescence from the bioluminescent complex correlates with the affinity of the drug or drug candidate for the drug target or drug target candidate.

69. 68. The bioluminescent complex of claim 67, wherein the first peptide tag is linked to a first interacting element, the second peptide tag is linked to a second interacting element, and the intensity of the bioluminescence from the bioluminescent complex correlates with the affinity of the first interacting element for the second interacting element under the conditions being analyzed.

70. 68. The bioluminescent complex of claim 67, wherein the first peptide tag is linked to a first co-localization element and the second peptide tag is linked to a second co-localization element, and a substantially increased bioluminescence indicates co-localization of the first co-localization element and the second co-localization element under the conditions being analyzed, but does not necessarily indicate an interaction.

71. (a)(i) a first peptide comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO:23 and less than 100% sequence identity to SEQ ID NO:6 and SEQ ID NO:9; (ii) a second peptide comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO: 25 and less than 100% sequence identity to SEQ ID NO: 7 and SEQ ID NO: 10; (iii) a polypeptide component comprising an amino acid sequence having 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) sequence identity to SEQ ID NO: 17, 21, and / or 302 and less than 100% sequence identity to SEQ ID NO: 5 and / or SEQ ID NO: 8, wherein the first peptide tag, the second peptide tag, and the polypeptide component are configured to generate a bioluminescent complex upon interaction of the first molecular entity and the second molecular entity; (iv) combining a coelenterazine or coelenterazine analog substrate; (b) detecting luminescence, wherein a higher level of luminescence compared to the level of luminescence produced by the polypeptide component and coelenterazine or a coelenterazine analog alone indicates the formation of a bioluminescent complex of the polypeptide component and the first and second peptides.

72. 72. The method of claim 71, wherein the polypeptide component and one or more of the first and second peptides are expressed intracellularly, exogenously added to a cell, and / or added to a sample.

73. 1. A method for detecting an interaction between a first molecular entity and a second molecular entity, comprising: (a) tagging the first molecular entity with a first peptide tag comprising an amino acid sequence having 40% or more sequence identity with SEQ ID NO: 23 and less than 100% sequence identity with SEQ ID NO: 6 and / or SEQ ID NO: 9; (b) tagging the second molecular entity with a second peptide tag comprising an amino acid sequence having 40% or more sequence identity with SEQ ID NO: 25 and less than 100% sequence identity with SEQ ID NO: 7 and / or SEQ ID NO: 10; (c) combining the tagged first molecular entity and the tagged second molecular entity; (d) adding a polypeptide component comprising an amino acid sequence having 40% or more sequence identity with SEQ ID NO:17, SEQ ID NO:21, and / or SEQ ID NO:302 and less than 100% sequence identity with SEQ ID NO:5 and / or SEQ ID NO:8, wherein the first peptide tag, the second peptide tag, and the polypeptide component are configured to generate a bioluminescent complex upon interaction of the first molecular entity and the second molecular entity; (e) adding a coelenterazine or coelenterazine analog substrate; (f) detecting a luminescent signal produced by the bioluminescent complex, wherein the magnitude of the luminescent signal correlates with the strength of the interaction between the first molecular entity and the second molecular entity.

74. 74. The method of claim 73, wherein the first molecular entity and / or the second molecular entity is a protein of interest or a peptide of interest, and wherein tagging comprises creating a fusion of the first molecular entity and / or the second molecular entity with the first peptide tag and / or the second peptide tag.

75. 74. The method of claim 73, wherein the first molecular entity and / or the second molecular entity is a small molecule, and wherein tagging comprises linking the first molecular entity and / or the second molecular entity directly or indirectly to the first peptide tag and / or the second peptide tag.

76. 74. The method of claim 73, wherein one of the first molecular entity and the second molecular entity is a drug or a drug candidate, and the other is a drug target or a drug target candidate, and the bioluminescent signal indicates binding of the drug or drug candidate to the other, which is a drug target or a drug target candidate.

77. 74. The method of Claim 73, wherein combining the tagged first molecular entity and the tagged second molecular entity comprises expressing one or both in a cell and / or adding one or both to a cell.

78. 1. A method for detecting an interaction in a cell between a first protein or peptide entity and a second protein or peptide entity, comprising: (a) expressing in the cell a fusion comprising the first protein or peptide entity and a first peptide tag comprising an amino acid sequence having 40% or more sequence identity with SEQ ID NO:23 and less than 100% sequence identity with SEQ ID NO:6 and SEQ ID NO:9; (b) expressing in the cell a fusion comprising the second protein or peptide entity and a second peptide tag comprising an amino acid sequence having 40% or more sequence identity to SEQ ID NO:25 and less than 100% sequence identity to SEQ ID NO:7 and / or SEQ ID NO:10; (c) expressing in the cell a polypeptide component comprising an amino acid sequence having 40% or more sequence identity to SEQ ID NO: 17, 21, and / or 302 and less than 100% sequence identity to SEQ ID NO: 5 and / or SEQ ID NO: 8, wherein the first peptide tag, the second peptide tag, and the polypeptide component are configured to generate a bioluminescent complex upon interaction of the first protein or peptide entity and the second protein or peptide entity; (d) adding a coelenterazine or coelenterazine analog substrate to said cells; (e) detecting a luminescent signal produced by the bioluminescent complex, wherein the magnitude of the luminescent signal correlates with the strength of the interaction between the first protein or peptide entity and the second protein or peptide entity.

