Complementary tags and reporters for dual modality labeling

Abstract

This specification provides compositions and systems comprising complementary tags and reporters for labeling and detecting targets by luminescence and a second mode (e.g., fluorescence), as well as methods for using them. In particular, a tag comprising a fusion of a first component of a bioluminescent complex and a first component of a modified dehalogenase complex is provided; a reporter comprising a second component of a bioluminescent complex and a second component of a modified dehalogenase complex is provided; and systems and methods comprising the tags and reporters described herein for dual-mode labeling and target detection are provided.

Description

Technical Field

[0001] This specification provides compositions and systems comprising complementary tags and reporters for labeling and detecting targets by luminescence and a second mode (e.g., fluorescence), and methods of using them. In particular, tags are provided that include a fusion of a first component of a bioluminescence complex and a first component of a modified dehalogenase complex, reporters are provided that include a second component of a bioluminescence complex and a second component of a modified dehalogenase complex, and systems and methods are provided that include the tags and reporters of this specification for dual-mode labeling and target detection.

[0002] Cross - reference to related applications This application claims the benefit of priority based on U.S. Patent Provisional Application No. 63 / 500,118, filed May 4, 2023, which is incorporated herein by reference in its entirety.

[0003] Sequence listing The text of the computer - readable sequence listing (title "PRMG_41898_601_SequenceListing.xml", created on May 3, 2024, file size 4,666,369 bytes) submitted with this specification is incorporated herein by reference in its entirety.

Background Art

[0004] Reporter technologies based on the complementation of split protein sequences have been demonstrated to be useful in a wide range of biomedical applications. In their most common configuration, these reporters function by producing little or no signal generation initially under conditions where their complementary sequences are kept separate or where their interactions are unfavorable. When conditions change to allow interactions such as chemical induction, physical proximity, changes in concentration, or affinity, the sequences interact with each other, fold into an active protein complex, and generate a signal measurable, for example, in the form of luminescence or fluorescence.

[0005] The utility of split protein complementation systems as research tools has been demonstrated, among other examples, in their applications to biosensing, diagnostics, drug discovery, target molecule deconvolution, cellular and molecular imaging, and the detection of energy transfer (BRET, FRET). The functions of these research tools can be directed towards specific molecular events by fusing split protein sequences to intracellular proteins, providing a simple and robust diversity in the methods and designs of their applications. The emergence of CRISPR genome editing technology has further increased the value of small (<35 amino acids) split protein sequences for use as fusion tags, as their small size enables efficient integration into the genome and reduces the likelihood of interfering with the function of target protein fusion partners.

[0006] Several examples of small-sequence asymmetric split protein reporters have been reported, including fluorescent proteins (GFP, RFP), luciferases (NanoLuc), and self-labeling proteins (HaloTag, SNAP-tag). It has been demonstrated that introducing these reporters as fusions into endogenous genes using CRISPR-based genome editing yields many of the functional capabilities outlined above. However, despite the usefulness and diversity of these split protein tags, users must select a single detection method (e.g., fluorescence or luminescence) and then focus on generating assays or modified cell lines limited to the advantages and disadvantages of the chosen detection type. For example, luminescence-based detection using NanoLuc binary technology (NANOBIT), while possessing exceptional sensitivity and linearity, is not widely adopted in cell imaging or flow cytometry due to the difficulty of luminescence detection with conventional microscopes or cytometry detectors. Additional applications where fluorescence is preferred over bioluminescence for imaging include: single-molecule tracking, localization, fixation, single-cell / intracellular resolution, and co-localization experiments using multiple colors. In contrast, small split fluorescent protein tags can be used in fluorescence microscopy or cytometry, but they are often limited in terms of the dynamic range and available emission wavelengths in measurements.

[0007] What is needed is a single split-protein technology that provides the use of both luminescence and fluorescence for the detection of complementarity between the tag and the complementary reporter sequence. Such a system would allow users to measure the functional dynamics of the target protein under conditions that minimize disruption while realizing the advantages of both detection methods. [Overview of the project]

[0008] This specification provides compositions and systems comprising complementary tags and reporters for labeling and detecting targets by luminescence and a second mode (e.g., fluorescence), as well as methods for using them. In particular, tags comprising a fusion of a first component of a bioluminescent complex and a first component of a modified dehalogenase complex are provided, reporters comprising a second component of a bioluminescent complex and a second component of a modified dehalogenase complex are provided, and systems and methods comprising the tags and reporters herein for dual-mode labeling and target detection are provided. In some embodiments, this specification provides a dual-luminescence-fluorescence detection technique based on the fusion of split NanoLuc® (LgBit and SmBiT / HiBiT) and split HaloTag® (LgHT and SmHT). Embodiments of such configurations maintain (1) a single miniature tag comprising a fusion between an SmHT sequence and an SmBiT / HiBiT sequence, and (2) a complementary reporter polypeptide comprising a fusion between an LgHT sequence and an LgBiT sequence. The advantages provided by such embodiments are first realized through the introduction of a small fusion tag, e.g., a SmHT-HiBiT tag, to the protein target of interest. Once tagged, a “detection complex” consisting of a reconstituted active NanoLuc® and HaloTag® complex is reconstituted by the detection of the protein target through spontaneous interaction via high affinity with the LgHT-LgBiT reporter polypeptide. In the non-complementary / non-complexed state, the dual peptide tag cannot generate a signal, resulting in a zero background state that is not obtainable when using a full-length fluorescent protein. The higher binding affinity between LgBiT and HiBiT facilitates the interaction between LgHT and SmHT via induced proximity. By introducing a luciferase substrate and a fluorescent HaloTag ligand, respectively, distinct luminescence and fluorescence signals are obtained, which can be configured to enable multiple functional measurements or assay types. The high affinity complementarity with the LgHT-LgBiT detection polypeptide provides the advantage of being able to select which detection mode is most beneficial depending on the situation, without changing the tag fused to the protein target.

