Multimolecular luciferase peptide and polypeptide

Abstract

Provided herein are compositions and methods for the assembly of trimolecular or multimolecular bioluminescent complexes. In certain embodiments, bioluminescent complexes are formed upon the interaction of three or more peptide and / or polypeptide components.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 941,255, filed on November 27, 2019, the entire disclosure of which is hereby incorporated by reference in its entirety.

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

Background Art

[0003] Biological processes and analyte detection rely on the co - localization and interaction between molecules, macromolecules, and molecular complexes. To understand such processes and to develop techniques and compounds for manipulating them for research, clinical, and other practical applications, it is necessary to have tools available for detecting and monitoring these co - localizations / interactions. In particular, high sensitivity is required for studying these interactions under physiological conditions (e.g., at normal expression levels for monitoring protein - protein interactions) or within complex sample matrices (e.g., blood samples, environmental samples).

Summary of the Invention

[0004] Provided herein are bioluminescent polypeptides, compositions, and methods for the assembly of tripartite or multipartite bioluminescent complexes. In certain embodiments, the bioluminescent complex is formed when three or more peptides and / or polypeptide components interact.

[0005] The experiments conducted during the development of the embodiments of this specification demonstrate the assembly of a bioluminescent complex that can generate luminescence in the presence of a suitable substrate (e.g., coelenterazine or a coelenterazine analog substrate) from complementary polypeptide(s) and peptide(s) that collectively span the length of the luciferase-based sequence (or more than 75% of the length, more than 80% of the length, more than 85% of the length, more than 90% of the length, more than 95% of the length, or more), or collectively include at least 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%, or more) sequence identity to the luciferase-based sequence. In some embodiments, the "complementary" polypeptide(s) and peptide(s) are separate molecules, each corresponding to a portion of the luciferase-based sequence. Due to structural complementarity, they assemble together to form a bioluminescent complex.

[0006] Additional experiments were conducted during the development of the embodiments herein for developing monomeric bioluminescent polypeptides with improved properties (e.g., stability, luminescence, etc.).

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

[0008] In some embodiments, the complementary polypeptide(s) and peptide(s) each comprise at least 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%, or more) sequence identity to the corresponding part of the luciferase-based sequence and, together, are variants of a part of the luciferase-based sequence that form a bioluminescent complex. In some embodiments, the complementary polypeptide(s) and peptide(s) collectively comprise at least 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%, or more) sequence identity to the entire luciferase-based sequence and, together, are variants of a part of the luciferase-based sequence that form a bioluminescent complex. In some embodiments, the fragment collectively comprises 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., a length greater than 75%, a length greater than 80%, a length greater than 85%, a length greater than 90%, a length greater than 95%, or more) of the full length of the luciferase-based sequence.

[0009] Examples of luciferase-based arrays 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 array (e.g., SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 788, and SEQ ID NO: 789) or variants thereof (e.g., having 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) collectively covering the remainder of the luciferase-based array. For example, if the luciferase-based array is 170 amino acid residues in length, exemplary polypeptide components can be, for example, 102, 124, 133, or 148 amino acids in length, and one, two, three, four, five or more complementary peptides can correspond to the remaining 68, 46, 37, or 22 amino acids. In some embodiments, each polypeptide component individually includes 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 array.

[0010] In some embodiments, a system or kit comprising: (a) a polypeptide component having at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to the polypeptide fragment of SEQ ID NO: 788 or SEQ ID NO: 789; and (b) one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively having at least 40% (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, wherein the bioluminescence signal generated by the bioluminescent complex assembled from the polypeptide component and the one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides in the presence of a coelenterazine or coelenterazine analog substrate is substantially increased compared to the bioluminescence signal generated by only the polypeptide component or the one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides and the coelenterazine substrate. In some embodiments, the polypeptide component has at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 790, and the one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively have at least 40% (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 component comprises at least 40% (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 at least 40% (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 component comprises at least 40% (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 at least 40% (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 component comprises at least 40% (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 at least 40% (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 component comprises at least 40% (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 at least 40% (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 component comprises at least 40% (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 at least 40% (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 component comprises at least 40% (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 at least 40% (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 component comprises at least 40% (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 at least 40% (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 bioluminescence signal substantially increases when the polypeptide component associates with one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides. In some embodiments, the polypeptide component 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 component and / or one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides include non-natural amino acids, amino acid analogs, and / or peptidomimetic amino acids. In some embodiments, the polypeptide component and / or one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides exist 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 interaction element, a co-localization element, and a binding moiety. In some embodiments, the additional amino acid sequences are 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, which is a binding moiety selected from the group consisting thereof. In some embodiments, the additional amino acid sequence is a first interaction polypeptide that is configured to form a complex with a second interaction polypeptide upon contact of the first and second interaction polypeptides. In some embodiments, the additional amino acid sequence is a first co-localization polypeptide that is configured to co-localize with a second co-localization polypeptide within an intracellular compartment, cell, tissue, or organism. In some embodiments, the additional amino acid sequence is a protein of interest and is a drug target candidate. In some embodiments, provided herein is a bioluminescent complex comprising a polypeptide component and one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides of the system or kit described herein.

[0011] In some embodiments, provided herein is a system or kit comprising two or more peptides, dipeptides, tripeptides, and / or polypeptide components that collectively have at least 40% (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 a coelenterazine or coelenterazine analog substrate, the bioluminescence signal generated by the bioluminescent complex is substantially increased compared to the bioluminescence signal generated by the polypeptide or only one or more complementary peptides and coelenterazine substrate. In some embodiments, the system or kit comprises a polypeptide component having at least 40% (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 at least 40% (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 at least 40% (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 at least 40% (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 at least 40% (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 at least 40% (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 at least 40% (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 at least 40% (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 at least 40% (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 at least 40% (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 at least 40% (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 at least 40% (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 at least 40% (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 at least 40% (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 at least 40% (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 at least 40% (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 bioluminescence signal substantially increases when the polypeptide associates with the one or more complementary peptides. In some embodiments, the polypeptide and / or the one or more complementary peptides comprise amino acid sequences that are not naturally occurring sequences or fragments thereof. In some embodiments, the polypeptide and / or the one or more complementary peptides comprise non-natural amino acids, amino acid analogs, and / or peptidomimetic amino acids. In some embodiments, the polypeptide and / or the one or more complementary peptides exist 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 interaction 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 the first interaction polypeptide configured to form a complex with the second interaction polypeptide upon contact between the first interaction polypeptide and the second interaction polypeptide.In some embodiments, the additional amino acid sequence is a first co-localized polypeptide configured to co-localize with a second co-localized polypeptide within an intracellular compartment, cell, tissue, or organism. In some embodiments, the additional amino acid sequence is a protein of interest and a drug target candidate. In some embodiments, provided herein are bioluminescent complexes comprising two or more peptide, dipeptide, tripeptide, and / or polypeptide components of the systems or kits described herein.

[0012] In some embodiments, (a) combining (i) a polypeptide component having at least 40% (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) one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively having at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to a complementary portion of SEQ ID NO: 788 or SEQ ID NO: 789, and (iii) coelenterazine or a coelenterazine analog substrate; and (b) detecting luminescence, wherein the detected luminescence indicates the formation of a bioluminescent complex of the polypeptide component and the one or more complementary peptides at a higher level of luminescence compared to the level of luminescence generated by the polypeptide component and coelenterazine or the coelenterazine analog alone. In some embodiments, one or more of the polypeptide component and the first and second peptides are expressed intracellularly, added exogenously to cells, and / or added to a sample. In some embodiments, (i) the polypeptide component has at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 790 and the one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively have at least 40% (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 component has at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 791 and the one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively have at least 40% (e.g., 40%, 45%, 50%,collectively include an array identity of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more, or (iii) the polypeptide component includes an array identity of 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) with respect to SEQ ID NO: 792, and one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively include an array identity of 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) with respect to SEQ ID NO: 796, or (iv) the polypeptide component includes an array identity of 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) with respect to SEQ ID NO: 793, and one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively include an array identity of 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) with respect to SEQ ID NO: 797, or (v) the polypeptide component includes an array identity of 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) with respect to SEQ ID NO: 790, and one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively include an array identity of 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) with respect to SEQ ID NO: 798, or (vi) the polypeptide component includes an array identity of 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,collectively include an array identity of 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) with respect to SEQ ID NO: 792, or one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively include an array identity of 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) with respect to SEQ ID NO: 800, or (viii) the polypeptide component includes an array identity of 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) with respect to SEQ ID NO: 793, and one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively include an array identity of 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) with respect to SEQ ID NO: 801.,

[0013] In some embodiments, (a) (i) combining two or more peptide, dipeptide, tripeptide, and / or polypeptide components that collectively have at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to the full length of SEQ ID NO: 788 or SEQ ID NO: 789, and (ii) a substrate of selenotetrazine or a selenotetrazine analog, and (b) detecting luminescence, wherein a higher level of luminescence compared to the level of luminescence generated by the peptide, dipeptide, tripeptide, and / or polypeptide components and selenotetrazine or a selenotetrazine analog indicates the formation of a bioluminescent complex of the peptide and polypeptide components, is provided herein. In some embodiments, one or more of the polypeptide components and the first and second peptides may be expressed intracellularly, added exogenously to cells, and / or added to a sample. In some embodiments, (i) the polypeptide component has at least 40% (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 have at least 40% (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 component has at least 40% (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 have at least 40% (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 component has at least 40% (e.g., 40%,including sequence identity of 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more, and one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively include sequence identity of 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) to SEQ ID NO: 796, or (iv) the polypeptide component includes sequence identity of 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) to SEQ ID NO: 793, and one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively include sequence identity of 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) to SEQ ID NO: 797, or (v) the polypeptide component includes sequence identity of 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) to SEQ ID NO: 790, and one or more complementary peptides, dipeptides, tripeptides, and / or polypeptides collectively include sequence identity of 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) to SEQ ID NO: 798, or (vi) the polypeptide component includes sequence identity of 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) to SEQ ID NO: 791, and one or more complementary peptides, dipeptides, tripeptides and / or polypeptides collectively include sequence identity of 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) to SEQ ID NO: 799, or (vii) the polypeptide component includes sequence identity of 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,or more) of sequence identity, 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) of sequence identity to SEQ ID NO: 800, or (viii) the polypeptide component comprises 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) of 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) of 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, 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 enabling the tagged first molecular entity and the tagged second molecular entity to contact each other; (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 peptide, dipeptide, tripeptide, and / or polypeptide components collectively comprise amino acid sequences having at least 40% (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 and can collectively form a bioluminescent complex, said adding; (e) adding coelenterazine or a coelenterazine analog substrate; and (f) detecting a luminescence signal generated by the bioluminescent complex, wherein the magnitude of the luminescence signal correlates with the strength of the interaction between the first molecular entity and the second molecular entity, said detecting, is provided herein. 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 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 directly or indirectly linking the first molecular entity and / or the second molecular entity to a first tag and / or a second tag.In some embodiments, one of the first molecular entity and the second molecular entity is a drug or drug candidate, the other is a drug target or drug target candidate, and the bioluminescence signal indicates binding of the drug or drug candidate to the other which is a drug target or drug target candidate. In some embodiments, combining the tagged first molecular entity and the tagged second molecular entity includes expressing one or both intracellularly and / or adding one or both to cells.

[0015] In some embodiments, a method for detecting an intracellular interaction between a first protein or peptide entity and a second protein or peptide entity, comprising: (a) expressing in the cell a fusion comprising a first peptide, dipeptide, or tripeptide tag having an amino acid sequence with at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to the first protein or peptide entity and a first portion of SEQ ID NO: 788 or 789; (b) expressing in the cell a fusion comprising a second peptide, dipeptide, or tripeptide tag having an amino acid sequence with at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to the second protein or peptide entity and a second portion of SEQ ID NO: 788 or 789; (c) expressing in the cell one or more peptide, dipeptide, tripeptide, and / or polypeptide components having an amino acid sequence with at least 40% (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 collectively have at least 40% (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 configured to generate a bioluminescent complex upon interaction 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 luminescence signal generated by the bioluminescent complex, wherein the magnitude of the luminescence signal correlates with the strength of the interaction between the first protein or peptide entity and the second protein or peptide entity. The method is provided herein.