79. 1. A method for detecting co-localization of a first molecular entity and a second molecular entity, comprising: (a) tagging the first molecular entity with a first peptide tag comprising an amino acid sequence having 40% or more sequence identity with SEQ ID NO: 23 and less than 100% sequence identity with SEQ ID NO: 6 and / or SEQ ID NO: 9; (b) tagging the second molecular entity with a second peptide tag comprising an amino acid sequence having 40% or more sequence identity with SEQ ID NO: 25 and less than 100% sequence identity with SEQ ID NO: 7 and / or SEQ ID NO: 10; (c) combining the tagged first molecular entity and the tagged second molecular entity in the same system; (d) adding a polypeptide component to the system, wherein the polypeptide component comprises an amino acid sequence having 40% or more sequence identity with SEQ ID NO: 17, 21, and / or 302 and less than 100% sequence identity with SEQ ID NO: 5 and SEQ ID NO: 8, and wherein the first peptide tag, the second peptide tag, and the polypeptide component are configured to generate a bioluminescent complex upon co-localization of the first molecular entity and the second molecular entity; (e) adding a coelenterazine or coelenterazine analog substrate to the system; (f) detecting a luminescent signal produced by the bioluminescent complex, wherein the presence of a luminescent signal above background indicates co-localization of the first molecular entity and the second molecular entity in the system, and / or the magnitude of the luminescent signal correlates with the amount of co-localization of the first molecular entity and the second molecular entity in the system.

80. 80. The method of claim 79, wherein the system comprises a cell, a tissue, an organ, a whole organism, a biochemical sample, or a non-cellular sample.

81. 80. The method of claim 79, wherein the first molecular entity and / or the second molecular entity is a protein of interest or a peptide of interest, and wherein tagging comprises creating a fusion of the first molecular entity and / or the second molecular entity with the first peptide tag and / or the second peptide tag.

82. 80. The method of claim 79, wherein the first molecular entity and / or the second molecular entity is a small molecule, and wherein tagging comprises linking the first molecular entity and / or the second molecular entity directly or indirectly to the first peptide tag and / or the second peptide tag.

83. 80. The method of claim 79, wherein combining the tagged first molecular entity and the tagged second molecular entity comprises expressing one or both in the system and / or adding one or both to the system.

84. 1. A method for detecting intracellular co-localization of a first protein or peptide entity with a second protein or peptide entity, comprising: (a) expressing in the cell a fusion comprising the first protein or peptide entity and a first peptide tag comprising an amino acid sequence having 40% or more sequence identity to SEQ ID NO:23 and less than 100% sequence identity to SEQ ID NO:6 and / or SEQ ID NO:9; (b) expressing in the cell a fusion comprising the second protein or peptide entity and a second peptide tag comprising an amino acid sequence having 40% or more sequence identity to SEQ ID NO:25 and less than 100% sequence identity to SEQ ID NO:7 and / or SEQ ID NO:10; (c) expressing in the cell a polypeptide component comprising an amino acid sequence having 40% or more sequence identity to SEQ ID NO: 17, 21, and / or 302 and less than 100% sequence identity to SEQ ID NO: 5 and / or SEQ ID NO: 8, wherein the first peptide tag, the second peptide tag, and the polypeptide component are configured to generate a bioluminescent complex upon co-localization of the first protein or peptide entity and the second protein or peptide entity; (d) adding a coelenterazine or coelenterazine analog substrate to said cells; (e) detecting a luminescent signal produced by the bioluminescent complex, wherein the presence of a luminescent signal above background indicates co-localization of the first protein or peptide entity and the second protein or peptide entity within the cell, and / or the magnitude of the luminescent signal correlates with the amount of co-localization of the first protein or peptide entity and the second protein or peptide entity in a system.

85. (a) a first binding moiety conjugated to a first peptide tag comprising an amino acid sequence having 40% or more sequence identity to SEQ ID NO:23 and less than 100% sequence identity to SEQ ID NO:6 and / or SEQ ID NO:9; (b) a second binding moiety conjugated to a second peptide tag comprising an amino acid sequence having 40% or more sequence identity with SEQ ID NO:25 and less than 100% sequence identity with SEQ ID NO:7 and / or SEQ ID NO:

10.

86. 86. The kit of claim 85, wherein the first binding moieties are independently selected from the group consisting of antibodies (polyclonal, monoclonal, and / or recombinant antibodies), antibody fragments, Protein A, the Ig-binding domain of Protein A, Protein G, the Ig-binding domain of Protein G, Protein A / G, the Ig-binding domain of Protein A / G, Protein L, the Ig-binding domain of Protein L, Protein M, the Ig-binding domain of Protein M, oligonucleotide probes, peptide nucleic acids, DARPins, aptamers, affimers, purified proteins (either the analyte itself or a protein that binds to the analyte), and analyte-binding domain(s) of a protein.

87. 87. The kit of claim 86, wherein the first and second binding moieties are primary binding moieties configured to bind to an antigen, epitope, or sequence on the same target entity.

88. 87. The kit of claim 86, wherein the first and second binding moieties are secondary binding moieties configured to bind to an antigen, epitope, or sequence on a primary binding moiety.

89. 87. The kit of claim 86, further comprising a polypeptide reagent comprising an amino acid sequence having 40% or more sequence identity with SEQ ID NO: 17, 21, and / or 302 and less than 100% sequence identity with SEQ ID NO: 5 and / or SEQ ID NO:

8.

90. 90. The kit of claim 89, further comprising coelenterazine or a coelenterazine analog.

91. 1. A method for detecting a target molecule, wherein the target molecule exhibits a first antigen, epitope, or sequence and a distinct second antigen, epitope, or sequence, the method comprising: (a) contacting a sample containing the target molecule with (i) a first primary binding moiety that recognizes the first antigen, epitope, or sequence and (ii) a second primary binding moiety that recognizes the second antigen, epitope, or sequence, and allowing the first and second primary binding moieties to bind to the first and second antigens, epitopes, or sequences; (b) contacting the sample with (i) a first secondary binding moiety conjugated to a first peptide tag and (ii) a second secondary binding moiety conjugated to a second peptide tag, wherein the first secondary binding moiety recognizes the first primary binding moiety and the second secondary binding moiety recognizes the second primary binding moiety, wherein the first or second peptide tag comprises an amino acid sequence having 40% or more sequence identity to SEQ ID NO:23 and less than 100% sequence identity to SEQ ID NO:6 and / or SEQ ID NO:9, and the other of the first or second peptide tag comprises an amino acid sequence having 40% or more sequence identity to SEQ ID NO:25 and less than 100% sequence identity to SEQ ID NO:7 and / or SEQ ID NO:10, and allowing the first and second secondary binding moieties to bind to the first and second primary binding moieties; (c) contacting the sample with a polypeptide component having 40% or more sequence identity to SEQ ID NO: 17, 21, and / or 302 and less than 100% sequence identity to SEQ ID NO: 5 and / or SEQ ID NO: 8, wherein the first peptide tag, the second peptide tag, and the polypeptide component are configured to generate a bioluminescent complex upon interaction; (d) contacting the sample with a coelenterazine or coelenterazine analog substrate; (e) detecting a luminescent signal produced by the bioluminescent complex, wherein the presence of a luminescent signal above background indicates the presence of the target molecule and / or the magnitude of the luminescent signal correlates to the amount of target molecule in the sample.