[0009] The fluorescence and emission signals can be detected separately and do not interfere with each other; the choice of detection mode is an example of the simplicity and versatility offered by this technology. Another advantage is that both signals can be measured sequentially or simultaneously in the same reaction or within the same cells contained in a single tube or microplate well. For example, the expression level of a tagged target protein can be quantified with high sensitivity using the emission of the NanoLuc® component of the complex's active split, and its intracellular localization was measured using a fluorescence microscope on the HaloTag component of the complex's active split. These measurements do not require lysis, washing, or purification steps and can be comprised of a multiplex, homogeneous live-cell assay. In these examples, LgBiT or LgHT, or fusions between the two, are produced within the cells in which the tag or double tag is detected. However, the system can also be used in in vitro biochemical or cytolytic forms, where the double peptide tag is produced as a fusion to the target gene and detected in lysis form by recombinant LgBiT, LgHT, or LgBiT-LgHT fusions.

[0010] Various embodiments of this specification include different substrates for detection complexes, particularly for HaloTag® ligands, where many modified chloroalkanes are described as dyes that fluoresce (in fluorescent or non-fluorescent forms) at wavelengths spanning the visible and near-infrared regions, as well as as non-fluorescent ligands with diverse functions (e.g., mechanical and biological sensors, chemically induced proximity, targeted degradation and post-translational modification, energy transfer, etc.). This offers a clear advantage over similar competing technologies using small tags such as split fluorescent proteins (GFP, RFP), because split fluorescent proteins can only emit light at one wavelength and do not provide the non-fluorescent labeling function of HaloTag. Taken together, the advantages of dual emission and fluorescence detection using small fusion tags and a single complementary detection polypeptide, in addition to the versatility provided by their substrates, confer a broad set of advantages underlying the individual technologies, while simultaneously providing novel capabilities that can only be realized by combining them.

[0011] In some embodiments, dual reporter systems are provided herein that include: (a) a tandem peptide tag comprising (i) a peptide component of a bioluminescent complex fused to (ii) a peptide component of a modified dehalogenase complex; and (b) a tandem polypeptide reporter comprising (i) a polypeptide component of a bioluminescent complex and (ii) a polypeptide component of a modified dehalogenase complex; where the peptide component and polypeptide component of the bioluminescent complex can interact to form a bioluminescent complex, which can produce bioluminescence in the presence of a substrate for the bioluminescent complex; and the peptide component and polypeptide component of the modified dehalogenase complex can interact to form a modified dehalogenase complex, which can form a covalent bond with a haloalkyl ligand. In some embodiments, the system further includes a substrate for the bioluminescent complex. In some embodiments, the system further includes a haloalkyl ligand. In some embodiments, the haloalkyl ligand includes a haloalkane moiety linked to a fluorophore. In some embodiments, the tandem peptide tag is linked to a target element (e.g., a cell target, protein, peptide, etc.). In some embodiments, the tandem peptide tag and target element are expressed intracellularly as a fusion.

[0012] In some embodiments, a tandem peptide tag is provided comprising a peptide component of a bioluminescent complex fused to a peptide component of a modified dehalogenase complex; where the peptide component of the bioluminescent complex can interact with the polypeptide component of the bioluminescent complex to form a bioluminescent complex, which can produce bioluminescence in the presence of a substrate for the bioluminescent complex; where the peptide component of the modified dehalogenase complex can interact with the polypeptide component of the modified dehalogenase complex to form a modified dehalogenase complex, which can form a covalent bond with a haloalkyl ligand. In some embodiments, a system comprising the polypeptide components of the tandem peptide tag and bioluminescent complex described herein is provided. In some embodiments, the system further comprises a substrate for the bioluminescent complex. In some embodiments, the system further comprises the polypeptide component of the modified dehalogenase complex. In some embodiments, the system further comprises a haloalkyl ligand. In some embodiments, the peptide components of the modified dehalogenase complex and the peptide components of the bioluminescent complex are present in the system as independent reporter polypeptides. In some embodiments, the peptide components of the modified dehalogenase complex and the peptide components of the bioluminescent complex are present in the system as a tandem polypeptide reporter.

[0013] In some embodiments, a system comprising polypeptide components of the tandem peptide tag and modified dehalogenase complex described herein is provided. In some embodiments, the system further comprises a substrate for the bioluminescent complex.

[0014] In some embodiments, a tandem polypeptide reporter is provided comprising a polypeptide component of a bioluminescent complex and a polypeptide component of a modified dehalogenase complex; where the polypeptide component of the bioluminescent complex interacts with the peptide component of the bioluminescent complex to form a bioluminescent complex, which can produce bioluminescence in the presence of a substrate for the bioluminescent complex; and the polypeptide component of the modified dehalogenase complex interacts with the peptide component of the modified dehalogenase complex to form a modified dehalogenase complex, which can form a covalent bond with a haloalkyl ligand. In some embodiments, a system comprising the peptide components of the tandem peptide reporter and bioluminescent complex described herein is provided. In some embodiments, the system further comprises a substrate for the bioluminescent complex. In some embodiments, the system further comprises the peptide components of the modified dehalogenase complex. In some embodiments, the system further comprises a haloalkyl ligand. In some embodiments, the polypeptide components of the modified dehalogenase complex and the polypeptide components of the bioluminescent complex are present in the system as independent reporter polypeptides. In some embodiments, the polypeptide components of the modified dehalogenase complex and the polypeptide components of the bioluminescent complex are present in the system as a tandem polypeptide reporter.

[0015] In some embodiments, a system comprising the peptide components of the tandem polypeptide reporter and modified dehalogenase complex described herein is provided. In some embodiments, the system further comprises a substrate for the bioluminescent complex.

[0016] In some embodiments, a method is provided for detecting a component of interest in a system, the method comprising: (a) linking the component of interest to a tandem peptide tag comprising a peptide component of a bioluminescent complex fused to a peptide component of a modified dehalogenase complex, wherein the peptide component of the bioluminescent complex interacts with the polypeptide component of the bioluminescent complex to form a bioluminescent complex, the bioluminescent complex can produce bioluminescence in the presence of a substrate for the bioluminescent complex, the peptide component of the modified dehalogenase complex interacts with the polypeptide component of the modified dehalogenase complex to form a modified dehalogenase complex, the modified dehalogenase complex can form a covalent bond with a haloalkyl ligand; (b) contacting the component of interest bound to the tandem peptide tag with the polypeptide component of the modified dehalogenase complex under conditions in which the modified dehalogenase complex is formed; (c) contacting the modified dehalogenase complex with a haloalkyl ligand comprising a haloalkane bound to a fluorophore; and (d) detecting fluorescence.