[0016] In some embodiments, a method for detecting the 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 at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity with 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 at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity with a second portion of SEQ ID NO: 788 or 789; (c) combining the tagged first molecular entity and the tagged second molecular entity within the same system; (d) adding to the system one or more peptide, dipeptide, tripeptide, and / or polypeptide components, wherein the components have at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity with a third portion of SEQ ID NO: 788 or 789, and the first tag, the second tag, and the components collectively have at least 40% (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, and the first peptide tag, the second peptide tag, and the components are configured to generate a bioluminescent complex upon co-localization of the first molecular entity and the second molecular entity, said adding; (e) adding coelenterazine or a coelenterazine analog substrate to the system; and (f) detecting the luminescence signal generated by the bioluminescent complex, wherein the presence of a luminescence signal above background indicates co-localization of the first molecular entity and the second molecular entity within the system and / or the magnitude of the luminescence signal correlates with the amount of co-localization of the first molecular entity and the second molecular entity within the system, said detecting.The method including the same is provided herein. In some embodiments, the system includes cells, tissues, organs, whole organisms, and / or non-cellular biochemical samples. In some embodiments, the first molecular entity and / or the second molecular entity is a protein or peptide of interest, and tagging includes 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 includes directly or indirectly linking the first molecular entity and / or the second molecular entity with a first tag and / or a second tag. In some embodiments, combining the tagged first molecular entity and the tagged second molecular entity includes expressing one or both in the system and / or adding one or both to the system.,

[0017] In some embodiments, a method for detecting intracellular co-localization of a first protein or peptide entity and a second protein or peptide entity, comprising: (a) expressing in the cell a fusion comprising a first peptide, dipeptide, or tripeptide tag having an amino acid sequence with at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity with the first protein or peptide entity and a first portion of SEQ ID NO: 788 or 789; (b) expressing in the cell a fusion comprising a second peptide, dipeptide, or tripeptide tag having an amino acid sequence with at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity with the second protein or peptide entity and 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 at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity with a third portion of SEQ ID NO: 788 or 789, wherein the first tag, the second tag, and the components collectively have at least 40% (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, and 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 coelenterazine or a coelenterazine analog substrate to the cell; and (e) detecting the luminescence signal generated by the bioluminescent complex, wherein the presence of a luminescence signal above background indicates co-localization of the first protein or peptide entity and the second protein or peptide entity in the cell, and / or the magnitude of the luminescence signal is,The method provided herein includes detecting, which correlates with the amount of co-localization of the first protein or peptide entity and the second protein or peptide entity within the system.

[0018] In some embodiments, a method of detecting a target molecule, wherein the target molecule presents 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 enabling 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 at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity with the first and second portions of SEQ ID NO: 788 or 789; (c) enabling 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 at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity with the third portion of SEQ ID NO: 788 or 789, wherein the first tag, the second tag, and the components collectively have at least 40% (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 and are configured to generate a bioluminescent complex upon interaction; (d) contacting the sample with coelenterazine or a coelenterazine analog substrate; and (e) detecting the luminescence signal generated by the bioluminescent complex.The method provided herein includes detecting that the presence of a luminescent signal above background indicates the presence of a target molecule and / or that the magnitude of the luminescent signal correlates with the amount of the target molecule in the sample. In some embodiments, the binding moiety is 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, 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, nucleic acid, or small molecule. In some embodiments, the sample is present in vitro or in vivo.,

[0019] In some embodiments, a method of detecting a target molecule, wherein the target molecule presents a first antigen, epitope, or sequence and a distinct second antigen, epitope, or sequence, the method comprising: (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, the first tag comprises an amino acid sequence having at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity with the first portion of SEQ ID NO: 788 or 789, and the second tag comprises an amino acid sequence having the 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 component having at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity with the third portion of SEQ ID NO: 788 or 789, wherein the first tag, the second tag, and the component collectively have at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity with the whole of SEQ ID NO: 788 or 789, and the first tag, the second tag, and the component are configured to generate a bioluminescent complex upon interaction; (d) contacting the sample with coelenterazine or a coelenterazine analog substrate; and (e) detecting the luminescence signal generated by the bioluminescent complex, wherein the presence of a luminescence signal above background indicates the presence of the target molecule and / or the magnitude of the luminescence signal correlates with the amount of the target molecule in the sample. The method is provided herein.In some embodiments, the binding moiety is 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, 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, 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, peptides, dipeptides, tripeptides, and / or polypeptides listed in Table 1, Table 9, or Table 10 are provided herein. 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 (2) or a set (e.g., 2, 3, 4, 5, or more) of peptides, dipeptides, tripeptides, and / or polypeptides listed in Table 1, Table 9, or Table 10 is provided. In particular, pairs or sets of peptides, dipeptides, tripeptides, and / or polypeptides that are complementary and can form a bioluminescent complex when interacting with each other (e.g., promoted, not promoted) are provided.

[0021] In some embodiments, the polypeptide component comprises at least 40% (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, 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, at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) 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,A polypeptide having sequence identity to one of 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, 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 is 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, 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, 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,A suitable fragment of a polypeptide of SEQ ID NO: 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, 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 at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to one of 829 is provided. In some embodiments, such fragments can form a bioluminescent complex with a combination of suitable peptides, dipeptides, tripeptides, polypeptides, etc. provided herein.,

[0022] In some embodiments, the peptide component comprises at least 40% (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 at least 40% (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 can form 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, provided is a single peptide, dipeptide, tripeptide, or polypeptide 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 (e.g., as a reagent, as a tag, etc.). In some embodiments, provided are a pair (2) or a set (e.g., 2, 3, 4, 5, or more) of 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 particular, provided are pairs or sets of peptides, dipeptides, tripeptides, and / or polypeptides that are complementary and can form a bioluminescent complex when interacting with each other (e.g., promoted, not promoted).

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

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

[0026] In some embodiments, β6-7-like dipeptides comprising SEQ ID NOs: 817 and 818 are provided herein. In some embodiments, β6-7-like dipeptides having at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NOs: 817 and 818 are provided herein. In some embodiments, the dipeptide is provided alone or as a pair with a complementary polypeptide and / or other peptide(s), dipeptide(s), and / or tripeptide(s) for the formation of a bioluminescent complex. In some embodiments, nucleic acids and vectors encoding the dipeptide and its fusions are provided. In some embodiments, a molecule and / or protein of interest is tagged with the dipeptides herein.

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

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

[0029] In some embodiments, β9-10-like dipeptides comprising SEQ ID NO: 23 / 13, 23 / 15, 25 / 13, or 25 / 15 are provided herein. In some embodiments, β8-9-like dipeptides having at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 23 / 13, 23 / 15, 25 / 13, or 25 / 15 are provided herein. In some embodiments, the dipeptide is provided alone or as a pair / set with a complementary polypeptide and / or other peptide(s), dipeptide(s), and / or tripeptide(s) for the formation of a bioluminescent complex. In some embodiments, the dipeptide is provided alone or as a pair / set with a complementary polypeptide and / or other peptide(s), dipeptide(s), and / or tripeptide(s) for the formation of a bioluminescent complex. In some embodiments, nucleic acids and vectors encoding the dipeptide and its fusions are provided. In some embodiments, a molecule and / or protein of interest is tagged with a dipeptide herein.

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

[0031] In some embodiments, β7-9-like tripeptides comprising SEQ ID NO: 818 / 819 / 23 or 818 / 819 / 25 are provided herein. In some embodiments, β7-9-like tripeptides having at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 818 / 819 / 23 or 818 / 819 / 25 are provided herein. In some embodiments, the tripeptide is provided alone or as a pair with a complementary polypeptide and / or other peptide(s), dipeptide(s), and / or tripeptide(s) for the formation of a bioluminescent complex. In some embodiments, the tripeptide is provided alone or as a pair with a complementary polypeptide and / or other peptide(s), dipeptide(s), and / or tripeptide(s) for the formation of a bioluminescent complex. In some embodiments, nucleic acids and vectors encoding the tripeptide and its fusions are provided. In some embodiments, a molecule and / or protein of interest is tagged with the tripeptides herein.

[0032] In some embodiments, β8-10-like tripeptides comprising SEQ ID NO: 819 / 23 / 13, 819 / 23 / 15, 819 / 25 / 13, or 819 / 25 / 15 are provided herein. In some embodiments, β7-9-like tripeptides having at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity to SEQ ID NO: 819 / 23 / 13, 819 / 23 / 15, 819 / 25 / 13, or 819 / 25 / 15 are provided herein. In some embodiments, the tripeptide is provided alone or as a pair / set with a complementary polypeptide and / or other peptide(s), dipeptide(s), and / or tripeptide(s) for the formation of a bioluminescent complex. In some embodiments, the tripeptide is provided alone or as a pair / set with a complementary polypeptide and / or other peptide(s), dipeptide(s), and / or tripeptide(s) for the formation of a bioluminescent complex. In some embodiments, the tripeptide is provided alone or as a pair / set with a complementary polypeptide and / or other peptide(s), dipeptide(s), and / or tripeptide(s) for the formation of a bioluminescent complex. In some embodiments, nucleic acids and vectors encoding the tripeptide and its fusions are provided. In some embodiments, a molecule and / or protein of interest is tagged with a tripeptide herein.

[0033] In some embodiments, a peptide comprising an amino acid sequence having at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 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, wherein the bioluminescence signal generated in the presence of coelenterazine or a coelenterazine analog substrate when the peptide contacts 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 bioluminescence signal generated by the peptide and coelenterazine or a coelenterazine analog substrate alone, is provided herein. In some embodiments, the bioluminescence signal is substantially increased when the peptide associates with the second peptide and the polypeptide complement. In some embodiments, the peptide exhibits enhancement of one or more traits compared to the peptide of SEQ ID NO: 6 and / or SEQ ID NO: 9, wherein the trait is selected from affinity for the second peptide and the polypeptide complement, or enhanced expression, solubility, stability, and / or bioluminescence activity when combined with the second peptide and the 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 non-natural amino acids, amino acid analogs, and / or peptidomimetic 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, fusion polypeptides (e.g., gene fusions (or alternatively, generated by chemical conjugation or synthesis)) are provided herein that include the peptides described in the previous paragraph and additional amino acid sequences or compounds (e.g., small molecule drugs). In some embodiments, the additional amino acid sequence is selected from the group consisting of a protein of interest, an interaction 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, 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 (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 interaction polypeptide that is configured to form a complex with a second interaction polypeptide upon contact of the first and second interaction polypeptides. In some embodiments, the additional amino acid sequence is a first co-localization polypeptide that is configured to co-localize with a second co-localization polypeptide within an intracellular compartment, cell, tissue, or organism. In some embodiments, the additional amino acid sequence is a protein of interest and is a drug target candidate.

[0035] In some embodiments, a peptide comprising an amino acid sequence having at least 40% (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, wherein when the peptide contacts 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 bioluminescence signal generated in the presence of coelenterazine or a coelenterazine analog substrate is substantially increased compared to the bioluminescence signal generated by the peptide and coelenterazine or a coelenterazine analog substrate alone. The peptide is provided herein. In some embodiments, the bioluminescence signal is substantially increased when the peptide associates with the second peptide and the polypeptide complement. In some embodiments, the peptide exhibits enhancement of one or more traits compared to the peptide of SEQ ID NO: 7 and / or SEQ ID NO: 10, wherein the trait is selected from affinity for the second peptide and the polypeptide complement, or enhanced expression, solubility, stability, and bioluminescence activity when combined with the second peptide and the 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 non-natural amino acids, amino acid analogs, and / or peptidomimetic 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, fusion polypeptides comprising the peptides described in the previous paragraph and additional amino acid sequences (e.g., gene fusions, synthetically produced fusions, chemical conjugates, enzyme conjugates, etc.) are provided herein. In some embodiments, the additional amino acid sequence is selected from the group consisting of a protein of interest, an interaction 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 interaction polypeptide configured to form a complex with a second interaction polypeptide upon contact of the first and second interaction 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 an intracellular compartment, cell, tissue, or organism. In some embodiments, the additional amino acid sequence is a protein of interest and is a drug target candidate.

[0037] In some embodiments, a composition comprising: (a) a first peptide having an amino acid sequence that has greater 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 second peptide having an amino acid sequence that has greater 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, less than 100% sequence identity with SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 29, wherein when the first peptide contacts the second peptide and a polypeptide complement consisting of SEQ ID NO: 17, SEQ ID NO: 21, or SEQ ID NO: 302, a bioluminescence signal generated in the presence of a coelenterazine or coelenterazine analog substrate is substantially increased compared to a bioluminescence signal generated by only the first peptide and / or the second peptide and a coelenterazine substrate. In some embodiments, the bioluminescence signal is substantially increased when the first peptide associates with the second peptide and the polypeptide complement. In some embodiments, the first peptide exhibits enhancement of one or more traits compared to the peptides of SEQ ID NO: 7 and / or SEQ ID NO: 10, and the second peptide exhibits enhancement of one or more traits compared to the peptides of SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 29, where the trait is selected from affinity for the second peptide and the polypeptide complement, or enhanced expression, solubility, stability, and bioluminescence activity when combined with the second peptide and the 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 non-natural amino acids, amino acid analogs, and / or peptoid amino acids.

[0038] In some embodiments, compositions are provided herein that include 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 interaction 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 interaction polypeptide that is configured to form a complex with the second interaction polypeptide upon contact of the first and second interaction polypeptides. In some embodiments, the additional amino acid sequence is a first co-localization polypeptide that is configured to co-localize with a second co-localization polypeptide within an intracellular compartment, cell, tissue, or organism. In some embodiments, the additional amino acid sequence is a protein of interest and is a drug target candidate.