92. 92. The method of claim 91, wherein the binding moieties are independently selected from the group consisting of antibodies (polyclonal, monoclonal, and / or recombinant antibodies), antibody fragments, Protein A, the Ig-binding domain of Protein A, Protein G, the Ig-binding domain of Protein G, Protein A / G, the Ig-binding domain of Protein A / G, Protein L, the Ig-binding domain of Protein L, Protein M, the Ig-binding domain of Protein M, oligonucleotide probes, peptide nucleic acids, DARPins, aptamers, affimers, purified proteins (either the analyte itself or a protein that binds to the analyte), and analyte-binding domain(s) of a protein.

93. 92. The method of claim 91, wherein the target molecule is a protein, a nucleic acid, or a small molecule.

94. 92. The method of claim 91, wherein the sample is in vitro or in vivo.

95. 1. A method for detecting a target molecule, wherein the target molecule exhibits a first antigen, epitope, or sequence and a distinct second antigen, epitope, or sequence, the method comprising: (a) contacting the sample with (i) a first binding moiety conjugated to a first peptide tag and (ii) a second binding moiety conjugated to a second peptide tag, wherein the first secondary binding moiety recognizes the first antigen, epitope, or sequence, and the second binding moiety recognizes the second antigen, epitope, or sequence, wherein the first or second peptide tag comprises an amino acid sequence having 40% or more sequence identity with SEQ ID NO:23 and less than 100% sequence identity with SEQ ID NO:6 and / or SEQ ID NO:9, and the other of the first or second peptide tag comprises an amino acid sequence having 40% or more sequence identity with SEQ ID NO:25 and less than 100% sequence identity with SEQ ID NO:7 and / or SEQ ID NO:10, and allowing the first and second binding moieties to bind to the first and second antigens, epitopes, or sequences; (c) contacting the sample with a polypeptide component having 40% or more sequence identity to SEQ ID NO: 17, 21, and / or 302 and less than 100% sequence identity to SEQ ID NO: 5 and / or SEQ ID NO: 8, wherein the first peptide tag, the second peptide tag, and the polypeptide component are configured to generate a bioluminescent complex upon interaction; (d) contacting the sample with a coelenterazine or coelenterazine analog substrate; (e) detecting a luminescent signal produced by the bioluminescent complex, wherein the presence of a luminescent signal above background indicates the presence of the target molecule and / or the magnitude of the luminescent signal correlates to the amount of target molecule in the sample.

96. 96. The method of claim 95, wherein the binding moieties are independently selected from the group consisting of antibodies (polyclonal, monoclonal, and / or recombinant antibodies), antibody fragments, Protein A, the Ig-binding domain of Protein A, Protein G, the Ig-binding domain of Protein G, Protein A / G, the Ig-binding domain of Protein A / G, Protein L, the Ig-binding domain of Protein L, Protein M, the Ig-binding domain of Protein M, oligonucleotide probes, peptide nucleic acids, DARPins, aptamers, affimers, purified proteins (either the analyte itself or a protein that binds to the analyte), and analyte-binding domain(s) of a protein.

97. 96. The method of claim 95, wherein the target molecule is a protein, a nucleic acid, or a small molecule.

98. 96. The method of claim 95, wherein the sample is an in vitro sample, an in vivo sample, or a biochemical sample.

99. A β9 / β10-like dipeptide comprising an amino acid sequence having more than 40% but less than 100% sequence identity with SEQ ID NO: 35 and less than 100% sequence identity with SEQ ID NO: 205 and SEQ ID NO: 206, wherein when the peptide is contacted with a polypeptide complement consisting of SEQ ID NO: 17, 21, and / or 302, the bioluminescent signal generated in the presence of coelenterazine or a coelenterazine analog substrate is substantially increased compared to the bioluminescent signal generated by the peptide and the coelenterazine substrate alone.

100. 100. The β9 / β10-like dipeptide of claim 99, wherein the bioluminescent signal is substantially increased when the peptide associates with the polypeptide complement.

101. The β9 / β10-like dipeptide of claim 99, wherein the peptide exhibits enhanced one or more traits compared to the peptides of SEQ ID NO: 205 and / or SEQ ID NO: 206, the traits being selected from affinity for the polypeptide complement, expression, solubility, stability, and bioluminescent activity when combined with the polypeptide complement.

102. 100. The β9 / β10-like dipeptide of claim 99, wherein the amino acid sequence is not a naturally occurring protein or a fragment thereof.

103. The β9 / β10-like dipeptide of claim 102, wherein the amino acid sequence contains unnatural amino acids, amino acid analogs, and / or peptoid amino acids.

104. A nucleic acid comprising a sequence encoding the β9 / β10-like dipeptide of claim 99.

105. A fusion polypeptide comprising the β9 / β10-like dipeptide of claim 99 and an additional amino acid sequence.

106. 106. The fusion polypeptide of claim 105, wherein the additional amino acid sequence is selected from the group consisting of a protein of interest, an interacting element, a co-localization element, and a binding moiety.

107. 107. The fusion polypeptide of claim 106, wherein the additional amino acid sequence is a binding moiety selected from the group consisting of an antibody (polyclonal, monoclonal, and / or recombinant antibody), an antibody fragment, Protein A, the Ig-binding domain of Protein A, Protein G, the Ig-binding domain of Protein G, Protein A / G, the Ig-binding domain of Protein A / G, Protein L, the Ig-binding domain of Protein L, Protein M, the Ig-binding domain of Protein M, an oligonucleotide probe, a peptide nucleic acid, a DARPin, an aptamer, an affimer, a purified protein (either the analyte itself or a protein that binds to the analyte), and an analyte-binding domain(s) of a protein.