[0017] In some embodiments, a method is provided for detecting a component of interest in a system, the method comprising: (a) linking the component of interest to a tandem peptide tag containing a peptide component of a bioluminescent complex fused to a peptide component of a modified dehalogenase complex, wherein the peptide component of the bioluminescent complex interacts with the polypeptide component of the bioluminescent complex to form a bioluminescent complex, the bioluminescent complex can produce bioluminescence in the presence of a substrate for the bioluminescent complex, the peptide component of the modified dehalogenase complex interacts with the polypeptide component of the modified dehalogenase complex to form a modified dehalogenase complex, the modified dehalogenase complex can form a covalent bond with a haloalkyl ligand; (b) contacting the component of interest bound to the tandem peptide tag with the polypeptide component of the bioluminescent complex under conditions in which the bioluminescent complex is formed; (c) contacting the bioluminescent complex with a substrate for the bioluminescent complex; and (d) detecting bioluminescence.

[0018] In some embodiments, a method is provided for detecting a target component in a system, the method comprising: (a) linking the target component to a tandem peptide tag comprising a peptide component of a bioluminescent complex fused to a peptide component of a modified dehalogenase complex, wherein the peptide component of the bioluminescent complex interacts with the polypeptide component of the bioluminescent complex to form a bioluminescent complex, the bioluminescent complex can produce bioluminescence in the presence of a substrate for the bioluminescent complex, and the peptide component of the modified dehalogenase complex interacts with the polypeptide component of the modified dehalogenase complex (b) a complex can be formed, and the modified dehalogenase complex can form a covalent bond with a haloalkyl ligand; (c) contact the desired component linked to the tandem peptide tag with a tandem polypeptide reporter containing (i) the polypeptide component of the bioluminescent complex and (ii) the polypeptide component of the modified dehalogenase complex, under conditions in which the bioluminescent complex and the modified dehalogenase complex are formed; (d) contact the bioluminescent complex with a substrate for a haloalkyl ligand containing a haloalkane linked to the bioluminescent complex and / or fluorophore; and (e) detect bioluminescence and / or fluorescence.

[0019] In some embodiments, a method is provided for detecting a target component in a system, the method comprising: (a) linking the target component to a tandem peptide tag comprising a peptide component of a bioluminescent complex fused to a peptide component of a modified dehalogenase complex, wherein the peptide component of the bioluminescent complex interacts with the polypeptide component of the bioluminescent complex to form a bioluminescent complex, the bioluminescent complex can produce bioluminescence in the presence of a substrate for the bioluminescent complex, and the peptide component of the modified dehalogenase complex interacts with the polypeptide component of the modified dehalogenase complex to form a modified dehalogenase complex (b) the formation of a bioluminescent complex, the modified dehalogenase complex being able to form a covalent bond with a haloalkyl ligand; (b) contacting the desired component linked to a tandem peptide tag with an independent polypeptide containing (i) the polypeptide component of the bioluminescent complex and (ii) the polypeptide component of the modified dehalogenase complex, under conditions in which the bioluminescent complex and the modified dehalogenase complex are formed; (c) contacting the bioluminescent complex with a substrate for a haloalkyl ligand containing a haloalkane linked to the bioluminescent complex and / or fluorophore; and (d) detecting bioluminescence and / or fluorescence. [Brief explanation of the drawing]