[0039] In some embodiments, a polypeptide comprising an amino acid sequence having at least 40% (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, wherein when the polypeptide contacts a first peptide consisting of SEQ ID NO: 23 and a second peptide consisting of SEQ ID NO: 25, the bioluminescence signal generated in the presence of coelenterazine or a coelenterazine analog substrate is substantially increased as compared to the bioluminescence signal generated by the peptide and the coelenterazine or coelenterazine analog substrate alone. The polypeptide is provided herein. In some embodiments, the bioluminescence signal is substantially increased when the polypeptide associates with the first and second peptides. In some embodiments, the polypeptide exhibits enhancement of one or more traits as compared to the polypeptide of SEQ ID NO: 5 and / or SEQ ID NO: 8, wherein the trait is selected from affinity for the first and / or second peptides, 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), 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 non-natural amino acids, amino acid analogs, and / or peptoid amino acids.

[0040] In some embodiments, fusion polypeptides are provided herein that include the polypeptides described in the previous paragraph and additional amino acid sequences, nucleic acid sequences, or other fused or appended molecules (e.g., gene fusions, synthetically produced fusions, chemical conjugates, enzyme conjugates, etc.). In some embodiments, the additional sequence or other molecule is selected from the group consisting of a protein of interest, an interaction 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, 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 sequence or other fused or appended molecule is a first interaction polypeptide that is configured to form a complex with a second interaction polypeptide upon contact of the first and second interaction polypeptides. In some embodiments, the additional sequence or other fused or appended molecule is a first co-localization polypeptide that is configured to co-localize with a second co-localization polypeptide within an intracellular compartment, cell, tissue, or organism. In some embodiments, the additional sequence or other fused or appended molecule is a protein of interest and is a drug target candidate.

[0041] In some embodiments, an amino acid sequence having at least 40% (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, wherein when the polypeptide contacts a first peptide consisting of SEQ ID NO: 23 and a second peptide consisting of SEQ ID NO: 25, the bioluminescence signal generated in the presence of coelenterazine or a coelenterazine analog substrate is substantially increased compared to the bioluminescence signal generated by the peptide and the coelenterazine or coelenterazine analog substrate alone. The polypeptide is provided herein. In some embodiments, the bioluminescence signal is substantially increased when the polypeptide associates with the first and second peptides. In some embodiments, the polypeptide exhibits enhancement of one or more traits compared to the polypeptide of SEQ ID NO: 5 and / or SEQ ID NO: 8, wherein the trait is selected from affinity for the first and / or second peptides, 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), 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 non-natural amino acids, amino acid analogs, and / or peptoid amino acids.

[0042] In some embodiments, fusion polypeptides (e.g., gene fusions, synthetically produced fusions, chemical conjugates, enzyme conjugates, etc.) comprising the peptides described in the previous paragraph and additional amino acid sequences are provided herein. In some embodiments, the additional amino acid sequence is selected from the group consisting of a protein of interest, an interaction 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 interaction polypeptide configured to form a complex with a second interaction polypeptide upon contact of the first and second interaction 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 an intracellular compartment, cell, tissue, or organism. In some embodiments, the additional amino acid sequence is a protein of interest and a drug target candidate.

[0043] In some embodiments, an amino acid sequence having greater 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 contacts a polypeptide complement consisting of SEQ ID NO: 17, SEQ ID NO: 21, or SEQ ID NO: 302, the bioluminescence signal generated in the presence of coelenterazine or a coelenterazine analog substrate is substantially increased compared to the bioluminescence signal generated by the peptide and coelenterazine or a coelenterazine analog substrate alone. The β9 / β10-like dipeptide is provided herein. In some embodiments, the dipeptide (e.g., β9 / β 10 -like dipeptide) associates (e.g., forms a bioluminescent complex) with the polypeptide components (e.g., β 1~8 -like polypeptides) described herein without facilitation (e.g., by an interaction element). In other embodiments, the dipeptide (e.g., β9 / β 10 -like dipeptide) and the polypeptide components (e.g., β 1~8 -like polypeptides) described herein do not form a bioluminescent complex without facilitation (e.g., by an interaction element), but associate (e.g., form a bioluminescent complex) with facilitation by an appropriate interaction element. In some embodiments, the bioluminescence signal is substantially increased when the peptide associates with the polypeptide complement. In some embodiments, the peptide exhibits enhancement of one or more properties compared to the peptides of SEQ ID NO: 205 and / or SEQ ID NO: 206, where the property is selected from affinity for the polypeptide complement, or enhanced expression, solubility, stability, and / or bioluminescence 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 non-natural amino acids, amino acid analogs, and / or peptidomimetic amino acids.

[0044] In some embodiments, fusion polypeptides (e.g., gene fusions, synthetically produced fusions, chemical conjugates, enzyme conjugates, etc.) comprising the β9 / β10-like dipeptides and additional amino acid sequences described herein are provided herein. In some embodiments, the additional amino acid sequence is selected from the group consisting of a protein of interest, an interaction element, a co-localization element, and a binding moiety. In some embodiments, the additional amino acid sequence or other fusion or added molecule is 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, which is a binding moiety selected from the group consisting of. In some embodiments, the additional amino acid sequence or other fusion or added molecule is a first interaction polypeptide that is configured to form a complex with the second interaction polypeptide upon contact with the second interaction polypeptide. In some embodiments, the additional amino acid sequence or other fusion or added molecule is a first co-localization polypeptide that is configured to co-localize with a second co-localization polypeptide within an intracellular compartment, cell, tissue, or organism. In some embodiments, the additional amino acid sequence or other fusion or added molecule is a protein of interest and a drug target candidate.

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

[0046] In some embodiments, a bioluminescent complex comprising: (a) a polypeptide comprising an amino acid sequence having at least 40% (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 at least 40% (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 second peptide comprising an amino acid sequence having at least 40% (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 the bioluminescent complex produces substantially increased bioluminescence in the presence of coelenterazine or a coelenterazine analog substrate as compared to 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, and the bioluminescent complex is provided herein. 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 interaction 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 drug candidate, and the other peptide tag is linked to a drug target or drug target candidate, wherein 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 interaction element and a second peptide tag is linked to a second interaction element, where the intensity of bioluminescence from the bioluminescent complex correlates with the affinity of the first interaction element for the second interaction element under the conditions being tested (e.g., in some embodiments, combinations of the first peptide, the second peptide, the polypeptide component, and the substrate do not form a bioluminescent complex in the absence of interaction between the interaction elements (and do not produce a significant light output (e.g., above background))). 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, where substantially increased bioluminescence indicates co-localization of the first co-localization element and the second co-localization element under the conditions being tested, but does not necessarily indicate interaction.

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

[0048] In some embodiments, (a) (i) a first peptide comprising an amino acid sequence having at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 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) a second peptide comprising an amino acid sequence having at least 40% (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, (iii) a polypeptide component comprising an amino acid sequence having at least 40% (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, 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, the polypeptide component, and (iv) combining a coelenterazine or coelenterazine analog substrate, and (b) detecting luminescence, wherein a higher level of luminescence is detected as compared to the level of luminescence generated by the polypeptide component and coelenterazine or coelenterazine analog alone, indicating formation of the bioluminescent complex of the polypeptide component and the first and second peptides, are provided herein. In some embodiments, one or more of the polypeptide component and the first and second peptides are expressed intracellularly, added exogenously to cells, and / or added to a sample.

[0049] In some embodiments, 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 at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity with SEQ ID NO: 23 and less than 100% sequence identity with SEQ ID NOs: 6 and 9; (b) tagging the second molecular entity with a second 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%, or more) sequence identity with SEQ ID NO: 25 and less than 100% sequence identity with SEQ ID NOs: 7 and 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 at least 40% (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 NOs: 5 and 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 luminescence signal generated by the bioluminescent complex, wherein the magnitude of the luminescence signal correlates (e.g., is proportional, directly proportional, etc.) with the number, strength, preference, 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 rate, and / or specific activity of the resulting bioluminescent complex correlates (e.g., is proportional, directly proportional, etc.) with the number, strength, preference, and / or stability of the interaction(s) 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 or peptide of interest, and tagging comprises creating a fusion (or synthetic conjugation) with a first peptide tag and / or a second peptide tag of the first molecular entity and / or the second molecular entity. 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 a first peptide tag and / or a second peptide 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 bioluminescence 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 intracellularly and / or adding one or both to cells. In some embodiments, combining the tagged first molecular entity and the tagged second molecular entity is performed in vitro, in a cell-free 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 untagged drug target or drug target candidate into the 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 individually.

[0050] In some embodiments, a method of performing a competitive assay for detecting an interaction between a first molecular entity and a second molecular entity, comprising: (a) combining (i) a tracer comprising a first molecular entity tagged with a first 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%, or more) sequence identity with SEQ ID NO: 23 and less than 100% sequence identity with SEQ ID NOs: 6 and 9, (ii) a second molecular entity tagged with a second 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%, or more) sequence identity with SEQ ID NO: 25 and less than 100% sequence identity with SEQ ID NOs: 7 and 10, (iii) coelenterazine or a coelenterazine analog substrate, (iv) a polypeptide component comprising an amino acid sequence having at least 40% (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 NOs: 5 and 8, and (v) a sample suspected of containing the untagged first molecular entity, wherein the first peptide tag, the second peptide tag, and the polypeptide component are configured to generate a bioluminescent complex and generate a bioluminescent signal in the presence of coelenterazine or a coelenterazine analog substrate; (b) detecting the bioluminescent signal generated by the bioluminescent complex; and (c) comparing the bioluminescent signal generated in the presence of the sample to 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 a peptide (e.g., a drug or a drug candidate).In some embodiments, the second molecular entity is a drug target or drug target candidate (e.g., a protein).

[0051] In some embodiments, a method for detecting an intracellular interaction between a first protein, peptide, or molecular entity and a second protein, peptide, or molecular entity, comprising: (a) expressing (or adding to a cell or other system, e.g., a cell-free sample) a fusion (e.g., a gene fusion, synthetic fusion, chemical conjugation, etc.) comprising the first protein, peptide, or molecular entity and a first 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%, or more) sequence identity with SEQ ID NO: 23 and less than 100% sequence identity with SEQ ID NOs: 6 and 9; (b) expressing (or adding to a cell or other system, e.g., a cell-free sample) a fusion (e.g., a gene fusion, synthetic fusion, chemical conjugation, etc.) comprising the second protein, peptide, or molecular entity and a second 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%, or more) sequence identity with SEQ ID NO: 25 and less than 100% sequence identity with SEQ ID NOs: 7 and 10; (c) expressing (or adding to a cell or other system, e.g., a cell-free sample) a polypeptide component comprising an amino acid sequence having at least 40% (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 NOs: 5 and 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, 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 luminescence signal generated by the bioluminescent complex,The method provided herein includes detecting that the magnitude of the luminescence 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)-(e) are performed simultaneously. In some embodiments, two or more of steps (a)-(e) are performed individually.,

[0052] In some embodiments, a method for detecting the 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 at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 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) tagging the second molecular entity with a second 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%, 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; (c) combining the tagged first molecular entity and the tagged second molecular entity within the same system; (d) adding to the system a polypeptide component comprising an amino acid sequence having at least 40% (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, 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 in the system; (e) adding to the system a coelenterazine or coelenterazine analog substrate; and (f) detecting a luminescence signal generated by the bioluminescent complex, wherein the presence of a luminescence 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 luminescence signal correlates with the amount of co-localization of the first molecular entity and the second molecular entity in the system. In some embodiments, the system comprises a cell, a tissue, an organ, or an entire organism.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 of the first molecular entity and / or the second molecular entity with the first peptide tag and / or the second peptide tag (e.g., a genetic fusion, a synthetic fusion, a chemical conjugation, an enzymatic conjugation, etc.). 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 cell-free sample, etc. 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. 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 individually.

[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, 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 at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity with SEQ ID NO: 23 and less than 100% sequence identity with SEQ ID NOs: 6 and 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 at least 40% (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 NOs: 7 and 10; (c) expressing in the cell a polypeptide component comprising an amino acid sequence having at least 40% (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 NOs: 5 and 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 luminescence signal generated by the bioluminescent complex, wherein the presence of a luminescence signal above background indicates co-localization of the first protein or peptide entity and the second protein or peptide entity in the cell and / or the magnitude of the luminescence 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. The method is provided herein.In some embodiments, two or more of steps (a) to (e) are executed simultaneously. In some embodiments, two or more of steps (a) to (e) are executed individually.