108. 107. The fusion polypeptide of claim 106, wherein the additional amino acid sequence is a first interacting polypeptide configured to form a complex with a second interacting polypeptide upon contact of the first interacting polypeptide and the second interacting polypeptide.

109. The fusion polypeptide of claim 106, wherein the additional amino acid sequence is a first co-localization polypeptide configured to co-localize with a second co-localization polypeptide within an intracellular compartment, cell, tissue, or organism.

110. The fusion polypeptide of claim 106, wherein the additional amino acid sequence is a protein of interest and a potential drug target.

111. A nucleic acid comprising a sequence encoding the fusion polypeptide of claim 105.

112. 1. A method of performing a competitive assay for detecting an interaction between a first molecular entity and a second molecular entity, comprising: (a)(i) a tracer comprising the first molecular entity tagged with a first peptide tag comprising an amino acid sequence having 40% or more sequence identity with SEQ ID NO: 23 and less than 100% sequence identity with SEQ ID NO: 6 and SEQ ID NO: 9; (ii) the second molecular entity tagged with a second peptide tag comprising an amino acid sequence having 40% or more sequence identity with SEQ ID NO: 25 and less than 100% sequence identity with SEQ ID NO: 7 and SEQ ID NO: 10; (iii) a coelenterazine or coelenterazine analog substrate; and (iv) a polypeptide component comprising an amino acid sequence having 40% or more sequence identity with SEQ ID NO: 17, 21, and / or 302 and less than 100% sequence identity with SEQ ID NO: 5 and / or SEQ ID NO: 8; (v) a sample suspected of containing an untagged first molecular entity, the combining, wherein the first peptide tag, the second peptide tag, and the polypeptide component form a bioluminescent complex configured to generate a bioluminescent signal in the presence of the coelenterazine or coelenterazine analog substrate; (b) detecting the bioluminescent signal produced by the bioluminescent complex; and (c) comparing the bioluminescent signal produced in the presence of the sample with a control bioluminescent signal produced in the absence of the sample, wherein a decrease in the bioluminescent signal indicates the presence or amount of an untagged first molecular entity in the sample.

113. 113. The method of claim 112, wherein the first molecular entity is a small molecule or a peptide.

114. 113. The method of claim 112, wherein the second molecular entity is a drug target or a drug target candidate.

115. (a) a polypeptide component comprising 40% or more sequence identity to a polypeptide fragment of SEQ ID NO: 788 or SEQ ID NO: 789; (b) one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides that collectively comprise 40% or more sequence identity to the complementary portion of SEQ ID NO:788 or SEQ ID NO:789, the system or kit, wherein in the presence of coelenterazine or a coelenterazine analog substrate, the bioluminescent signal generated by a bioluminescent complex assembled from the polypeptide component and one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides is substantially increased as compared to the bioluminescent signal generated by the polypeptide component or one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides and the coelenterazine substrate alone.

116. The system or kit of claim 115, wherein the polypeptide component comprises 40% or more sequence identity to SEQ ID NO: 790, and the one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or more sequence identity to SEQ ID NO:

794.

117. The system or kit of claim 115, wherein the polypeptide component comprises 40% or more sequence identity to SEQ ID NO: 791 and the one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or more sequence identity to SEQ ID NO:

795.

118. The system or kit of claim 115, wherein the polypeptide component comprises 40% or more sequence identity to SEQ ID NO:792 and the one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or more sequence identity to SEQ ID NO:

796.

119. The system or kit of claim 115, wherein the polypeptide component comprises 40% or more sequence identity to SEQ ID NO: 793 and the one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or more sequence identity to SEQ ID NO:

797.

120. The system or kit of claim 115, wherein the polypeptide component comprises 40% or more sequence identity to SEQ ID NO: 790 and the one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or more sequence identity to SEQ ID NO:

798.

121. The system or kit of claim 115, wherein the polypeptide component comprises 40% or more sequence identity to SEQ ID NO:791 and the one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or more sequence identity to SEQ ID NO:

799.

122. The system or kit of claim 115, wherein the polypeptide component comprises 40% or more sequence identity to SEQ ID NO:792 and the one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or more sequence identity to SEQ ID NO:

800.

123. The system or kit of claim 115, wherein the polypeptide component comprises 40% or more sequence identity to SEQ ID NO:793 and the one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or more sequence identity to SEQ ID NO:

801.

124. 116. The system or kit of claim 115, wherein the bioluminescent signal is substantially increased when the polypeptide component associates with the one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides.

125. 116. The system or kit of claim 115, wherein the polypeptide component and / or one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides comprise an amino acid sequence that is not a naturally occurring sequence or a fragment thereof.

126. 126. The system or kit of claim 125, wherein the polypeptide component and / or one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides comprise unnatural amino acids, amino acid analogs, and / or peptoid amino acids.

127. 116. The system or kit of claim 115, wherein the polypeptide component and / or one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides are present as fusions with one or more additional amino acid sequences.

128. 128. The system or kit of claim 127, wherein the additional amino acid sequence is selected from the group consisting of a protein of interest, an interacting element, a co-localization element, and a binding moiety.

129. 128. The system or kit of claim 127, wherein the additional amino acid sequence is a binding moiety selected from the group consisting of an antibody (polyclonal, monoclonal, and / or recombinant antibody), an antibody fragment, Protein A, the Ig-binding domain of Protein A, Protein G, the Ig-binding domain of Protein G, Protein A / G, the Ig-binding domain of Protein A / G, Protein L, the Ig-binding domain of Protein L, Protein M, the Ig-binding domain of Protein M, an oligonucleotide probe, a peptide nucleic acid, a DARPin, an aptamer, an affimer, a purified protein (either the analyte itself or a protein that binds to the analyte), and an analyte-binding domain(s) of a protein.