[0020] [Figure 1] This is an illustration of an exemplary embodiment of the dual-tag / reporter technology described herein. [Figure 2] Complementation of split HaloTag using SmHT-HiBiT fusions. E. coli lysates expressing HaloTag[23-297]-FRB were mixed with lysates expressing FKBP-SmHT with the indicated modification (+ / -HiBiT tag) and incubated for 2 hours with or without rapamycin. JF646 HaloTag ligand was added to the mixture and incubated for 1 hour, after which the fluorescence activity of each combination was measured. SmHT refers to the HaloTag[3-19] fragment. [Figure 3]Complementation of split HaloTag using SmHT-HiBiT fusions. E. coli lysates expressing HaloTag[22-297](Q145H+P154)-FRB were mixed with lysates expressing FKBP-SmHT with the indicated modification (+ / -HiBiT tag) and incubated with or without rapamycin for 2 hours. JF646 HaloTag ligand was added to the mixture and incubated for 1 hour, after which the fluorescence activity of each combination was measured. SmHT refers to the HaloTag[3-19] fragment. [Figure 4] Complementation of split HaloTag using SmHT-HiBiT fusions. E. coli lysates expressing HaloTag[23-297]-FRB were mixed with lysates expressing FKBP-SmHT with the indicated modification (+ / -HiBiT tag) and incubated for 2 hours with or without rapamycin. JF646 HaloTag ligand was added to this mixture and incubated for 1 hour, after which the fluorescence activity of each combination was measured. The response factor was calculated by dividing the fluorescence with rapamycin by the fluorescence without rapamycin. SmHT refers to the HaloTag[3-19] fragment. [Figure 5] Complementation of split HaloTag using SmHT-HiBiT fusions. E. coli lysates expressing HaloTag[22-297](Q145H+P154)-FRB were mixed with lysates expressing FKBP-SmHT with the indicated modification (+ / -HiBiT tag) and incubated for 2 hours with or without rapamycin. JF646 HaloTag ligand was added to the mixture and incubated for 1 hour, after which the fluorescence activity of each combination was measured. The response factor was calculated as the fluorescence with rapamycin minus the fluorescence without rapamycin. SmHT refers to the HaloTag[3-19] fragment. [Figure 6]Luminescence activity of SmHT-HiBiT fusion tags when complemented with LgBiT. Purified LgBiT protein and flimazine substrate were incubated with or without rapamycin for 2 hours, then added to E. coli lysates expressing FKBP-SmHT with the indicated modification (+ / -HiBiT tag). After 20 minutes of incubation, the luminescence activity of each combination was measured. SmHT refers to the HaloTag[3-19] fragment. [Figure 7] Detection of SmHT-HiBiT fusion tags by LgHT-LgBiT detection polypeptides via facilitated complementation. E. coli lysates expressing HaloTag[22-297(M2F)]-FRB or HaloTag[22-297(M2F)]-LgBiT were mixed with lysates expressing FKBP-SmHT with the indicated modification (+ / -HiBiT tag) and incubated with or without rapamycin for 2 hours. JF646 HaloTag ligand was added to the mixture, and after incubation for 1 hour, the fluorescence activity of each combination was measured. SmHT refers to the HaloTag[3-19] fragment. [Figure 8] Detection of SmHT-HiBiT fusion tags by LgHT-LgBiT detection polypeptides via facilitated complementation. E. coli lysates expressing HaloTag[22-297(Q145H+P154R)]-FRB or HaloTag[22-297](Q145H+P154R)-LgBiT were mixed with lysates expressing FKBP-SmHT with the indicated modification (+ / -HiBiT tag) and incubated with or without rapamycin for 2 hours. JF646 HaloTag ligand was added to the mixture and incubated for 1 hour, after which the fluorescence activity of each combination was measured. SmHT refers to the HaloTag[3-19] fragment. [Figure 9]Detection of SmHT-HiBiT fusion tags by luminescence after enhanced complementation with LgHT-LgBiT detection polypeptides. E. coli lysates expressing the indicated LgHT-FRB or LgHT-LgBiT fusion were mixed with lysates expressing FKBP-SmHT with the indicated modification (+ / -HiBiT tag) and incubated with or without rapamycin for 2 hours. The fluorescence activity was measured by adding JF646 HaloTag ligand to the mixture and incubating for 1 hour. The mixture was then diluted 1:10, furimazine was added, and the luminescence activity was measured. SmHT refers to the HaloTag[3-19] fragment. [Figure 10] Detection of synthetic SmHT-HiBiT peptides using LgHT-LgBiT detection polypeptides. E. coli lysates expressing HaloTag[22-297(M2F)]-LgBiT or HaloTag[22-297(Q145H+P174R)]-LgBiT were mixed with synthetic SmHT-HiBiT peptides within a specified concentration range and incubated for 2 hours. JF646 HaloTag ligand was added to the mixture, and after incubation for 1 hour, the fluorescence activity of each combination was measured. For each combination, the lowest fluorescence intensity was used as the baseline for subtracting all data points to enable nonlinear curve fitting and affinity calculations. SmHT refers to the HaloTag[3-19] fragment. [Figure 11] Detection of synthetic SmHT-HiBiT peptides using LgHT-LgBiT detection polypeptides. E. coli lysates expressing HaloTag[22-297(M2F)]-LgBiT or HaloTag[22-297(Q145H+P174R)]-LgBiT were mixed with synthetic SmHT-HiBiT peptides within a specified concentration range and incubated for 2 hours. Flimazine was added to the mixture, and after incubation for 20 minutes, the fluorescence activity of each combination was measured. SmHT refers to the HaloTag[3-19] fragment. [Figure 12]Effect of changes in the internal linker length of HaloTag[3-19]-HiBiT as a C-terminal fusion to FKBP on the luminescence activity in E. coli lysates. Constructs with various internal linker lengths separating HaloTag[3~19] and HiBiT sequences were expressed as C-terminal FKBP fusions in E. coli lysates. The FKBP fusion-expressing lysates were combined with E. coli lysates expressing LgBiT, HaloTag[22-297](M2F)-12xGly / Ser-LgBiT, or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). The reaction was incubated at room temperature for 30 minutes. Fluoroflimazine substrate was added to the reaction 5 minutes before luminescence detection. [Figure 13] Effect of changes in HaloTag[3-19]-HiBiT internal linker length on luminescence activity in E. coli lysates. Constructs with various internal linker lengths separating HaloTag[3-19] and HiBiT sequences were expressed as C-terminal FKBP fusions in E. coli lysates. The FKBP fusion-expressing lysates were combined with E. coli lysates expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). The reaction was incubated at room temperature for 30 minutes. JF646 HaloTag ligand was added to the lysate mixture, and the reaction was incubated at room temperature for a further 2 hours before fluorescence detection. [Figure 14]Effect of changes in HaloTag[3-19]-HiBiT internal linker length on the response factor of luminescence activity in E. coli lysates. Constructs with various internal linker lengths separating HaloTag[3-19] and HiBiT sequences were expressed as C-terminal FKBP fusions in E. coli lysates. The FKBP fusion-expressing lysates were combined with E. coli lysates expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). The reaction was incubated at room temperature