[0054] In some embodiments, a kit is provided herein that includes: (a) a first binding moiety conjugated to a first peptide tag that includes 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: 23 and less than 100% sequence identity with SEQ ID NOs: 6 and 9; and (b) a second binding moiety conjugated to a second peptide tag that includes 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 NOs: 7 and 10. In some embodiments, the first and second binding moieties are 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, 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 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 includes a polypeptide reagent that includes 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 NOs: 5 and 8. In some embodiments, the kit further includes coelenterazine or a coelenterazine analog.

[0055] In some embodiments, a method of detecting a target molecule, wherein the target molecule presents 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 enabling 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 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, the first or second peptide tag comprises an amino acid sequence having at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) sequence identity with SEQ ID NO: 23 (and less than 100% sequence identity with SEQ ID NOs: 6 and 9), the other of the first or second peptide tags comprises an amino acid sequence having at least 40% (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 NOs: 7 and 10), and enabling 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 at least 40% (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 NOs: 5 and 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 substrate of coelenterazine or a coelenterazine analog, and (e) detecting a luminescence signal generated by the bioluminescent complex, wherein the presence of a luminescence signal above background indicates the presence of the target molecule and / or the magnitude of the luminescence signal correlates with the amount of the target molecule in the sample, the method comprising such detecting is provided herein. In some embodiments, the binding moiety is 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, 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, a peptide, a nucleic acid, a chemical, or a drug. In some embodiments, the sample is present in vitro or in vivo.,

[0056] In some embodiments, a method for detecting a target molecule, wherein the target molecule presents 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 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, the second binding moiety recognizes the second antigen, epitope, or sequence, and the first or second peptide tag comprises an amino acid sequence having at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 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), and the other of the first or second peptide tags comprises an amino acid sequence having at least 40% (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 enabling 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 at least 40% (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), 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 coelenterazine or a coelenterazine analog substrate; and (e) detecting the luminescence signal generated by the bioluminescent complex, wherein the presence of a luminescence signal above background indicates the presence of the target molecule and / or the magnitude of the luminescence signal isThe present specification provides such a method that includes detecting the amount of a target molecule in the sample, which correlates with the amount of the target molecule in the sample. In some embodiments, the binding moiety is 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, 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, a peptide, a nucleic acid, a chemical substance, or a drug. In some embodiments, the sample is present in vitro or in vivo.,

[0057] In some embodiments, (a) (i) a peptide component comprising an amino acid sequence having at least 40% (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) a polypeptide component comprising an amino acid sequence having at least 40% (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) combining a coelenterazine or coelenterazine analog substrate, wherein the peptide component and the polypeptide component are configured to form a bioluminescent complex upon interaction, the combining, and (b) detecting luminescence, wherein a higher level of luminescence is detected compared to the level of luminescence produced by the polypeptide component and the coelenterazine or coelenterazine analog alone, indicating the formation of a bioluminescent complex between the polypeptide component and the peptide, the detecting luminescence, are provided herein. In some embodiments, the peptide is a fusion with a first interaction element (e.g., a gene fusion, a synthetic fusion, a chemical conjugate, an enzyme conjugate, etc.), and the polypeptide component is a fusion with a second interaction element (a gene fusion, a synthetic fusion, a chemical conjugate, an enzyme conjugate, etc.), wherein the peptide and the polypeptide component form a bioluminescent complex upon interaction of the interaction elements, but do not form a bioluminescent complex in the absence of interaction between the interaction elements. In some embodiments, the peptide and the polypeptide component form a bioluminescent complex in the absence of facilitation (e.g., by the interaction elements).In some embodiments, the peptide is a fusion or conjugate (e.g., a gene fusion, a synthetic fusion, a chemical conjugate, an enzyme conjugate, etc.) with a protein, peptide, or molecule of interest (e.g., not an interaction element), and / or the polypeptide component is a fusion or conjugate (e.g., a gene fusion, a synthetic fusion, a chemical conjugate, an enzyme conjugate, etc.) with a protein, peptide, or molecule of interest (e.g., not an interaction element). In some embodiments, the peptide component and the polypeptide component form a bioluminescent complex without facilitation by an interaction element upon co-localization (e.g., within a sample, within a cell, within a tissue, within a subject, etc.). In some embodiments, the peptide component and the polypeptide component form a bioluminescent complex when facilitated by an interaction element, but do not form a bioluminescent complex without facilitation.

[0058] In some embodiments, provided herein is a monomeric polypeptide comprising an amino acid sequence having greater 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, and that exhibits luminescence in the presence of coelenterazine or a coelenterazine analog substrate. In some embodiments, a peptide comprising an amino acid sequence having greater than 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity to one or more of SEQ ID NO: 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, provided is a fusion protein of the polypeptides described herein.

[0059] In some embodiments, provided herein are nucleic acids comprising a sequence encoding an amino acid sequence having greater than 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or in the range between) sequence identity to SEQ ID NO: 788 or 789, but less than 100% sequence identity, 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 in the range between) sequence identity to one or more of SEQ ID NO: 780, 782, 784, 786, 802, 804, 806, 808, 813, 815, or 829. In some embodiments, provided herein are nucleic acids encoding a fusion protein of the polypeptides described herein.

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

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

[0062] In some embodiments, as used herein, a method is provided that comprises: (a) contacting a polypeptide comprising an amino acid sequence having more than 40% sequence identity to SEQ ID NO: 15, linked to the N-terminus of an amino acid sequence having more than 40% sequence identity to one of SEQ ID NO: 17, 21, or 302, with a coelenterazine or coelenterazine analog substrate and a peptide or 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) sequence identity to SEQ ID NO: 23; and (b) detecting luminescence.

[0063] In some embodiments, as used herein, (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 a range therebetween) sequence identity to SEQ ID NO: 11, linked to an amino acid sequence localized 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 a range therebetween) sequence identity to SEQ ID NO: 23, wherein a bioluminescent complex is provided that forms between the polypeptide and the peptide tag upon co-localization 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, as used herein, (a) expressing the 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 co-localization of the polypeptide and peptide tag. In some embodiments, the method further comprises inducing a translocation protein of interest linked to the peptide tag to a specific cellular location.

[0064] This patent or application file contains drawings executed in at least one color. Copies of this patent or patent application publication with color drawings will be provided by the Patent and Trademark Office upon request and payment of the necessary fees.

Brief Description of the Drawings

[0065]

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Mode for Carrying Out the Invention

[0066] Definition Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the embodiments described herein, but some 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 present invention is not limited to the specific molecules, compositions, methodologies, or protocols described herein, because these can be changed by routine experimentation and optimization. Also, the terms used herein are for the purpose of describing only specific versions or embodiments and are 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 one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the specification of the present invention including definitions shall prevail. Accordingly, the following definitions apply in relation to 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" includes reference to one or more peptides and their equivalents known to those of ordinary skill in the art, and so on.

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

[0070] As used herein, the term "comprise" and its linguistic variations mean that the recited features (if any), components (if any), method steps (if any), etc. are present and the presence of additional features (if any), components (if any), method steps (if any), etc. is not excluded. Conversely, the term "consisting of" and its linguistic variations mean that the recited features (if any), components (if any), method steps (if any), etc. are present and any features (if any), components (if any), method steps (if any), etc. not recited are excluded, except for impurities that are normally incidental. The expression "consisting essentially of" means the recited features (if any), components (if any), method steps (if any), etc., and any additional features (if any), components (if any), method steps (if any), etc. that do not substantially affect the basic nature of the composition, system, or method. Many embodiments herein are described using the open language "comprising". Such embodiments include a plurality of closed "consisting of" and / or "consisting essentially of" embodiments, and alternatively, these may be claimed or described using such language.

[0071] As used herein, the term "substantially" means that the recited characteristics, parameters, and / or values need not be achieved exactly, and 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 preclude the effect that the feature is intended to provide. A characteristic or feature that is substantially absent (e.g., substantially non-luminescent) may be one that is within the range of noise, lower than the background, below the detection ability of the assay being used, or 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 prominent characteristic (e.g., the luminescence intensity of a bioluminescent protein or bioluminescent complex).

[0072] As used herein, the term "bioluminescence" refers to the generation and emission of light by a chemical reaction catalyzed or enabled by an enzyme, protein, protein complex, or other biomolecule (e.g., a bioluminescent complex). In typical embodiments, the substrate of a bioluminescence entity (e.g., a bioluminescent protein or bioluminescent complex) is converted to an unstable form by the bioluminescence entity, and the substrate then 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.) being able to hybridize, dimerize, or otherwise form a complex with each other. For example, "complementary peptides and polypeptides" can assemble to form a complex. Complementary components may require assistance (facilitation), for example, to bring the components into a three-dimensional structure suitable for complementarity, to place the components in proximity suitable for complementarity, to co-localize complementary components, to reduce the interaction energy for complementarity, 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 aggregate of molecules (e.g., peptides, polypeptides, etc.) that are in direct and / or indirect contact with each other. In one embodiment, "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 the interaction of the molecules. In such an embodiment, a complex of molecules (e.g., peptides and polypeptides) is formed 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, the term "complex" refers to an assembly of two or more molecules (e.g., peptides, polypeptides, or combinations thereof), unless otherwise indicated.

[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 emission 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 emission is small enough that it does not create a 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, 1×10 ) when compared to a noticeable 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, 1×10 4 1 / 10, 1×10 5 1 / 10, 1×106 one - tenth, 1×10 7 refers to peptides and polypeptides such as one - tenth. In some embodiments, such non - luminescent peptides and polypeptides aggregate under the reference conditions described herein to form a bioluminescent complex.

[0077] As used herein, the term "interaction element" refers to a moiety that aids or facilitates the aggregation of non - luminescent elements to form a bioluminescent complex. In some embodiments, a pair of interaction elements (also referred to as an "interaction pair") is attached to a pair of non - luminescent elements (e.g., non - luminescent peptides), and the attractive interaction between the two interaction elements promotes the formation of a bioluminescent complex. However, the present invention is not limited to such a mechanism, and an understanding of such a mechanism is not required to practice the present invention. The interaction element can promote the formation of a bioluminescent complex by any suitable mechanism (e.g., bringing non - luminescent elements into proximity, putting non - luminescent elements into a steric structure suitable for stable interaction, reducing the activation energy for complex formation, combinations thereof, etc.). The interaction element can be a protein, polypeptide, peptide, small molecule, cofactor, nucleic acid, lipid, carbohydrate, antibody, etc. The interaction pair can consist of two identical interaction elements (i.e., a homogenous pair) or two different interaction elements (i.e., a heterogenous pair). In the case of a heterogenous pair, the interaction elements can be of the same type of moiety (e.g., polypeptide) or two different types of moieties (e.g., polypeptide and small molecule). In some embodiments where complex formation by an interaction pair is investigated, the interaction pair can be referred to as a "target pair" or a "pair of interest", and the individual interaction elements can be 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 intermolecular interactions between two or more (e.g., three) entities that are too weak to result in significant complex formation between the entities, except at concentrations that are substantially higher (e.g., two-fold, five-fold, ten-fold, one-hundred-fold, one-thousand-fold or more) than physiological or assay conditions, or where there is promotion by formation of a second complex of the attached elements (e.g., interacting elements).

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

[0080] As used herein, the term "co-localizing element" refers to a moiety that promotes co-localization of non-emissive elements. In some embodiments, a set of non-emissive elements has an affinity sufficient to form a complex when the non-emissive elements co-localize at a sufficient concentration. In such embodiments, a set (e.g., a pair) of co-localizing elements (also referred to as a "co-localizing pair") is attached to a pair of non-emissive elements (e.g., non-emissive peptides), and co-localization of the two co-localizing elements (e.g., within an intracellular compartment, within a tissue, in solution, on a solid matrix support, etc.) promotes co-localization of the non-emissive elements, thereby promoting formation of a bioluminescent complex. However, the invention is not limited to such mechanisms, and understanding of such mechanisms is not required to practice the invention. In some embodiments, due to the ability of non-emissive elements to self-assemble into a luminescent complex, the co-localizing elements do not need to directly interact to promote complex formation. The co-localizing elements can be proteins, polypeptides, peptides, small molecules, cofactors, nucleic acids, lipids, carbohydrates, antibodies, and the like. The co-localizing pair may consist of two identical co-localizing elements (i.e., a homogenous pair) or two different co-localizing elements (i.e., a heterogenous pair). In the case of a heterogenous pair, the co-localizing elements may be of the same type of moiety (e.g., polypeptide) or two different types of moieties (e.g., polypeptide and small molecule). In some embodiments where the localization of the co-localizing pair is investigated, the co-localizing pair may be referred to as a "target pair" or a "pair of interest", and the individual co-localizing elements may be 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 "selentrazine" refers to naturally occurring ("natural") selentrazine. As used herein, the term "selentrazine analog" or "selentrazine derivative" refers to synthetic analogs (e.g., derivatives or variants) and natural analogs of selentrazine, including, in addition to those disclosed in WO2003 / 040100; US Patent Application No. 12 / 056073 (paragraph

[0086] ); US Patent No. 8,669,103; WO2012 / 061529, US Patent Publication No. 2017 / 0233789, and US Patent Publication No. 2018 / 0030059 (the disclosures of which are incorporated herein by reference in their entireties), frimazine, selentrazine-n, selentrazine-f, selentrazine-h, selentrazine-hcp, selentrazine-cp, selentrazine-c, selentrazine-e, selentrazine-fcp, bisdeoxyselentrazine ("selentrazine-hh"), selentrazine-i, selentrazine-icp, selentrazine-v, and 2-methylselentrazine. In some embodiments, selentrazine analogs include, for example, pro-substrates such as those described in US Patent Application No. 12 / 056073; US Publication No. 2012 / 0707849; US Publication No. 2014 / 0099654 (the disclosures of which are incorporated herein by reference in their entireties).