130. 128. The system or kit of claim 127, wherein the additional amino acid sequence is a first interacting polypeptide configured to form a complex with a second interacting polypeptide upon contact of the first interacting polypeptide and the second interacting polypeptide.

131. The system or kit of claim 127, wherein the additional amino acid sequence is a first co-localization polypeptide configured to co-localize with a second co-localization polypeptide within an intracellular compartment, cell, tissue, or organism.

132. The system or kit of claim 127, wherein the additional amino acid sequence is a protein of interest and a potential drug target.

133. 133. A bioluminescent complex comprising a polypeptide component of the system or kit of any one of claims 115 to 132 and one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides.

134. A system or kit comprising two or more peptide, dipeptide, tripeptide, and / or polypeptide components collectively having 40% or more sequence identity to SEQ ID NO:788 or SEQ ID NO:789, wherein in the presence of a coelenterazine or coelenterazine analog substrate, the bioluminescent signal generated by the bioluminescent complex is substantially increased compared to the bioluminescent signal generated by the polypeptide or one or more complementary peptides and the coelenterazine substrate alone.

135. 135. The system or kit of claim 134, comprising a polypeptide component having 40% or greater sequence identity to SEQ ID NO: 790, and one or more complementary peptides, dipeptides, and / or tripeptides collectively having 40% or greater sequence identity to SEQ ID NO:

794.

136. The system or kit of claim 134, wherein the polypeptide comprises 40% or more sequence identity to SEQ ID NO: 791 and the one or more complementary peptides collectively comprise 40% or more sequence identity to SEQ ID NO:

795.

137. The system or kit of claim 134, wherein the polypeptide comprises 40% or more sequence identity to SEQ ID NO: 792 and the one or more complementary peptides collectively comprise 40% or more sequence identity to SEQ ID NO:

796.

138. The system or kit of claim 134, wherein the polypeptide comprises 40% or more sequence identity to SEQ ID NO: 793 and the one or more complementary peptides collectively comprise 40% or more sequence identity to SEQ ID NO:

797.

139. The system or kit of claim 134, wherein the polypeptide comprises 40% or more sequence identity to SEQ ID NO: 790 and the one or more complementary peptides collectively comprise 40% or more sequence identity to SEQ ID NO:

798.

140. The system or kit of claim 134, wherein the polypeptide comprises 40% or more sequence identity to SEQ ID NO: 791 and the one or more complementary peptides collectively comprise 40% or more sequence identity to SEQ ID NO:

799.

141. The system or kit of claim 134, wherein the polypeptide comprises 40% or more sequence identity to SEQ ID NO: 792 and the one or more complementary peptides collectively comprise 40% or more sequence identity to SEQ ID NO:

800.

142. The system or kit of claim 134, wherein the polypeptide comprises 40% or more sequence identity to SEQ ID NO: 793 and the one or more complementary peptides collectively comprise 40% or more sequence identity to SEQ ID NO:

801.

143. 135. The system or kit of claim 134, wherein the bioluminescent signal is substantially increased when the polypeptide associates with the one or more complementary peptides.

144. 135. The system or kit of claim 134, wherein the polypeptide and / or one or more complementary peptides comprise an amino acid sequence that is not a naturally occurring sequence or a fragment thereof.

145. 145. The system or kit of claim 144, wherein the polypeptide and / or one or more complementary peptides comprise unnatural amino acids, amino acid analogs, and / or peptoid amino acids.

146. 135. The system or kit of claim 134, wherein the polypeptide and / or one or more complementary peptides are present as a fusion with one or more additional amino acid sequences.

147. 147. The system or kit of claim 146, wherein the additional amino acid sequence is selected from the group consisting of a protein of interest, an interacting element, a co-localization element, and a binding moiety.

148. 147. The system or kit of claim 146, wherein the additional amino acid sequence is a binding moiety selected from the group consisting of an antibody (polyclonal, monoclonal, and / or recombinant antibody), an antibody fragment, Protein A, the Ig-binding domain of Protein A, Protein G, the Ig-binding domain of Protein G, Protein A / G, the Ig-binding domain of Protein A / G, Protein L, the Ig-binding domain of Protein L, Protein M, the Ig-binding domain of Protein M, an oligonucleotide probe, a peptide nucleic acid, a DARPin, an aptamer, an affimer, a purified protein (either the analyte itself or a protein that binds to the analyte), and an analyte-binding domain(s) of a protein.

149. 147. The system or kit of claim 146, wherein the additional amino acid sequence is a first interacting polypeptide configured to form a complex with a second interacting polypeptide upon contact of the first interacting polypeptide and the second interacting polypeptide.

150. The system or kit of claim 146, wherein the additional amino acid sequence is a first co-localization polypeptide configured to co-localize with a second co-localization polypeptide within an intracellular compartment, cell, tissue, or organism.

151. The system or kit of claim 146, wherein the additional amino acid sequence is a protein of interest and a potential drug target.

152. (a) (i) a polypeptide component comprising 40% or more sequence identity to a polypeptide fragment of SEQ ID NO: 788 or SEQ ID NO: 789; (ii) one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides that collectively comprise 40% or greater sequence identity to the complementary portion of SEQ ID NO:788 or SEQ ID NO:789; and (iii) combining a coelenterazine or coelenterazine analog substrate; (b) detecting luminescence, wherein a higher level of luminescence compared to the level of luminescence produced by the polypeptide component and coelenterazine or a coelenterazine analog alone indicates the formation of a bioluminescent complex of the polypeptide component and the one or more complementary peptides.

153. 153. The method of claim 152, wherein one or more of the polypeptide components and the first and second peptides are expressed within a cell, exogenously added to a cell, and / or added to a sample.