for 30 minutes. JF646 HaloTag ligand was added to the lysate mixture, and the reaction was incubated at room temperature for a further 2 hours before fluorescence detection. The response factor was calculated by dividing the raw fluorescence readings of each reactant, including the FKBP fusion and polypeptide reporter lysate, by the fluorescence readings of the polypeptide reporter lysate alone. [Figure 15] Effect of changes in the internal linker length of the N-terminal tag HaloTag[3-19]-HiBiT on the luminescence activity in E. coli lysates. Constructs with various internal linker lengths separating HaloTag[3-19] and HiBiT sequences were expressed as N-terminal FKBP fusions in E. coli lysates. The FKBP fusion-expressing lysates were combined with E. coli lysates expressing LgBiT, HaloTag[22-297](M2F)-12xGly / Ser-LgBiT, or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). The reaction was incubated at room temperature for 30 minutes. The reaction was diluted 10-fold in buffer and added to a diluted reaction mixture containing fluoroflimazine substrate 5 minutes before luminescence detection. [Figure 16]Effect of changes in the internal linker length of the HaloTag[3-19]-HiBiT fusion to FKBP on the luminescence activity in E. coli lysates. Constructs with various internal linker lengths separating HaloTag[3-19] and HiBiT sequences were expressed as N-terminal FKBP fusions in E. coli lysates. The FKBP fusion-expressing lysates were combined with E. coli lysates expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). The reaction was incubated at room temperature for 30 minutes. JF646 HaloTag ligand was added to the lysate mixture, and the reaction was incubated at room temperature for a further 2 hours before fluorescence detection. [Figure 17] Effect of changes in the internal linker length of the HaloTag[3-19]-HiBiT fusion to FKBP on the response factor of luminescence activity in E. coli lysates. Constructs with various internal linker lengths separating HaloTag[3-19] and HiBiT sequences were expressed as N-terminal FKBP fusions in E. coli lysates. The FKBP fusion-expressing lysates were combined with E. coli lysates expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). The reaction was incubated at room temperature for 30 minutes. JF646 HaloTag ligand was added to the lysate mixture, and the reaction was incubated at room temperature for a further 2 hours before fluorescence detection. The response factor was calculated by dividing the raw fluorescence readings of each reactant, including the FKBP fusion and polypeptide reporter lysate, by the fluorescence readings of the polypeptide reporter lysate alone. [Figure 18]Effects of changes in HaloTag[3-19]-HiBiT internal linker length and composition on luminescence activity in E. coli lysates. Constructs with various internal linker lengths and compositions that separate HaloTag[3-19] and HiBiT sequences were expressed as C-terminal FKBP fusions in E. coli lysates. The FKBP fusion-expressing lysates were combined with E. coli lysates expressing LgBiT, HaloTag[22-297](M2F)-12xGly / Ser-LgBiT, or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). The reaction was incubated at room temperature for 30 minutes. Fluoroflimazine substrates were added to the reaction 5 minutes before luminescence detection. [Figure 19] Effects of changes in HaloTag[3-19]-HiBiT internal linker length and composition on fluorescence activity in E. coli lysates. Constructs with various internal linker lengths separating HaloTag[3-19] and HiBiT sequences were expressed as C-terminal FKBP fusions in E. coli lysates. The FKBP fusion-expressing lysates were combined with E. coli lysates expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). The reaction was incubated at room temperature for 30 minutes. JF646 HaloTag ligand was added to the lysate mixture, and the reaction was incubated at room temperature for a further 2 hours before fluorescence detection. [Figure 20] Effect of various linker lengths between the C-terminal fusions of FKBP and HaloTag[3-19]-HiBiT on luminescence activity in E. coli lysates. Constructs with various linker lengths separating FKBP and HaloTag[3-19]-HiBiT were expressed in E. coli lysates. The FKBP fusion-expressing lysates were combined with E. coli lysates expressing LgBiT, HaloTag[22-297](M2F)-12xGly / Ser-LgBiT, or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). The reaction was incubated at room temperature for 30 minutes. Fluoroflimazine substrates were added to the reaction 5 minutes before luminescence detection. [Figure 21] Effect of various linker lengths between the C-terminal fusions of FKBP and HaloTag[3-19]-HiBiT on the fluorescence activity in E. coli lysates. Constructs with various linker lengths separating FKBP and HaloTag[3-19]-HiBiT were expressed in E. coli lysates. The FKBP fusion-expressing lysates were combined with E. coli lysates expressing LgBiT, HaloTag[22-297](M2F)-12xGly / Ser-LgBiT, or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). The reaction was incubated at room temperature for 30 minutes. JF646 HaloTag ligand was added to the lysate mixture, and the reaction was incubated at room temperature for a further 2 hours before fluorescence detection. [Figure 22] The effect of various linker lengths between FKBP and the HaloTag[3-19]-HiBiT fusion fused to the N-terminus on the luminescence activity in E. coli lysates. Constructs with various linker lengths separating FKBP and HaloTag[3-19]-HiBiT were expressed in E. coli lysates. The FKBP fusion-expressing lysates were combined with E. coli lysates expressing LgBiT, HaloTag[22-297](M2F)-12xGly / Ser-LgBiT, or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). The reaction was incubated at room temperature for 30 minutes. The reaction was diluted 10-fold in buffer and added to the reaction mixture containing diluted fluoroflimazine substrate 5 minutes before luminescence detection. [Figure 23]The effect of various linker lengths between FKBP and the HaloTag[3-19]-HiBiT fusion fused to the N-terminus on the fluorescence activity in E. coli lysates. Constructs with various linker lengths separating FKBP and HaloTag[3-19]-HiBiT were expressed in E. coli lysates. The FKBP fusion-expressing lysates were combined with E. coli lysates expressing LgBiT, HaloTag[22-297](M2F)-12xGly / Ser-LgBiT, or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). The reaction was incubated at room temperature for 30 minutes. JF646 HaloTag ligand was added to the lysate mixture, and the reaction was incubated at room temperature for a further 2 hours before fluorescence detection. [Figure 24] Effect of HaloTag[3-19] cleavage on luminescence activity in E. coli lysates. HaloTag[3-19]-HiBiT constructs containing HaloTag[3-19] cleavage were expressed in E. coli lysates as C-terminal fusions to FKBP. The FKBP fusion-expressing lysates were combined with E. coli lysates expressing LgBiT, HaloTag[22-297](M2F)-12xGly / Ser-LgBiT, or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). The reactions were incubated at room temperature for 30 minutes. Fluoroflimazine substrates were added to the reaction 5 