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

[0083] As used herein, the term "fragment" refers to a peptide or polypeptide resulting from the cleavage or "fragmentation" of a larger entity as a whole (e.g., a protein, polypeptide, enzyme, etc.), or a peptide or polypeptide prepared to have the same sequence as them. Thus, a fragment is a partial sequence of an entity as a whole (e.g., a protein, polypeptide, enzyme, etc.), and is made and / or designed from the entity as a whole. A peptide or polypeptide that is not a partial sequence 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 partial sequence of a protein (e.g., natural or synthetic) that exhibits high bioluminescence activity.

[0084] As used herein, the term "subsequence" refers to a peptide or polypeptide having 100% sequence identity with a part of another larger peptide or polypeptide. A subsequence has a completely matching sequence with a part of a longer amino acid chain.

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

[0086] Examples of natural 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] Examples of non-natural amino acids include, but are not limited to, 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-hydroxyproline ("4Hyp"), isodesmosine, allo-isoleucine, N-methylalanine ("MeAla" or "Nime"), N-alkylglycine containing N-methylglycine ("NAG"), N-methylisoleucine, N-alkylpentylglycine containing N-methylpentylglycine ("NAPG"), 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-natural reactive amino acids are described, for example, in Boutureira, O. and G. J. 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 non-natural amino acid in which one or more of the C-terminal carboxy group, N-terminal amino group, and side chain bioactive group are chemically blocked, either reversibly or irreversibly, or otherwise modified to 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 having various protecting groups (Isidro-Llobet, A., et al. (2009). "Amino Acid-Protecting Groups." Chemical Reviews 109(6):2455-2504 (which is hereby incorporated 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 peptide amide bond (--C(O)NH--). The term "peptide" typically refers to a short amino acid polymer (e.g., a chain having less than 30 amino acids), while the term "polypeptide" typically refers to a longer amino acid polymer (e.g., a chain 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.) containing 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 two β - strands of luciferase (e.g., the "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.) containing 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 three β - strands of luciferase (e.g., the "β8 - 10 tripeptide" corresponding to the β8 - 10 strands of the OgLuc luciferase polypeptide)).

[0092] As used herein, the terms "peptide mimetic" and "peptide analogue" refer to peptidomimetic or polypeptidomimetic molecules that mimic sequences derived from proteins or peptides. Peptide mimetics can contain amino acid analogues, peptoid amino acids, and / or non - amino acid components, either alone or in combination with amino acids (e.g., natural or non - natural amino acids). Examples of peptide mimetics include chemically modified peptides / polypeptides, peptoids (where the side chain is attached to the nitrogen atom of the peptide backbone rather than the α - carbon), β - peptides (where the amino group is attached to the β - carbon rather than the α - carbon), etc.

[0093] As used herein, the term "peptoid" refers to a class of peptide mimetics in which the nitrogen atom of the peptide backbone, rather than the α - carbon, is functionalized with a side chain.

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

[0095] As used herein, a "conservative" amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid having similar chemical properties, e.g., size or charge. For the purposes of the present 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 classified into the following classes, for example, based on the nature of their common side chains: 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 can also include non-naturally occurring amino acid residues having chemical properties similar to those of natural residues. These non-natural residues are typically incorporated by chemical peptide synthesis rather than synthesis in a biological system. These include, but are not limited to, peptidomimetics, and other inverted or reverse forms of amino acid moieties. Embodiments herein can, in some embodiments, be limited to natural amino acids, non-natural amino acids, and / or amino acid analogs.

[0098] Non-conservative substitutions can involve the exchange of a member of one class with 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, e.g., acidic (e.g., aspartic acid, glutamic acid), basic (e.g., lysine, arginine, histidine), non-polar (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 that contain identical (similar) monomers (e.g., positions where the same amino acid is found in both sequences, positions where similar amino acids are found in both sequences) to obtain the number of matching positions; (3) dividing the number of matching 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 in length and have the same amino acids except for one position, Peptide A and Peptide B have 95% sequence identity. If the amino acids at the 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 in length and Peptide D is 15 amino acids in length, and 14 out of the 15 amino acids in Peptide D are identical to some of the amino acids in Peptide C, then Peptide C and Peptide D have 70% sequence identity, but Peptide D has 93.3% sequence identity with respect to the optimal comparison window of Peptide C.For the purpose of calculating the "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 sequence number can be expressed as having the 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 sequence number Z (e.g., 100 amino acids) can have up to X substitutions (e.g., 10) compared to sequence number Z, and thus can also be expressed as having X (e.g., 10) or fewer substitutions compared to sequence number Z.

[0101] As used herein, the term "wild-type" refers to a gene or gene product (e.g., protein, polypeptide, peptide, etc.) that has the characteristics (e.g., sequence) of that gene or gene product isolated from a naturally occurring source and is the most frequently observed in the population. In contrast, the term "variant" or "variant" refers to a gene or gene product that shows a modification in sequence when compared to the wild-type gene or gene product. It should be noted that a "naturally occurring variant" is a gene or gene product that occurs naturally but has a modified sequence when compared to the wild-type gene or gene product, and they are not the most frequently occurring sequences. An "artificial variant" is a gene or gene product that has a modified sequence when compared to the wild-type gene or gene product and does not occur naturally. Variant genes or gene products can be naturally occurring sequences that exist in nature but are not the most commonly observed variants of the gene or gene product, or "synthetic products" made by human or experimental intervention.

[0102] As used herein, the term "physiological conditions" encompasses any conditions compatible with living cells, such as temperature, pH, salinity, and predominantly aqueous chemical composition, that are compatible with living cells.

[0103] As used herein, the term "sample" is used in the broadest sense. In a sense, this term means that it includes specimens or cultures obtained from any source, as well as biological samples and environmental samples. Biological samples can be obtained from animals (including humans) and include liquids, solids, tissues, and gases. Biological samples include blood preparations such as plasma and serum. A sample can also refer to a cell lysate or a purified form of an enzyme, peptide, and / or polypeptide described herein. Cell lysates can include cells lysed by 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 substances such as surface substances, 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-fluorescent peptide) that is linked to a second different peptide, polypeptide, or protein (e.g., an interaction element).

[0105] As used herein, the terms "conjugated" and "conjugation" refer to the covalent bond of two molecular entities (e.g., during or after synthesis and / or synthetic production). 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 different epitopes or antigen-binding domains. The 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 the 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.), and the whole antibody molecule or a fragment thereof can be a polyclonal antibody or a monoclonal antibody or a recombinant antibody, a chimeric antibody, a humanized antibody, a 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 a substantially higher affinity compared to other entities that do not present the antigen or epitope. In this regard, "substantially higher affinity" means an affinity high enough to enable the detection of an antigen or epitope that can be distinguished from a plurality of entities using a desired assay or measuring 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 etc.) binding constant (K a ) means a binding affinity. In certain such embodiments, an antibody can bind to different antigens so long as the different antigens contain its particular epitope. In certain examples, for instance, homologous proteins from different species can contain the same epitope.

[0108] As used herein, the term "antibody fragment" refers to a portion of a full-length antibody that includes 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, which is incorporated herein by reference in its entirety. In certain embodiments, antibody fragments are generated by enzymatic or chemical cleavage of intact antibodies (e.g., papain and pepsin digestion of antibodies) generated by recombinant DNA techniques or chemical polypeptide synthesis. For example, a "Fab" fragment includes one light chain as well as the C H1 and variable regions. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A "Fab´" fragment includes one light chain and the C H1 domain and C H2It comprises one heavy chain that includes an additional constant region extending between domains. An inter-chain disulfide bond can be formed between the two heavy chains of the Fab´ fragment to form an "F(ab´)2" molecule. The "Fv" fragment includes variable regions from both the heavy and light chains but lacks the constant regions. A single-chain Fv (scFv) fragment includes a heavy-chain variable region and a light-chain variable region linked by a flexible linker to form a single polypeptide chain having an antigen-binding region. Exemplary single-chain antibodies are described in detail in WO88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203, which are hereby incorporated by reference in their entirety. In certain instances, a single variable region (e.g., a heavy-chain variable region or a light-chain variable region) may have the ability to recognize and bind an antigen. Other antibody fragments will be understood by those of ordinary skill in the art.

[0109] As used herein, the term "peptide tag" refers to a peptide that can be attached (e.g., post-synthetically or during synthesis) or fused to another entity (e.g., a protein of interest, a molecule of interest, an interaction element, a co-localization element, etc.). A peptide tag may or may not be attached to another entity. As used herein, a peptide tag can typically form a bioluminescent complex with another peptide tag and a polypeptide under appropriate conditions. In embodiments where 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, or 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 can typically form a bioluminescent complex with a pair of peptide tags under appropriate conditions.

[0111] As used herein, the term "Oplophorus luciferase" ("OgLuc") refers to a luminescent polypeptide that is produced by or derived from Oplophorus gracilirostris, a deep-sea shrimp, and that has significant sequence identity, structural conservation, and / or functional activity with luciferases. In particular, the OgLuc polypeptide refers to a luminescent polypeptide having significant sequence identity, structural conservation, and / or functional activity with the mature 19 kDa subunit of the Oplophorus luciferase protein complex (e.g., lacking a signal sequence), e.g., SEQ ID NO: 1 (WT OgLuc) and 3 (NanoLuc) that includes 10 β-strands (β1, β2, β3, β4, β5, β6, β7, β8, β9, β10), and that utilizes a substrate such as coelenterazine or a coelenterazine derivative to produce luminescence.

[0112] As used herein, the term "β9-like peptide" refers to a peptide (or peptide tag) that includes significant sequence identity, structural conservation, and / or functional activity with the β (beta) 9-strand of the OgLuc polypeptide. In particular, a β9-like peptide is a peptide that can structurally complement an OgLuc polypeptide lacking the β9-strand and result in enhanced luminescence of the complex as compared to the OgLuc polypeptide in the absence of the β9-like peptide. Other "βX-like peptides" can be named similarly (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 includes significant sequence identity, structural conservation, and / or functional activity with the β (beta) 10-strand of the OgLuc polypeptide. In particular, a β10-like peptide is a peptide that can structurally complement an OgLuc polypeptide lacking the β10-strand and result in enhanced luminescence of the complex as compared to the OgLuc polypeptide in the absence of the β10-like peptide. Other "βX-like peptides" can be named similarly (e.g., β1-like, β2-like, β3-like, β4-like, β5-like, β6-like, β7-like, β8-like, β9-like).

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

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

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

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

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

[0119] As used herein, the term "LgTrip" corresponds to SEQ ID NO: 17 and refers to, for example, a combination of β9-like and β 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 that corresponds 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 di- or tripeptides) interact.

[0123] Experiments conducted during the development of embodiments herein have demonstrated that two small peptide components (e.g., a β10-like peptide and a β9-like peptide) and one polypeptide component (e.g., a β 1~8It is demonstrated that the three - molecule luciferase containing the -like polypeptide) aggregates to form a luminescent complex. Experiments conducted during the development of the embodiments herein further demonstrate the formation of bioluminescent complexes from up to five fragments of luciferase (or variants of such fragments), for example, polypeptide fragments (or variants thereof) and one or more peptide, dipeptide, or tripeptide fragments (or variants of such fragments).

[0124] The commercially available NANOLUC luciferase (Promega Corporation) contains 10 β (beta) strands (β1, β2, β3, β4, β5, β6, β7, β8, β9, β10). U.S. Patent No. 9,797,889, which is hereby incorporated by reference in its entirety, describes the development and use of a complementary system containing -like polypeptides and -like peptides (the actual polypeptide and peptide sequences in U.S. Patent No. 9,797,889 are different from the corresponding sequences in NANOLUC and wild - type native OgLuc). 1~9 -like polypeptides and 10 -like peptides.