154. (i) the polypeptide component comprises 40% or greater sequence identity to SEQ ID NO:790, and the one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or greater sequence identity to SEQ ID NO:794; or (ii) the polypeptide component comprises 40% or greater sequence identity to SEQ ID NO:791, and the one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or greater sequence identity to SEQ ID NO:795; or (iii) the polypeptide component comprises 40% or greater sequence identity to SEQ ID NO:792, and the one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or greater sequence identity to SEQ ID NO:796; or (iv) the polypeptide component comprises 40% or greater sequence identity to SEQ ID NO:793, and the one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or greater sequence identity to SEQ ID NO:797; or (v) the polypeptide component comprises 40% or greater sequence identity to SEQ ID NO:790, and the one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or greater sequence identity to SEQ ID NO:798; or (vi) the polypeptide component comprises 40% or greater sequence identity to SEQ ID NO:791, and the one or more complementary peptides, dipeptides, tripeptides and / or polypeptides collectively comprise 40% or greater sequence identity to SEQ ID NO:799; or (vii) the polypeptide component comprises 40% or greater sequence identity to SEQ ID NO:792, and the one or more complementary peptides, dipeptides, tripeptides and / or polypeptides collectively comprise 40% or greater sequence identity to SEQ ID NO:800; or (viii) the polypeptide component comprises 40% or greater sequence identity to SEQ ID NO: 793, and the one or more complementary peptides, dipeptides, tripeptides and / or polypeptides collectively comprise 40% or greater sequence identity to SEQ ID NO:

801.

155. (a) (i) two or more peptide, dipeptide, tripeptide, and / or polypeptide components that collectively comprise 40% or more sequence identity to the entire length of SEQ ID NO:788 or SEQ ID NO:789; (ii) combining a coelenterazine or coelenterazine analog substrate; (b) detecting luminescence, wherein a higher level of luminescence compared to the level of luminescence produced by the peptide, dipeptide, tripeptide, and / or polypeptide components and coelenterazine or a coelenterazine analog indicates the formation of a bioluminescent complex of the peptide and polypeptide components.

156. 156. The method of claim 155, wherein one or more of the polypeptide components and the first and second peptides are expressed intracellularly, exogenously added to a cell, and / or added to a sample.

157. (i) the polypeptide component comprises 40% or greater sequence identity to SEQ ID NO:790, and the one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or greater sequence identity to SEQ ID NO:794; or (ii) the polypeptide component comprises 40% or greater sequence identity to SEQ ID NO:791, and the one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or greater sequence identity to SEQ ID NO:795; or (iii) the polypeptide component comprises 40% or greater sequence identity to SEQ ID NO:792, and the one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or greater sequence identity to SEQ ID NO:796; or (iv) the polypeptide component comprises 40% or greater sequence identity to SEQ ID NO:793, and the one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or greater sequence identity to SEQ ID NO:797; or (v) the polypeptide component comprises 40% or greater sequence identity to SEQ ID NO:790, and the one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively comprise 40% or greater sequence identity to SEQ ID NO:798; or (vi) the polypeptide component comprises 40% or greater sequence identity to SEQ ID NO:791, and the one or more complementary peptides, dipeptides, tripeptides and / or polypeptides collectively comprise 40% or greater sequence identity to SEQ ID NO:799; or (vii) the polypeptide component comprises 40% or greater sequence identity to SEQ ID NO:792, and the one or more complementary peptides, dipeptides, tripeptides and / or polypeptides collectively comprise 40% or greater sequence identity to SEQ ID NO:800; or (viii) the polypeptide component comprises 40% or greater sequence identity to SEQ ID NO: 793, and the one or more complementary peptides, dipeptides, tripeptides and / or polypeptides collectively comprise 40% or greater sequence identity to SEQ ID NO:

801.

158. 1. A method for detecting an interaction between a first molecular entity and a second molecular entity, comprising: (a) tagging the first molecular entity with a first peptide, dipeptide, or tripeptide tag; (b) tagging the second molecular entity with a second peptide, dipeptide, or tripeptide tag; (c) combining the tagged first molecular entity and the tagged second molecular entity and / or allowing the tagged first molecular entity and the tagged second molecular entity to contact each other; (d) adding peptide, dipeptide, tripeptide, and / or polypeptide components, wherein the first peptide, dipeptide, or tripeptide tag, the second peptide, dipeptide, or tripeptide tag, and the peptide, dipeptide, tripeptide, and / or polypeptide components collectively comprise an amino acid sequence having 40% or greater sequence identity to the entirety of SEQ ID NO: 788 or 789, and are capable of assembling to form a bioluminescent complex; (e) adding a coelenterazine or coelenterazine analog substrate; (f) detecting a luminescent signal produced by the bioluminescent complex, wherein the magnitude of the luminescent signal correlates with the strength of the interaction between the first molecular entity and the second molecular entity.

159. 159. The method of claim 158, wherein the first molecular entity and / or the second molecular entity is a protein of interest or a peptide of interest, and wherein tagging comprises creating a fusion of the first molecular entity and / or the second molecular entity with the first tag and / or the second tag.

160. 159. The method of claim 158, wherein the first molecular entity and / or the second molecular entity is a small molecule, and wherein tagging comprises linking the first molecular entity and / or the second molecular entity directly or indirectly to the first tag and / or the second tag.

161. The method of claim 158, wherein one of the first molecular entity and the second molecular entity is a drug or a candidate drug, and the other is a drug target or a candidate drug target, and the bioluminescent signal indicates binding of the drug or candidate drug to the other, which is the drug target or candidate drug target.

162. 159. The method of Claim 158, wherein combining the tagged first molecular entity and the tagged second molecular entity comprises expressing one or both in a cell and / or adding one or both to a cell.