minutes before luminescence detection. [Figure 25]Effect of HaloTag[3-19] cleavage on the luminescence activity of HaloTag[3-19]-HiBiT in E. coli lysates. HaloTag[3-19]-HiBiT constructs containing HaloTag[3-19] cleavage were expressed in E. coli lysates as C-terminal fusions to FKBP. The FKBP fusion-expressing lysates were combined with E. coli lysates expressing either HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). The reactions were incubated at room temperature for 30 minutes. JF646 HaloTag ligand was added to the lysate mixture, and the reaction was incubated at room temperature for a further 2 hours before fluorescence detection. [Figure 26] Effect of HiBiT mutations on the luminescence activity of HaloTag[3-19]-HiBiT in E. coli lysates. HaloTag[3-19]-HiBiT constructs containing HiBiT mutations were expressed as FKBP fusions in E. coli lysates. The FKBP fusion-expressing lysates were combined with E. coli lysates expressing LgBiT, HaloTag[22-297](M2F)-12xGly / Ser-LgBiT, or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). The reactions were incubated at room temperature for 30 minutes. Fluoroflimazine substrates were added to the reaction 5 minutes before luminescence detection. [Figure 27] Effect of HiBiT mutations on the luminescence activity of HaloTag[3-19]-HiBiT in E. coli lysates. HaloTag[3-19]-HiBiT constructs containing HiBiT mutations were expressed in E. coli lysates as a C-terminal fusion to FKBP. The FKBP fusion-expressing lysates were combined with E. coli lysates expressing either HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). The reactions were incubated at room temperature for 30 minutes. JF646 HaloTag ligand was added to the lysates, and the reactions were incubated at room temperature for a further 2 hours before fluorescence detection. [Figure 28] Effect of HaloTag[3-19]-HiBiT mutants on the enhancement of LgBiT luminescence across a range of synthetic peptide concentrations. HaloTag[3-19]-HiBiT synthetic peptide mutants containing mutations from the native sequence (EIGTGFPFDPHYVEVLG) (bold) were added stepwise to purified LgBiT-6xHis protein. The reaction was incubated at 4°C for 30 minutes. A flimazine substrate was added to the reaction immediately before measuring luminescence. The peptides in this experiment were conjugated to biotin at the N-terminus. [Figure 29] Effect of HaloTag[3-19]-HiBiT mutants on the enhancement of HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT-6xHis luminescence across a range of synthetic peptide concentrations. Synthetic HaloTag[3-19]-HiBiT mutant peptides containing mutations (bold) from the natural sequence (EIGTGFPFDPHYVEVLG) were added stepwise to purified HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT-6xHis protein. The reaction was incubated at 4°C for 30 minutes. A flimazine substrate was added to the reaction immediately before measuring luminescence. The peptides in this experiment were conjugated to biotin at the N-terminus. [Figure 30] Effect of HaloTag[3-19]-HiBiT mutants on the enhancement of HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT-6xHis luminescence across a range of synthetic peptide concentrations. HaloTag[3-19]-HiBiT synthetic peptides containing mutations (bold) from the native sequence (EIGTGFPFDPHYVEVLG) were added stepwise to purified HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT-6xHis protein. The reaction was incubated at 4°C for 30 minutes. A flimazine substrate was added to the reaction immediately before measuring luminescence. The peptides in this experiment were conjugated to biotin at the N-terminus. [Figure 31]Effect of HaloTag[3-19]-HiBiT mutants on the enhancement of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis luminescence across a range of synthetic peptide concentrations. HaloTag[3-19]-HiBiT mutant synthetic peptides containing sequence mutations from the native sequence (EIGTGFPFDPHYVEVLG) or additional linker residues (both in bold) were added stepwise to purified HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis protein. The reaction was incubated at 4°C for 30 minutes. A flimazine substrate was added to the reaction immediately before measuring luminescence. [Figure 32] Effect of HaloTag[3-19]-HiBiT mutants on the enhancement of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis across a range of synthetic peptide concentrations. HaloTag[3-19]-HiBiT mutant synthetic peptides containing mutations from the natural sequence (EIGTGFPFDPHYVEVLG) (bold) were added stepwise to purified HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis protein. The reaction was incubated at 4°C for 30 minutes. A flimazine substrate was added to the reaction immediately before measuring luminescence. [Figure 33] Effect of HaloTag[3-19]-HiBiT mutants on the enhancement of HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT-6xHis fluorescence across a range of synthetic peptide concentrations. HaloTag[3-19]-HiBiT mutant synthetic peptides containing mutations (bold) from the native sequence (EIGTGFPFDPHYVEVLG) were added stepwise to purified HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT-6xHis protein. The reactions were incubated at 4°C for 30 minutes. JF646 HaloTag ligand was added to each reaction, and fluorescence was measured after incubation with the ligand at room temperature for 30 minutes. The peptides in this experiment were conjugated to biotin at the N-terminus. [Figure 34]The effect of HaloTag[3-19]-HiBiT mutants on the enhancement of HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT luminescence when complemented by synthetic peptides across a range of synthetic peptide concentrations. Synthetic HaloTag[3-19]-HiBiT mutant peptides containing mutations from the natural sequence (EIGTGFPFDPHYVEVLG) (bold) were added stepwise to purified HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT-6xHis protein. The reactions were incubated at 4°C for 30 minutes. JF646 HaloTag ligand was added to each reaction, and fluorescence was measured after incubation with the ligand at room temperature for 30 minutes. The peptides in this experiment were conjugated to biotin at the N-terminus. [Figure 35] The effect of HaloTag[3-19]-HiBiT mutants on the enhancement of HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT luminescence across a range of synthetic peptide concentrations. HaloTag[3-19]-HiBiT mutant synthetic peptides containing mutations from the native sequence (EIGTGFPFDPHYVEVLG) (bold) were added stepwise to purified HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT-6xHis protein. The reaction was incubated at 4°C for 30 minutes. JF646 HaloTag ligand was added to each reaction, and fluorescence was measured after incubation with the ligand at room temperature for 30 minutes. [Figure 36]Effect of HaloTag[3-19]-HiBiT mutants on the fluorescence of HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT-6xHis across a range of synthetic peptide concentrations. HaloTag[3-19]-HiBiT mutant synthetic peptides containing mutations from the natural sequence (EIGTGFPFDPHYVEVLG) (bold) were added stepwise to purified HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT-6xHis