[0125] In the experiments conducted during the development of the embodiments herein, the -like polypeptide was further divided by the removal of the β9 strand. The remaining portion ( 1~9 -like 1~8The polypeptide (hereinafter referred to as LgTrip 2098 in the present specification) has the sequence of SEQ ID NO: 17 or SEQ ID NO: 31 (with a His tag). Experiments were conducted 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 a high-affinity two-molecule system). Experiments demonstrated that LgTrip 2098 (SEQ ID NO: 17 or SEQ ID NO: 31 with a His tag), which was poorly expressed in E. coli, was unstable and sensitive to surface deactivation. During the development of the embodiments herein, experiments were carried out to develop artificial variants that exhibit one or more (e.g., all) of enhanced stability, enhanced expression, enhanced activity, enhanced molecular interactions, etc., and can be used in a system for reconstituting a bioluminescent complex with peptides corresponding to the β9 chain (e.g., a β9-like peptide such as SmTrip9 pep245; SEQ ID NO: 23) and the β10 chain (e.g., a β10-like peptide such as SmTrip10 pep86, HiBiT, SEQ ID NO: 25). The experiments conducted during the development of the embodiments herein demonstrate, for example, that LgTrip 3092 (SEQ ID NO: 19) or LgTrip 3546 (SEQ ID NO: 51) can form a luminescent complex 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 described 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) having enhanced properties for the reconstitution of luminescent complexes.

[0126] Additional experiments conducted during the development of the embodiments described herein were other polypeptide components (e.g., β 1~5 -like, β 1~6 -like, β 1~7 -like, etc.) and complementary peptides (e.g., β6-like, β7-like, β8-like, β9-like, β 10 -like), dipeptides (e.g., β 6~7 -like, β 7~8 -like, β 8~9 -like, β 9~10 -like), tripeptides (e.g., β 6~8 -like, β 7~9 -like, β 8~10 -like), polypeptides (e.g., β 6~10 -like, β 6~9 -like, β 7~10Demonstrate that a NANOLUC-based bioluminescent complex can be formed using constructs that include corresponding combinations (such as etc.). Experiments conducted during the development of the embodiments herein have demonstrated the formation of bioluminescent complexes from two or more (such as 2, 3, 4, 5, etc.) peptide and polypeptide components that collectively include the full length of a luciferase construct (such as a full-length luciferase polypeptide that includes sequence identity of 40% or more (such as 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) with SEQ ID NO: 788 or 789).

[0127] In some embodiments, compositions and methods are provided herein for the assembly of bioluminescent complexes from two peptide tags (such as a β9-like peptide (such as SmTrip9) and a β10-like peptide (such as SmTrip10)) and polypeptide components (such as a β 1~8 -like polypeptide (such as LgTrip)).

[0128] In some embodiments, polypeptide components (such as β 1~5 -like, β 1~6 -like, β 1~7 -like, or β 1~8 -like polypeptides) as well as complementary peptide(s) (such as β6-like, β7-like, β8-like, β9-like, β 10 -like), dipeptide(s) (such as β 6~7 -like, β 7~8 -like, β 8~9 -like, β 9~10 -like), tripeptide (such as β 6~8 -like, β 7~9 -like, β 8~10 -like), and / or polypeptide(s) (such as β 6~10 -like, β 6~9 -like, β 7~10 -like, etc.). Compositions and methods are provided herein for the assembly of bioluminescent complexes from these components.

[0129] In some embodiments, one or more (e.g., two, three, four, five, etc.) of the peptide tags and polypeptide components are not fragments of existing proteins (e.g., not sub-sequences that are structurally complementary to known polypeptide sequences). However, in other embodiments, one or more of the peptide tags and polypeptide components can be fragments of known or existing proteins, polypeptides, or peptides. In certain embodiments, the bioluminescence activity of the polypeptide component (of the bioluminescent complex) is enhanced (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 10 2 -fold, 10 3 -fold, 10 4 -fold, 10 5 -fold, 10 6times, or more). In some embodiments, for example, a peptide (peptide tag) / polypeptide element that can be assembled into a bioluminescent complex for 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.) is provided herein. In some embodiments, a peptide / dipeptide / tripeptide tag (e.g., β6-like, β7-like, β8-like, β9-like (e.g., SmTrip9), and / or β10-like peptide (e.g., SmTrip10), and / or their dipeptides and tripeptides) is fused to or otherwise linked to an interaction element. In certain embodiments, the peptide / dipeptide / tripeptide tag and the polypeptide component do not form a complete bioluminescent complex without the facilitation by the interaction between the interaction elements for the purpose of detecting / monitoring molecular interactions. However, the interaction (e.g., binding) of the interaction elements with each other (or with a target molecule or complex) facilitates the formation of the bioluminescent complex. In some embodiments, the bioluminescence signal from the bioluminescent complex (or the ability to generate such a signal in the presence of a substrate) functions as a reporter of the formation of the complex by the interaction elements. When an interaction complex is formed, a bioluminescent complex is formed and the bioluminescence signal is detected / measured / monitored (e.g., in the presence of a substrate). When an interaction complex cannot be formed (e.g., due to unfavorable conditions, unstable interactions between the interaction elements, incompatible interaction elements), no bioluminescent complex is formed and no bioluminescence signal is generated (e.g., in the presence of a substrate). In some embodiments, the bioluminescence signal from the bioluminescent complex (or the ability to generate such a signal in the presence of a substrate) functions as a reporter of the binding of the interaction element to a target. When binding to the target occurs, a bioluminescent complex is formed and the bioluminescence signal is detected / measured / monitored (e.g., in the presence of a substrate).When it is unable to bind to the target (for example, due to unfavorable conditions, an unstable interaction between the interacting element and the target, or the absence of the target), the bioluminescent complex is not formed and no bioluminescence signal is generated.

[0130] In certain embodiments, the interacting elements are two molecules of interest (e.g., a protein(s) of interest, a small molecule(s) of interest, etc.). For example, the assay can be performed 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) peptides, and / or their dipeptides and tripeptides) to detect the interaction between the two molecules of interest. When the molecules of interest interact (e.g., transiently, stably, etc.), the peptide / dipeptide / tripeptide tags are in proximity in a favorable conformation and a bioluminescent complex is formed between the peptide / dipeptide / tripeptide tag and the polypeptide component of the bioluminescent complex (and a bioluminescence signal is generated / detected (e.g., in the presence of a substrate)). When the molecules of interest do not interact, the peptide / dipeptide / tripeptide tags are not in proximity and / or are not arranged in an orientation that promotes complex formation with the polypeptide component of the bioluminescent complex, the bioluminescent complex is not formed, and no bioluminescence signal is generated (in the presence of a substrate). Such embodiments can be used to study the effect of an inhibitor on complex formation, the effect of a mutation on complex formation, the effect of conditions (e.g., temperature, pH, etc.) on complex formation, the interaction of a small molecule (e.g., a therapeutic candidate) with a target molecule, 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 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) that can assemble into a bioluminescent complex without the facilitation of interaction elements are provided. In such embodiments, when the peptide / dipeptide / tripeptide tags and polypeptide components are present (e.g., co-localize) simultaneously within the same sample, within an intracellular compartment, intracellularly, within a tissue, etc., a bioluminescent complex is formed. In some embodiments, peptide / dipeptide / tripeptide (tag) / polypeptide elements are provided herein for use in detecting and monitoring the co-localization of molecular elements (e.g., protein(s), nucleic acid(s), small molecule(s), lipid, carbohydrate, cell structure, etc.) (e.g., without molecular interactions). In some embodiments, the bioluminescent complex is formed from peptide / dipeptide / tripeptide tags and polypeptide components that collectively span the entire β1-like to β10-like sequences. In some embodiments, the peptide / dipeptide / tripeptide tags are fused or otherwise linked to the co-localizing element. In certain embodiments, particularly for detecting / monitoring co-localization (e.g., without molecular interactions), the peptide / dipeptide / tripeptide tags and polypeptide components can form a bioluminescent complex without facilitation (e.g., without interaction elements). Upon co-localization of the co-localizing element (e.g., fused to the peptide / dipeptide / tripeptide tag) (e.g., within the same cell, on the same surface, within the same intracellular compartment, within the same tissue, etc.), the formation of the bioluminescent complex (from the peptide / dipeptide / tripeptide tag and polypeptide component) is facilitated regardless of the presence or absence of interaction of the co-localizing element. In some embodiments, the bioluminescence signal from the bioluminescent complex (or the ability to generate such a signal in the presence of a substrate) functions as a reporter of the co-localization of the co-localizing element.When the co-localized elements are co-localized, a bioluminescent complex of the polypeptide component and the peptide / dipeptide / tripeptide tag fused to the co-localized element is formed, and the bioluminescent signal is detected / measured / monitored (e.g., in the presence of a substrate). When the co-localized elements are not co-localized, no bioluminescent complex is formed and no bioluminescent signal is generated (e.g., in the presence of a substrate).

[0132] In certain embodiments, the co-localized pair includes two molecules of interest (e.g., a protein(s) of interest, a small molecule(s) of interest, etc.). For example, the assay can be performed by tethering each to a separate 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) to detect co-localization of the two molecules of interest (e.g., within a cell, within a cellular compartment, within a tissue, etc.). When the molecules of interest are co-localized, the peptide tags are in proximity in a favorable conformation and a bioluminescent complex is formed with the polypeptide component (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). When the molecules of interest are not co-localized, the polypeptide component and the peptide / dipeptide / tripeptide tag do not interact to form a complex and no bioluminescent signal is generated (e.g., in the presence of a substrate). Such embodiments can be used to study co-localization of molecules of interest under various conditions.

[0133] In some embodiments, provided are systems, assays, and devices 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 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) 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, a detection / binding agent (e.g., a binding moiety, binding sequence, etc.) that recognizes a secondary analyte bound by the target analyte, a secondary binding agent that binds to the primary binding agent, etc. In some embodiments, various combinations of peptide / dipeptide / tripeptide tags tethered / fused to the detection / binding agents described above are used in assays and devices for detecting / quantifying / identifying analytes in a sample. Exemplary systems that are useful in the assays and devices are shown, for example, in FIGS. 51-56 and described herein.

[0134] In some embodiments, provided herein are compositions and methods for the assembly of bioluminescent complexes from dipeptides (e.g., β9 / β10-like dipeptides) and polypeptide components (e.g., β 1~8 -like (e.g., LgTrip) polypeptides). In some embodiments, the dipeptide and polypeptide components are not fragments of existing proteins (e.g., not sub-sequences that are structurally complementary to 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 bioluminescence activity of the polypeptide component (of the bioluminescent complex) is enhanced (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 10 2 -fold, 10 3 -fold, 10 4 -fold, 10 5times, 10 6 times, or more). In some embodiments, β 1~8 -like polypeptide exhibits a background luminescence weaker than that of the β 1~9 -like polypeptide. In some embodiments, the β 1~8 -like polypeptide exhibits increased thermal and chemical stability as compared to the β 1~9 -like polypeptide.

[0135] In some embodiments, provided herein are bioluminescent complexes that include, but are not limited to, 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 tripeptide + 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 + 8 / 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 peptides, dipeptides, tripeptides, and polypeptide components are within the scope of this specification.

[0136] In some embodiments, the β1-5-like polypeptide comprises positions 1-102 of SEQ ID NO: 788. In some embodiments, the β1-6-like polypeptide comprises positions 1-124 of SEQ ID NO: 788. In some embodiments, the β1-7-like polypeptide comprises positions 1-133 of SEQ ID NO: 788. In some embodiments, the 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 the bioluminescent complex span partial beta strands of the base luciferases 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 seen at the division point between 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 that span the entire sequence of the base luciferases described herein (e.g., OgLuc, NANOLUC, SEQ ID NO: 788, SEQ ID NO: 789, etc.) can form a bioluminescent complex even when the division point of the components is not between beta strands.

[0139] For example, the cleavage position between β5 and β6 may be between positions 102 and 103 of SEQ ID NO: 788, or in some embodiments, such cleavage position may be at positions up to 5 residues before and after that position (e.g., after positions 96, 97, 98, 99, 100, 101, 103, 104, 105, 106, 107). In some embodiments, the cleavage position between β6 and β7 may be between positions 124 and 125 of SEQ ID NO: 788, or in some embodiments, such cleavage position may be at positions up to 5 residues before and after that position (e.g., after positions 118, 119, 120, 121, 122, 123, 125, 126, 127, 128, 129). In some embodiments, the cleavage position between β7 and β8 may be between positions 133 and 134 of SEQ ID NO: 788, or in some embodiments, such cleavage position may be at positions up to 5 residues before and after that position (e.g., after positions 127, 128, 129, 130, 131, 132, 134, 135, 136, 137, 138). In some embodiments, the cleavage position between β8 and β9 may be between positions 148 and 149 of SEQ ID NO: 788, or in some embodiments, such cleavage position may be at positions up to 5 residues before and after that position (e.g., after positions 142, 143, 144, 145, 146, 147, 149, 150, 151, 152, 153).

[0140] In some embodiments, two consecutive 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.) include all of the 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 cleavage point in the base sequence are not present in the corresponding peptide, dipeptide, tripeptide, and / or polypeptide component.