163. 1. A method for detecting an interaction in a cell between a first protein or peptide entity and a second protein or peptide entity, comprising: (a) expressing in the cell a fusion comprising the first protein or peptide entity and a first peptide, dipeptide, or tripeptide tag comprising an amino acid sequence having 40% or more sequence identity to a first portion of SEQ ID NO: 788 or 789; (b) expressing in the cell a fusion comprising the second protein or peptide entity and a second peptide, dipeptide, or tripeptide tag comprising an amino acid sequence having 40% or more sequence identity to a second portion of SEQ ID NO: 788 or 789; (c) expressing in the cell a peptide, dipeptide, tripeptide, and / or polypeptide component comprising an amino acid sequence having 40% or greater sequence identity to a third portion of SEQ ID NO: 788 or 789, wherein the first tag, the second tag, and the component collectively comprise 40% or greater sequence identity to the entirety of SEQ ID NO: 788 or 789, and are configured to generate a bioluminescent complex upon interaction of the first protein or peptide entity and the second protein or peptide entity; (d) adding a coelenterazine or coelenterazine analog substrate to said cells; (e) detecting a luminescent signal produced by the bioluminescent complex, wherein the magnitude of the luminescent signal correlates with the strength of the interaction between the first protein or peptide entity and the second protein or peptide entity.

164. 1. A method for detecting co-localization of a first molecular entity and a second molecular entity, comprising: (a) tagging the first molecular entity with a first peptide, dipeptide, or tripeptide tag comprising an amino acid sequence having 40% or more sequence identity to a first portion of SEQ ID NO: 788 or 789; (b) tagging the second molecular entity with a second peptide, dipeptide, or tripeptide tag comprising an amino acid sequence having 40% or more sequence identity to a second portion of SEQ ID NO: 788 or 789; (c) combining the tagged first molecular entity and the tagged second molecular entity in the same system; (d) adding a peptide, dipeptide, tripeptide, and / or polypeptide component to the system, wherein the component has 40% or greater sequence identity to a third portion of SEQ ID NO: 788 or 789, and the first tag, the second tag, and the component collectively comprise 40% or greater sequence identity to the entirety of SEQ ID NO: 788 or 789, and wherein the first peptide tag, the second peptide tag, and the component are configured to generate a bioluminescent complex upon co-localization of the first molecular entity and the second molecular entity; (e) adding a coelenterazine or coelenterazine analog substrate to the system; (f) detecting a luminescent signal produced by the bioluminescent complex, wherein the presence of a luminescent signal above background indicates co-localization of the first molecular entity and the second molecular entity in the system, and / or the magnitude of the luminescent signal correlates with the amount of co-localization of the first molecular entity and the second molecular entity in the system.

165. 165. The method of claim 164, wherein the system comprises a cell, a tissue, an organ, a whole organism, a biochemical sample, or a non-cellular sample.

166. 165. The method of claim 164, wherein the first molecular entity and / or the second molecular entity is a protein of interest or a peptide of interest, and wherein tagging comprises creating a fusion of the first molecular entity and / or the second molecular entity with the first tag and / or the peptide tag.

167. 165. The method of claim 164, wherein the first molecular entity and / or the second molecular entity is a small molecule, and wherein tagging comprises linking the first molecular entity and / or the second molecular entity directly or indirectly to the first tag and / or the second tag.

168. 165. The method of Claim 164, wherein combining the tagged first molecular entity and the tagged second molecular entity comprises expressing one or both in the system and / or adding one or both to the system.

169. 1. A method for detecting intracellular co-localization of a first protein or peptide entity with a second protein or peptide entity, comprising: (a) expressing in the cell a fusion comprising the first protein or peptide entity and a first peptide, dipeptide, or tripeptide tag comprising an amino acid sequence having 40% or more sequence identity to a first portion of SEQ ID NO: 788 or 789; (b) expressing in the cell a fusion comprising the second protein or peptide entity and a second peptide, dipeptide, or tripeptide tag comprising an amino acid sequence having 40% or more sequence identity to a second portion of SEQ ID NO: 788 or 789; (c) expressing in the cell one or more peptide, dipeptide, tripeptide, or polypeptide components having 40% or greater sequence identity to a third portion of SEQ ID NO: 788 or 789, wherein the first tag, the second tag, and the components collectively comprise 40% or greater sequence identity to the entirety of SEQ ID NO: 788 or 789, and wherein the first tag, the second tag, and the components are configured to generate a bioluminescent complex upon co-localization of the first protein or peptide entity and the second protein or peptide entity; (d) adding a coelenterazine or coelenterazine analog substrate to said cells; (e) detecting a luminescent signal produced by the bioluminescent complex, wherein the presence of a luminescent signal above background indicates co-localization of the first protein or peptide entity and the second protein or peptide entity within the cell, and / or the magnitude of the luminescent signal correlates with the amount of co-localization of the first protein or peptide entity and the second protein or peptide entity in a system.

170. 1. A method for detecting a target molecule, wherein the target molecule exhibits a first antigen, epitope, or sequence and a distinct second antigen, epitope, or sequence, the method comprising: (a) contacting a sample containing the target molecule with (i) a first primary binding moiety that recognizes the first antigen, epitope, or sequence and (ii) a second primary binding moiety that recognizes the second antigen, epitope, or sequence, and allowing the first and second primary binding moieties to bind to the first and second antigens, epitopes, or sequences; (b) contacting the sample with (i) a first secondary binding moiety conjugated to a first tag and (ii) a second secondary binding moiety conjugated to a second tag, wherein the first secondary binding moiety recognizes the first primary binding moiety, the second secondary binding moiety recognizes the second primary binding moiety, and the first or second tag comprises an amino acid sequence having 40% or greater sequence identity to the first and second moieties of SEQ ID NO: 788 or 789; (c) allowing the first and second secondary binding moieties to bind to the first and second primary binding moieties; (d) contacting the sample with one or more peptide, dipeptide, tripeptide, and / or polypeptide components having 40% or greater sequence identity to a third portion of SEQ ID NO: 788 or 789, wherein the first tag, the second tag, and the components collectively comprise 40% or greater sequence identity to the entirety of SEQ ID NO: 788 or 789, and wherein the first tag, the second tag, and the components are configured to generate a bioluminescent complex upon interaction; (d) contacting the sample with a coelenterazine or coelenterazine analog substrate; (e) detecting a luminescent signal produced by the bioluminescent complex, wherein the presence of a luminescent signal above background indicates the presence of the target molecule and / or the magnitude of the luminescent signal correlates to the amount of target molecule in the sample.