[0141] In some embodiments, peptides are provided herein that 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 the following: ● β6-like - GVTPNKLNYFGRPYEGIAVFDG (SEQ ID NO: 802), ● β7-like - KKITTTGTL (SEQ ID NO: 803), ● β8-like - WNGNKIIDERLITPD (SEQ ID NO: 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, peptides are provided herein that 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 the following: ● β6 / 7-like - GVTPNKLNYFGRPYEGIAVFDGKKITTTGTL (SEQ ID NO: 808), ● β7 / 8-like - KKITTTGTLWNGNKIIDERLITPD (SEQ ID NO: 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, tripeptides are provided herein that 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 the following: ● β6 / 7 / 8-like - GVTPNKLNYFGRPYEGIAVFDGKKITTTGTLWNGNKIIDERLITPD (SEQ ID NO: 813), ● β7 / 8 / 9-like - KKITTTGTLWNGNKIIDERLITPDGSMLFRVTINS (SEQ ID NO: 814), ● Β8 / 9 / 10-like (high affinity) - WNGNKIIDERLITPDGSMLFRVTINSVSGWRLFKKIS (SEQ ID NO: 815), and ● Β8 / 9 / 10-like (low affinity) - WNGNKIIDERLITPDGSMLFRVTINSVTGYRLFEEIL (SEQ ID NO: 816).

[0144] In some embodiments, polypeptides are provided herein that include at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) sequence identity with 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 (such as, for example, a set 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 with one of SEQ ID NOs: 788, 789, 790, 791, 792, and 793.

[0146] In some embodiments, a peptide / dipeptide / tripeptide component (such as, for example, a set of peptides / polypeptides or a bioluminescent complex) (e.g., a tag) collectively comprises 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, a set of components and complexes of the peptides, dipeptides, tripeptides, and polypeptides listed above are provided herein. In certain embodiments, a set of components spanning all 10 beta strands of the underlying luciferase sequence is selected.

[0148] In some embodiments, the systems, interactions, co-localizations, detections, and other methods, assays, and techniques described herein for use with two peptide tags (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 for use with the dipeptide systems (e.g., β9 / 10-like dipeptides and polypeptide components) described herein. In some embodiments, the dipeptide has a high affinity for the polypeptide component, and in such embodiments, when the dipeptide and the polypeptide component come into contact without facilitation (e.g., co-localize, are added to a sample, etc.), a bioluminescent complex is formed. In some embodiments, the dipeptide has a low affinity for the polypeptide component, and in such embodiments, when the dipeptide and the polypeptide component come into contact without facilitation (e.g., co-localize, are added to a sample, etc.), a bioluminescent complex is not formed. Dipeptide / polypeptide pairs of various affinities, such as the systems of two peptide tags (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)) described herein, can be selected for different uses. In some embodiments, systems, methods, and assays for complementary systems of two components are described in U.S. Patent No. 9,797,890, which is hereby incorporated 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 interactions, co-localizations, detections, and other methods, assays, and techniques described herein for use with a system of two peptide tags (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)) are also useful for use with a system comprising any suitable combination of the peptides, dipeptides, tripeptides, and polypeptides described herein. In some embodiments, the components have high affinity for each other, and in such embodiments, when the components contact each other without facilitation (e.g., co-localize, are added to a sample, etc.), a bioluminescent complex is formed. In some embodiments, one or more of the components have low affinity for one or more of the other components, and in such embodiments, when the components contact each other without facilitation (e.g., co-localize, are added to a sample, etc.), a bioluminescent complex is not formed. Other systems having various affinities as described herein, such as the system of two peptide tags 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)), can be provided for different uses. In some embodiments, systems, methods, and assays for a complementary system of two components are described in U.S. Patent No. 9,797,890, which is 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, a complementary panel of compatible 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) is provided herein, which has variable affinities and luminescence due to the formation of bioluminescent complexes therefrom (e.g., high affinity / high luminescence, medium affinity / high luminescence, low affinity / medium luminescence, etc.). By utilizing different combinations of peptide / dipeptide / tripeptide tags and polypeptide components, an adaptable system is provided that includes various sets ranging from low affinity, luminescence, expression level, stability, solubility, and other variable properties to higher affinity, luminescence, expression level, 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 finely tuned to the particular molecule(s) and / or conditions of interest being studied, and extends the range of molecular interactions and / or co-localizations that can be detected / monitored / identified / quantified to include interactions with very high or very low affinities. Also 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 herein (e.g., peptide tags) (e.g., as compared to larger polypeptides and proteins), they exhibit resistance to denaturation (they do not have the tertiary structure required for function).

[0152] In some embodiments, the peptide / dipeptide / tripeptide tag and the polypeptide component 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 interaction elements) of different strengths, durations, and / or stabilities to result in the formation of a bioluminescent complex. In some embodiments, a very stable interaction complex is required to generate a detectable bioluminescent signal (e.g., in the presence of a substrate). In other embodiments, even a weak or transient interaction complex results in the formation of a bioluminescent complex. In still other embodiments, the bioluminescent complex is formed in the absence of an interaction complex as long as the peptide / dipeptide / tripeptide tag and the polypeptide component are co-localized. In some embodiments, the strength or degree of the interaction complex is directly proportional to the strength of the resulting bioluminescent signal. Some combinations of peptide / dipeptide / tripeptide tags / polypeptide components produce a detectable signal when combined with an interaction complex with a high millimolar dissociation constant (e.g., K d > 100 mM). Other combinations of peptide / dipeptide / tripeptide tags / polypeptide components require an interaction pair with a low millimolar (e.g., K d < 100 mM), micromolar (e.g., K d < 1 mM), nanomolar (e.g., K d < 1 μM), or even picomolar (e.g., K d < 1 nM) dissociation constant to produce a bioluminescent complex with a detectable signal.

[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, none of the peptide / dipeptide / tripeptide tags and 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, peptide tags (e.g., β9-like (e.g., SmTrip9) and β10-like (e.g., SmTrip10) peptides; β9 / β10-like dipeptides, etc.) that assemble with each other to form a bioluminescent complex are also polypeptide components (e.g., β 1~8 -like (e.g., LgTrip) polypeptides) are not fragments of existing proteins (e.g., the same existing protein, an existing bioluminescent protein, etc.). In some embodiments, the peptide / dipeptide / tripeptide tags or polypeptide components of the bioluminescent complex for use in embodiments of the present invention are not partial sequences of existing proteins. In some embodiments, the non-luminescent elements for use in the embodiments described herein do not contain structurally complementary partial sequences of existing proteins.

[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 their dipeptides and tripeptides) are non-luminescent or substantially non-luminescent alone (e.g., in the presence or absence of a substrate). In some embodiments, the peptide / dipeptide / tripeptide tags herein are non-luminescent or substantially non-luminescent (e.g., in the presence or absence of a substrate) when they associate with each other in the absence of 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 are non-luminescent or substantially non-luminescent alone (e.g., in the presence or absence of a substrate). In some embodiments, a single 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 their dipeptides and tripeptides) and a polypeptide component are non-luminescent or substantially non-luminescent (e.g., in the presence or absence of a substrate) in the absence of a second or third or fourth peptide / dipeptide / tripeptide tag. 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 and generate a bioluminescence signal in the presence of a substrate.

[0155] In certain embodiments, the interaction element and / or co-localization element and the peptide / dipeptide / tripeptide tag are attached, fused, linked, conjugated, etc. In typical embodiments, the first peptide / dipeptide / tripeptide tag and the first interaction element (or the first co-localization element) are attached to each other, and the second peptide / dipeptide / tripeptide tag and the second interaction element (or the second co-localization element) are attached to each other. Attachment of the peptide / dipeptide / tripeptide tag to the interaction element (or co-localization element) can be achieved by any suitable mechanism, chemistry, linker, etc. The interaction element (or co-localization 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 interaction element (or co-localization element) are directly conjugated, and in other embodiments, they are conjugated by a linker. In some embodiments, the peptide / dipeptide / tripeptide tag and the interaction element (or co-localization element) are provided as a gene / recombinant fusion. In some embodiments, endogenous tagging with the peptide / dipeptide / tripeptide tags herein (e.g., under endogenous control regulation) enables monitoring of normal cellular functions with 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 a variant thereof) is monitored in cells, animals, lysates, etc.In other embodiments, the peptide tag and the interaction element (or co-localization element) are linked by chemical modification / chemical conjugation, such as native chemical ligation, Staudinger ligation, "traceless" Staudinger ligation, amide coupling, methods using activated esters, methods targeting lysine, tyrosine and cysteine residues, imine bond formation (with or without orthoboric acid), boronic acid / diol interaction, disulfide bond formation, copper-using / copper-free azide, diazo, tetrazine "click" chemistry, UV-promoted thiol-ene conjugation, photo-labeling with diazirine, Diels-Alder cycloaddition, metathesis reaction, Suzuki cross-coupling, thiazolidine coupling (step-4), streptavidin / biotin complementation, HaloTag / chloroalkane substrate complementation, etc. In some embodiments, the peptide / dipeptide / tripeptide tag and the interaction element (or co-localization element) are generated synthetically (e.g., solid-phase synthesis, liquid-phase synthesis, etc.). In some embodiments, the interaction element (or co-localization element) is generated by any suitable means (e.g., synthetically or recombinantly) or obtained (e.g., from crude lysates, extracted proteins, purified proteins, etc.).

[0156] In some embodiments where the interacting element (or co-localizing 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-localizing 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-localizing 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-localizing element), optionally one or more N-terminal sequences, C-terminal sequences, regulatory elements (e.g., promoter, translation initiation site, etc.), and linker sequences. In some embodiments, the peptide / dipeptide / tripeptide tag and the interacting element (or co-localizing element) are contained within a fusion polypeptide. In some embodiments, a first fusion of the peptide / dipeptide / tripeptide tag and the interacting element (or co-localizing element) and a second fusion of the peptide / dipeptide / tripeptide tag and the interacting element (or co-localizing element) are expressed separately, while in other embodiments, a fusion protein comprising or consisting of both the interaction (or co-localization) and the peptide / dipeptide / tripeptide tag is expressed.

[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 their dipeptides and tripeptides) and a first interaction 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 their dipeptides and tripeptides) and a second interaction 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 the cells. In some embodiments, the interaction and / or co-localization of the fusion proteins is analyzed in the cell. In some embodiments, the interaction and / or co-localization of the fusion proteins is analyzed in the 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 the cells or parts of the cells, by transfer of the fusion protein from one cell to the other, or by secretion of one or more fusion proteins into the extracellular medium of the cells). 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 cytopathic agent.In some embodiments, the polypeptide components (e.g., β1-5-like, β1-6-like, β1-7-like, β1-8-like (e.g., LgTrip) polypeptides) for complex formation (with the first and second fusion proteins) 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 their dipeptides and tripeptides) are expressed in the same cell or cell lysate as one or both of the fusion proteins containing the tags. In some embodiments, the peptide / dipeptide / tripeptide / polypeptide components for complex formation with the peptide / dipeptide / tripeptide tags (within the first and second fusion proteins) are expressed in a cell or cell lysate different from one or both of the fusion proteins containing the peptide tags. In some embodiments, the peptide / dipeptide / tripeptide / polypeptide components for complex formation with the peptide / dipeptide / tripeptide tags (within the first and second fusion proteins) are added to a cell, cell lysate, or other sample containing the fusion protein 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 the analysis of samples (e.g., co-localization, molecular interactions, target detection / quantification / identification / monitoring, etc.). In some embodiments, one or more of the components of the systems herein are added and / or provided to a sample or expressed within the sample. Suitable samples useful in the embodiments herein include, but are not limited to: blood, plasma, serum, urine, saliva, cells, cell lysates, tissues, tissue homogenates, purified nucleic acids, feces, vaginal secretions, cerebrospinal fluid, allantoic fluid, water, biofilms, soil, dust, food, beverages, agricultural products, plants, etc.

[0159] In certain embodiments, provided are nucleic acids, DNAs, RNAs, vectors, etc. that encode 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), fusion polypeptides, fusion proteins, etc. Such nucleic acids and vectors can be used for expression, transformation, gene introduction, injection, etc.

[0160] In some embodiments, 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 their dipeptides and tripeptides) and an interaction element, a co-localization element, or a binding agent are linked by a linker. In some embodiments, the linker binds a signal element and an interaction element or a co-localization element and provides a desired amount of space / distance between the elements. In some embodiments, the linker enables both the signal element and the interaction element to simultaneously form their respective complexes (e.g., a luminescent complex and an interaction complex). In some embodiments, the linker assists the interaction element in facilitating the formation of the luminescent complex. In some embodiments, when the interaction complex is formed, the linker that binds each peptide / dipeptide / tripeptide tag to its respective interaction element positions the peptide tag at an appropriate distance and conformation to form a bioluminescent complex. In some embodiments, the interaction element or co-localization element and the peptide / dipeptide / tripeptide tag are held proximally by the linker (e.g., less than 4 monomer units). 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 interaction element (e.g., to prevent an undesired interaction between the peptide / dipeptide / tripeptide tag and the interaction element or co-localization element due to steric issues, to enable an appropriate orientation of non-luminescent elements during the formation of the interaction complex, to enable the propagation of complex formation from the interaction complex to the luminescent complex, etc.). In certain embodiments, the linker provides an appropriate attachment chemistry between the peptide / dipeptide / tripeptide tag and the interaction element. The linker may improve the synthetic process for manufacturing the peptide / dipeptide / tripeptide tag and the interaction element or co-localization element (e.g., enabling them to be synthesized as a single unit, enabling the binding after the synthesis of the two elements, etc.).

[0161] In some embodiments, the linker is any suitable chemical moiety that can link, bind, or tether 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 their dipeptides and tripeptides) or a polypeptide component (e.g., β1-5-like, β1-6-like, β1-7-like, β1-8-like (e.g., LgTrip) polypeptides) to an interaction element or a co-localization element. In some embodiments, the linker is a polymer of one or more repeating or non-repeating monomer units (e.g., a nucleic acid strand, an amino acid strand, a carbon-containing polymer strand, a carbon strand, etc.). When the peptide / dipeptide / tripeptide tag and the interaction element, co-localization element, or binding agent are part of a fusion protein, the linker (if present) is typically an amino acid strand. When the peptide / dipeptide / tripeptide tag and the interaction element, co-localization element, or binding agent are tethered to each other after expression of the individual elements, the linker can include any chemical moiety having a functional group (or reactive group) that is reactive with the functional groups of the peptide tag and the interaction element or co-localization element, respectively, at either end. A signal element, as well as any suitable moiety that can tether an interaction element, co-localization element, and / or binding agent, can be useful as a linker.

[0162] A 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 from 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 from 1 to 50 non-hydrogen atoms (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) selected from the group consisting of C, N, P, O, and S (in addition to hydrogen atoms).

[0163] The scope of the embodiments herein is not limited by the types of linkers available. Peptide / dipeptide / tripeptide tags, polypeptide components, and interacting, co-localizing, or binding agents are either directly linked (e.g., a linker consisting of a single covalent bond) or linked via a suitable linker. 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 PEG, polysaccharides, glycosaminoglycans, dendrimers (WO93 / 06868 and Angew. Chem. Int. Ed. Engl. 29:138-175 (1990) by Tomalia et al., which are incorporated herein by reference in their entirety), PEG-chelate agent polymers (W94 / 08629, WO94 / 09056, and WO96 / 26754, which are incorporated herein by reference in their entirety), oligonucleotide linkers, phospholipid derivatives, alkenyl chains, alkynyl chains, disulfides, or combinations thereof. In some embodiments, the linker is cleavable (e.g., enzymatically (e.g., TEV protease site), chemically, photoinducible, 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 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), recognition elements, interaction elements, co-localization elements, binding agents, analytes, substrates, etc. are attached to or contained within a solid surface or matrix (e.g., by any suitable chemical action). In some embodiments, one or more system components are attached to or contained within a solid surface or matrix (e.g., by any suitable chemical action), and 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 is made of any suitable material, e.g., Ahlstrom CytoSep, nitrocellulose, cellulose acetate, cellulose (e.g., Whatman FTA-DMPK-A, B, and C cards; Whatman ET 3 / Chr; Whatman Protein Saver 903 card; Whatman grade 1 filter paper; Whatman FTA Elute;Made from Ahlstrom 226 specimen collection paper, Noviplex Plasma Prep Cards, polypropylene membrane, PVDF, nitrocellulose membrane (Millipore Nitrocellular Hi Flow Plus), polytetrafluoroethylene membrane, cellulose mixed ester, glass fiber media (e.g., Whatman Unifilter plate, glass fiber filter membrane, Agilent dry matrix spotting card, Ahlstrom grade 8950, etc.), plastics (e.g., polyester, polypropylene, polyethersulfone, poly(methacrylate), acrylic polymer, polytetrafluoroethylene, etc.), natural and synthetic polymers (e.g., polymer mixtures, block copolymers, etc.), sugars (e.g., pullulan, trehalose, maltose, sucrose, cellulose, etc.), polyamides (e.g., natural (e.g., wool, silk, etc.), synthetic (e.g., aramid, nylon, etc.), 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, Almel, duralumin, magnox, Zamak, etc.), iron alloys (e.g., steel, stainless steel, surgical stainless steel, silicon steel, tool steel, cast iron, specular iron, etc.), cobalt alloys (e.g., stellite, talonite, etc.), nickel alloys (e.g., German silver, chromel, mu-metal, Monel metal, nichrome, Nicrosil, Nisil, nitinol, etc.), copper alloys (e.g., beryllium copper, biron, brass, bronze, phosphor bronze, constantan, cupronickel, bell metal, Devarda alloy, gold plating alloy, white metal, Nordic Gold, king gold, tumbaga, etc.), silver alloys (e.g., sterling silver, etc.), tin alloys (e.g., Britannium, pewter, solder, etc.), gold alloys (electrum, white gold, etc.), amalgams, etc.), 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 their dipeptides and tripeptides) and polypeptide components of the luminescence complex having less than 100% sequence identity and / or similarity to any portion of an existing luciferase (e.g., firefly luciferase, Renilla luciferase, Oplophorus luciferase, highly sensitive Oplophorus luciferase as described in U.S. Patent Application No. 2010 / 0281552 and U.S. Patent Application No. 2012 / 0174242, which are hereby incorporated by reference in their entirety) are provided. Certain embodiments involve the formation of a bioluminescent complex of peptide / dipeptide / tripeptide tags and polypeptide components having less than 100% sequence identity with the whole or a part (e.g., 8 or more amino acids, less than about 25 amino acids for a peptide) of SEQ ID NO: 1 (e.g., the full sequence of wild-type Oplophorus luciferase) and / or SEQ ID NO: 3 (e.g., the full sequence of NANOLUC). Certain embodiments involve the formation of a bioluminescent complex from peptide / dipeptide / tripeptide tags and polypeptide components having 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 with the whole or a part (e.g., 8 or more amino acids, less than about 25 amino acids for a peptide) of SEQ ID NO: 1 (e.g., the full sequence of wild-type Oplophorus luciferase) and / or SEQ ID NO: 3 (e.g., the full sequence of NANOLUC). In some embodiments, peptide / dipeptide / tripeptide tags and polypeptide components having less than 100% sequence similarity with a part (e.g., 8 or more amino acids, less than about 25 amino acids for a peptide) of SEQ ID NO: 1 (e.g., the full sequence of wild-type Oplophorus luciferase) and / or SEQ ID NO: 3 (e.g., the full sequence of NANOLUC) are provided.In some embodiments, there are provided peptide / dipeptide / tripeptide tags and polypeptide components having 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 of SEQ ID NO: 1 (e.g., the full sequence of wild-type Oplophorus luciferase) and / or SEQ ID NO: 3 (e.g., the full sequence of NANOLUC) (e.g., 8 or more amino acids, less than about 25 amino acids for a peptide). In some embodiments, there are provided peptide / dipeptide / tripeptide tags having less than 100% sequence identity and / or similarity to a portion of SEQ ID NO: 1 (e.g., the full sequence of wild-type Oplophorus luciferase) and / or SEQ ID NO: 3 (e.g., the full sequence of NANOLUC) that is about 25 amino acids or less, wherein two of such peptides, when combined under appropriate conditions (e.g., stabilized by an interaction pair, brought into proximity by a co-localization element, etc.), form a bioluminescent complex with a polypeptide component having 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 full sequence of wild-type Oplophorus luciferase) and / or SEQ ID NO: 3 (e.g., the full sequence of NANOLUC).In some embodiments, provided are peptide / dipeptide / tripeptide tags having less than 100% sequence identity and / or similarity with a portion of about 25 amino acids or less of SEQ ID NO: 1 (e.g., the full sequence of wild-type Oplophorus luciferase) and / or SEQ ID NO: 3 (e.g., the full sequence of NANOLUC), wherein a pair of such peptide tags, when combined under appropriate conditions (e.g., stabilized by an interaction pair, brought into proximity by a co-localization element, etc.), forms a bioluminescent complex with a polypeptide component having 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 with another portion of SEQ ID NO: 1 (e.g., the full sequence of wild-type Oplophorus luciferase) and / or SEQ ID NO: 3 (e.g., the full sequence of NANOLUC). In some embodiments, provided are peptide / dipeptide / tripeptide tags having 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 with a portion of about 25 amino acids or less of SEQ ID NO: 1 (e.g., the full sequence of wild-type Oplophorus luciferase) and / or SEQ ID NO: 3 (e.g., the full sequence of NANOLUC), wherein a pair of such peptides, when combined under appropriate conditions (e.g., stabilized by an interaction pair, brought into proximity by a co-localization element, etc.), forms a bioluminescent complex with a polypeptide having 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 with another portion of SEQ ID NO: 1 (e.g., the full sequence of wild-type Oplophorus luciferase) and / or SEQ ID NO: 3 (e.g., the full sequence of NANOLUC).Similarly, provided are polypeptide components having 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 or similarity to a part of SEQ ID NO: 1 (e.g., the full sequence of wild-type Oplophorus luciferase) and / or SEQ ID NO: 3 (e.g., the full sequence of NANOLUC), wherein such polypeptide components, when combined under appropriate conditions, form a bioluminescent complex with a pair of peptide tags having less than 100% but optionally 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 ...

Claims

**Claim 1** A system or kit comprising two or more peptide and / or polypeptide components collectively having at least 90% sequence identity to SEQ ID NO: 788 or SEQ ID NO: 789, wherein the two or more peptide and / or polypeptide components are capable of forming a bioluminescent complex when interacting with each other, in the presence of coelenterazine or a coelenterazine analog substrate, the bioluminescence signal generated by the bioluminescent complex is substantially increased compared to the bioluminescence signals generated by the two or more peptide and / or polypeptide components and the coelenterazine or coelenterazine analog substrate alone, wherein the two or more peptide and / or polypeptide components structurally correspond to 8 or fewer β-strands of Oplophorus gracilirostris luciferase, wherein the coelenterazine analog is selected from the group consisting of furimazine, 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, said system or kit. **Claim 2** The system or kit according to claim 1, wherein the two or more peptide and / or polypeptide components have at least 90% sequence identity to SEQ ID NO: 790 or SEQ ID NO:

794. **Claim 3** The system or kit according to claim 1, wherein the two or more peptide and / or polypeptide components have at least 90% sequence identity to SEQ ID NO: 791 or SEQ ID NO:

795. **Claim 4** The system or kit according to claim 1, wherein the two or more peptide and / or polypeptide components have at least 90% sequence identity to SEQ ID NO: 792 or SEQ ID NO:

796. **Claim 5** The system or kit according to claim 1, wherein the two or more peptide and / or polypeptide components have at least 90% sequence identity to SEQ ID NO: 793 or SEQ ID NO:

797. **Claim 6** The system or kit according to claim 1, wherein the two or more peptide and / or polypeptide components have at least 90% sequence identity to SEQ ID NO: 790 or SEQ ID NO:

798.

7. The system or kit according to claim 1, wherein the two or more peptide and / or polypeptide components have at least 90% sequence identity to SEQ ID NO: 791 or SEQ ID NO:

799.

8. The system or kit according to claim 1, wherein the two or more peptide and / or polypeptide components have at least 90% sequence identity to SEQ ID NO: 792 or SEQ ID NO:

800.

9. The system or kit according to claim 1, wherein the two or more peptide and / or polypeptide components have at least 90% sequence identity to SEQ ID NO: 793 or SEQ ID NO:

801.

10. The system or kit according to claim 1, wherein the two or more peptide and / or polypeptide components comprise non-natural amino acids, amino acid analogs, and / or peptidomimetic amino acids.

11. The system or kit according to claim 1, wherein the two or more peptide and / or polypeptide components exist as a fusion with one or more additional amino acid sequences.

12. The system or kit according to claim 11, wherein the additional amino acid sequence is selected from the group consisting of a protein of interest, an interaction element, a co-localization element, and a binding moiety.

13. The system or kit according to claim 11, 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.

14. The system or kit according to claim 11, wherein the additional amino acid sequence is the first interaction polypeptide configured to form a complex with the second interaction polypeptide upon contact between the first interaction polypeptide and the second interaction polypeptide.

15. The system or kit according to claim 11, wherein the additional amino acid sequence is the first co-localization polypeptide configured to co-localize with a second co-localization polypeptide within an intracellular compartment, cell, tissue, or organism.

16. The system or kit according to claim 11, wherein the additional amino acid sequence is a protein of interest and a drug target candidate.

17. A bioluminescent complex comprising the two or more peptide and / or polypeptide components of the system or kit according to claim 1.

18. A method comprising the step of contacting the bioluminescent complex according to claim 17 with a substrate of the bioluminescent complex.

19. The method according to claim 18, wherein the substrate is coelenterazine or furimazine.

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