171. 171. The method of claim 170, wherein the binding moieties are independently selected from the group consisting of antibodies (polyclonal, monoclonal, and / or recombinant antibodies), antibody fragments, Protein A, the Ig-binding domain of Protein A, Protein G, the Ig-binding domain of Protein G, Protein A / G, the Ig-binding domain of Protein A / G, Protein L, the Ig-binding domain of Protein L, Protein M, the Ig-binding domain of Protein M, oligonucleotide probes, peptide nucleic acids, DARPins, aptamers, affimers, purified proteins (either the analyte itself or a protein that binds to the analyte), and analyte-binding domain(s) of a protein.

172. 171. The method of claim 170, wherein the target molecule is a protein, a nucleic acid, or a small molecule.

173. 171. The method of claim 170, wherein the sample is in vitro or in vivo.

174. 1. A method for detecting a target molecule, wherein the target molecule exhibits a first antigen, epitope, or sequence and a distinct second antigen, epitope, or sequence, the method comprising: (a) contacting the sample with (i) a first binding moiety conjugated to a first tag and (ii) a second binding moiety conjugated to a second tag, wherein the first secondary binding moiety recognizes the first antigen, epitope, or sequence, the second binding moiety recognizes the second antigen, epitope, or sequence, the first tag comprises an amino acid sequence having 40% or more sequence identity to a first portion of SEQ ID NO: 788 or 789, and the second tag comprises an amino acid sequence having a first portion of SEQ ID NO: 788 or 789; (b) enabling the first and second binding moieties to bind to the first and second antigens, epitopes, or sequences; (c) contacting the sample with a peptide, dipeptide, tripeptide, or polypeptide entity having 40% or greater sequence identity to a third portion of SEQ ID NO: 788 or 789, wherein the first tag, the second tag, and the entity collectively comprise 40% or greater sequence identity to the entirety of SEQ ID NO: 788 or 789, and wherein the first tag, the second tag, and the entity are configured to generate a bioluminescent complex upon interaction; (d) contacting the sample with a coelenterazine or coelenterazine analog substrate; (e) detecting a luminescent signal produced by the bioluminescent complex, wherein the presence of a luminescent signal above background indicates the presence of the target molecule and / or the magnitude of the luminescent signal correlates to the amount of target molecule in the sample.

175. 175. The method of claim 174, wherein the binding moieties are independently selected from the group consisting of antibodies (polyclonal, monoclonal, and / or recombinant antibodies), antibody fragments, Protein A, the Ig-binding domain of Protein A, Protein G, the Ig-binding domain of Protein G, Protein A / G, the Ig-binding domain of Protein A / G, Protein L, the Ig-binding domain of Protein L, Protein M, the Ig-binding domain of Protein M, oligonucleotide probes, peptide nucleic acids, DARPins, aptamers, affimers, purified proteins (either the analyte itself or a protein that binds to the analyte), and analyte-binding domain(s) of a protein.

176. 175. The method of claim 174, wherein the target molecule is a protein, nucleic acid, or small molecule.

177. 175. The method of claim 174, wherein the sample is an in vitro sample, an in vivo sample, or a biochemical sample.

178. A composition comprising a polypeptide having an amino acid sequence that has more than 40% but less than 100% sequence identity to SEQ ID NO: 788 or 789 and less than 100% sequence identity to SEQ ID NO: 1 or 3, wherein the polypeptide exhibits luminescence in the presence of coelenterazine or a coelenterazine analog substrate.

179. 179. The composition of claim 178, wherein the peptide comprises an amino acid sequence that has greater than 40% sequence identity to one or more of SEQ ID NOs: 780, 782, 784, 786, 802, 804, 806, 808, 813, 815, or 829.

180. A nucleic acid comprising a sequence encoding the polypeptide of claim 178.

181. A fusion polypeptide comprising the polypeptide of claim 178 and an additional amino acid sequence.

182. A nucleic acid comprising a sequence encoding the fusion polypeptide of claim 181.

183. (a) contacting the composition of claim 178 with a coelenterazine or coelenterazine analog substrate; (b) detecting the emitted light; A method comprising:

184. A polypeptide comprising an amino acid sequence having greater than 40% sequence identity to SEQ ID NO: 15 linked to the N-terminus of an amino acid sequence having greater than 40% sequence identity to one of SEQ ID NOs: 17, 21, or 302, wherein when the polypeptide is contacted with a peptide or polypeptide having the amino acid sequence of SEQ ID NO: 23, the bioluminescent signal generated in the presence of coelenterazine or a coelenterazine analog substrate is substantially increased compared to the bioluminescent signal generated by the polypeptide and the coelenterazine substrate alone.

185. A nucleic acid comprising a sequence encoding the polypeptide of claim 184.

186. A fusion polypeptide comprising the polypeptide of claim 184 and an additional amino acid sequence.

187. A nucleic acid comprising a sequence encoding the fusion polypeptide of claim 181.

188. (a) contacting the polypeptide of claim 184 with a coelenterazine or coelenterazine analog substrate and a peptide or polypeptide comprising an amino acid sequence having 40% or greater sequence identity to SEQ ID NO: 23; (b) detecting the emitted light; A method comprising:

189. (a) a sensor polypeptide comprising a first amino acid sequence having at least 40% sequence identity to SEQ ID NO: 11 linked to an amino acid sequence that localizes to a specific cellular location; (b) a protein of interest linked to a peptide tag comprising an amino acid sequence having at least 40% sequence identity to SEQ ID NO: 23; Including, A system in which a bioluminescent complex is formed between the polypeptide and the peptide tag upon co-localization within the specific cellular location.

190. 190. The system of claim 189, wherein the specific cellular location is selected from the plasma membrane, the nucleus, the mitochondria, and the endoplasmic reticulum.

191. (a) expressing the system of claim 189 in a cell; (b) contacting the cell with a coelenterazine or coelenterazine analog substrate; (c) detecting the emitted light; Including, An increase in luminescence indicates the formation of said bioluminescent complex and co-localization of said polypeptide and peptide tag.

192. 192. The method of claim 191, further comprising the step of inducing translocation of a protein of interest linked to a peptide tag to said specific cellular location.

Images