protein. The reaction was incubated at 4°C for 30 minutes. JF646 HaloTag ligand was added to each reaction, and fluorescence was measured after incubation with the ligand at room temperature for 30 minutes. [Figure 37] Effect of HaloTag[3-19]-HiBiT variants on the luminescence intensity of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis across a range of synthetic peptide concentrations. HaloTag[3-19]-HiBiT synthetic peptides containing mutations (bold) or deletions (indicated by dashes) from the natural sequence (SEQ ID NO: 3061:EIGTGFPFDPHYVEVLG) were added stepwise to E. coli lysates expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. The reaction was incubated at room temperature for 2 hours. JF646 HaloTag ligand was added to the synthetic peptide / lysate mixture, and the reaction was incubated at room temperature for a further 1.5 hours, after which fluorescence detection was performed. [Figure 38]Effect of HaloTag[3-19] mutants on the luminescence intensity of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT across a range of synthetic peptide concentrations. HaloTag[3-19] synthetic peptides containing mutations (bold) from the natural sequence (SEQ ID NO: 3061:EIGTGFPFDPHYVEVLG) were added stepwise to E. coli lysates expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. The reaction was incubated at room temperature for 2 hours. JF646 HaloTag ligand was added to the synthetic peptide / lysate mixture, and the reaction was incubated at room temperature for a further 1.5 hours, after which fluorescence detection was performed. [Figure 39] Effect of HaloTag[3-19] mutants on the enhancement of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT luminescence across a range of synthetic peptide concentrations. HaloTag[3-19] synthetic peptides containing mutations (bold) from the natural sequence (SEQ ID NO: 3061:EIGTGFPFDPHYVEVLG) were added stepwise to E. coli lysates expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. The reaction was incubated at room temperature for 2 hours. JF646 HaloTag ligand was added to the synthetic peptide / lysate mixture, and the reaction was incubated at room temperature for a further 1.5 hours, after which fluorescence detection was performed. [Figure 40] Effect of HaloTag[3-19] mutants on the luminescence intensity of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT across a range of synthetic peptide concentrations. HaloTag[3-19] synthetic peptides containing mutations (bold) from the natural sequence (SEQ ID NO: 3061:EIGTGFPFDPHYVEVLG) were added stepwise to E. coli lysates expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. The reaction was incubated at room temperature for 2 hours. JF646 HaloTag ligand was added to the synthetic peptide / lysate mixture, and the reaction was incubated at room temperature for a further 1.5 hours, after which fluorescence detection was performed. [Figure 41]Binding of HaloTag[22-297](M2F+F148M+V177E)-12xGly / Ser-LgBiT-6xHis to biotin-HaloTag[3-19](I2R)-4xGly / Ser-VS-HiBiT synthetic peptide. The association and dissociation of the polypeptide reporter HaloTag[22-297](M2F+F148M+V177E)-12xGly / Ser-LgBiT-6xHis (shown to the right of the dotted line) to the biotin-HaloTag[3-19](I2R)-4xGly / Ser-VS-HiBiT synthetic peptide, immobilized on a streptavidin probe, was measured (shown to the right of the dotted line) using biolayer interferometry in PBST at 25°C. [Figure 42]Yeast surface presentation of HaloTag[3-19]-4xGly / Ser-VS-HiBiT. HaloTag[3-19]-4xGly / Ser-VS-HiBiT was fused to the yeast cell junction factor Aga2 for surface presentation. The resulting construct was transformed into S. cerevisiae and expressed. Cells were incubated with HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis at room temperature for 45 minutes. Unbound HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis was washed off, and cells were incubated with JF549 HaloTag ligand at room temperature for 45 minutes. Unbound ligand was washed off the cells, and cells were incubated with mouse-derived anti-HA tag antibody at room temperature for 30 minutes. Excess primary antibody was washed away, and cells were incubated at room temperature for 30 minutes with a secondary antibody, goat anti-mouse IgG conjugated to AlexaFluor488. Cells were washed and analyzed using a Sony Cell Sorter. Histograms represent 10,192 recorded events from single-cell positive gates. Cells in the AF488+ gate represent a population of cells positive for surface display of HaloTag[3-19]-4xGly / Ser-VS-HiBiT (left histogram, 51.23%). Cells in the JF549+ gate represent a population of cells expressing HaloTag[3-19]-4xGly / Ser-VS-HiBiT and complemented by JF549 HaloTag ligand-bound HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis (right histogram, 48.88%). [Figure 43]Yeast surface presentation of EGFP-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT. EGFP-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT was fused to the yeast cell junction factor Aga2 for surface presentation. The resulting construct was transformed into S. cerevisiae and expressed. Cells were incubated with HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis at room temperature for 45 minutes. Unbound HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis was washed off, and cells were incubated with JF549 HaloTag ligand at room temperature for 45 minutes. Cells were washed and analyzed using a Sony Cell Sorter. The histogram represents 10,569 recorded events. Cells in the EGFP+ gate represent a population of cells positive for surface display of EGFP-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT (histogram on the left, 48.07%). Cells in the JF549+ gate represent a population of cells complemented by JF549 HaloTag ligand-bound HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis (histogram on the right, 46.41%). [Figure 44]Yeast surface presentation of EGFP-GGSG-HaloTag[3-19]-GGSG-NVSGWRLFKKISN. The Aga2-EGFP-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT surface presentation construct was modified by replacing the VS-HiBiT sequence with NVSGWRLFKKISN, a sequence that complements LgBiT with lower affinity than HiBiT. The resulting construct was transformed into S. cerevisiae and expressed. Cells were incubated with HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis at room temperature for 45 minutes. Unbound HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis was washed off, and cells were incubated with JF549 HaloTag ligand at room temperature for 45 minutes. Cells were washed and analyzed using Sony Cell Sorter. The histogram represents 11,216 recorded events. Cells in the EGFP+ gate represent a population of cells positive for surface presentation of EGFP-GGSG-HaloTag[3-19]-NVSGWRLFKKISN (histogram on the left, 44.57%). Cells in the JF549+ gate represent a population of cells complemented by JF549 HaloTag ligand-bound HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis (histogram on the right, 44.24%). [Figure 45]Yeast surface presentation of EGFP-GGSG-HaloTag[3-19]-GGSG-NVTGYRLFKKISN. The Aga2-EGFP-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT surface presentation construct was modified by replacing the VS-HiBiT sequence with NVTGYRLFKKISN, a sequence that complements LgBiT with lower affinity than HiBiT. The resulting construct was transformed into S. cerevisiae and expressed. Cells were incubated with HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis at room temperature for 45 minutes. Unbound HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis was washed off, and cells were incubated with JF549 HaloTag ligand at room temperature for 45 minutes. Cells were washed and analyzed using Sony Cell Sorter. The histogram represents 11,203 recorded events. Cells in the EGFP+ gate represent a population of cells positive for surface presentation of EGFP-GGSG-HaloTag[3-19]-GGSG-NVTGYRLFKKISN (histogram on the left, 46.47%). Cells in the JF549+ gate represent a population of cells complemented by JF549 HaloTag ligand-bound HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis (histogram on the right, 44.84%). [Figure 46]Yeast surface presentation of EGFP-GGSG-HaloTag[3-19]-GGSG-VTGYRLFEKIS. The Aga2-EGFP-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT surface presentation construct was modified by replacing the VS-HiBiT sequence with VTGYRLFEKIS, a sequence that complements LgBiT with lower affinity than HiBiT. The resulting construct was transformed into S. cerevisiae and expressed. Cells were incubated with HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis at room temperature for 45 minutes. Unbound HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis was washed off, and cells were incubated with JF549 HaloTag ligand at room temperature for 45 minutes. Cells were washed and analyzed using Sony Cell Sorter. The histogram represents 11,230 recorded events. Cells in the EGFP+ gate represent a population of cells positive for surface presentation of EGFP-GGSG-HaloTag[3-19]-GGSG-VTGYRLFEKIS (left histogram, 49.51%). Cells in the JF549+ gate represent a population of cells complemented by JF549 HaloTag ligand-bound HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis (right histogram, 6.96%). [Figure 47] Complementation of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutants in E. coli lysates using gene fusion of FKBP and HaloTag[3-19] peptide. Mutants with either the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or M49F+D53G mutations were expressed in E. coli lysates and combined with lysates expressing FKBP fused to different tag sequences. The reaction mixtures were incubated at room temperature for 2 hours, then labeled with 50 nM JF646 HaloTag ligand, and subsequently detected by fluorescence detection. Data points represent independent cultures of each construct combined during the experiment, and error bars represent one standard deviation from the mean. [Figure 48]Complementation and stability of HaloTag[22-297](M2F+D53S+D56P)-12xGly / Ser-LgBiT in E. coli lysates using synthetic HaloTag[3-19] peptide. HaloTag[22-297](M2F+D53S+D56P)-12xGly / Ser-LgBiT was expressed in E. coli lysates and combined with excess synthetic HaloTag[3-19] peptide. The reaction mixture was incubated for the indicated time and temperature, then labeled with 50 nM JF646 HaloTag ligand, and detected by fluorescence. [Figure 49] Stability of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT in E. coli lysates after complementation with different synthetic peptides. HaloTag[22-297](M2F)-12xGly / Ser-LgBiT was expressed in E. coli lysates and combined with the indicated synthetic peptides at a concentration of 10 μM or higher. The reaction mixture was incubated at the indicated temperature for 10 minutes, then labeled with 50 nM JF646 HaloTag ligand, and subsequently detected by fluorescence. [Figure 50] Stability of HaloTag[22-297](M2F+D53G+V177E)-12xGly / Ser-LgBiT in E. coli lysates after complementation with different synthetic peptides. HaloTag[22-297](M2F+D53G+V177E)-12xGly / Ser-LgBiT was expressed in E. coli lysates and combined with the indicated synthetic peptides at a concentration of 10 μM or higher. The reaction mixture was incubated at the indicated temperature for 10 minutes, then labeled with 50 nM JF646 HaloTag ligand, and subsequently detected by fluorescence. [Figure 51]Stability of HaloTag[22-297](M2F+M49F+S89A)-12xGly / Ser-LgBiT in E. coli lysates after complementation with different synthetic peptides. HaloTag[22-297](M2F+M49F+S89A)-12xGly / Ser-LgBiT was expressed in E. coli lysates and combined with the indicated synthetic peptides at a concentration of 10 μM or higher. The reaction mixture was incubated at the indicated temperature for 10 minutes, then labeled with 50 nM JF646 HaloTag ligand, and subsequently detected by fluorescence. [Figure 52] Stability of HaloTag[22-297](M2F+M49F+L57I)-12xGly / Ser-LgBiT in E. coli lysates after complementation with different synthetic peptides. HaloTag[22-297](M2F+M49F+L57I)-12xGly / Ser-LgBiT was expressed in E. coli lysates and combined with the indicated synthetic peptides at a concentration of 10 μM or higher. The reaction mixture was incubated at the indicated temperature for 10 minutes, then labeled with 50 nM JF646 HaloTag ligand, and subsequently detected by fluorescence. [Figure 53] Labeling reaction rate of HaloTag[22-297](M2F+D53G+V177E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis mutants in E. coli lysates after complementation with synthetic peptide. HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis or its mutant with the D53G+V177E mutation was expressed in E. coli lysates and combined with 200 μM HaloTag[3-19] synthetic peptide. The reaction mixture was incubated at room temperature for 3 hours and then added to 10 nM TMR HaloTag ligand. The fluorescence polarization of the ligand was continuously read to monitor labeling. Lysates were added at 3 minutes in the graph, and the baseline fluorescence polarization of the ligand and the subsequent increase in binding upon addition of lysates were measured first. [Figure 54]Labeling reaction rate of HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis mutants in E. coli lysates after complementation with synthetic peptide. HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis or its K140E mutant was expressed in E. coli lysates and combined with 200 μM HaloTag[3-19] synthetic peptide. The reaction mixture was incubated at room temperature for 3 hours and then added to 10 nM TMR HaloTag ligand. The fluorescence polarization of the ligand was continuously read to monitor labeling. Lysates were added at 3 minutes in the graph, and the baseline fluorescence polarization of the ligand and the subsequent increase in binding upon addition of lysates were measured first. [Figure 55] Labeling reaction rate of HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis mutants in E. coli lysates after complementation with synthetic peptide. Mutants containing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis, K140E, or D53G+K140E were expressed in E. coli lysates and combined with 200 μM HaloTag[3-19] synthetic peptide. The reaction mixture was incubated at room temperature for 3 hours and then added to 10 nM TMR HaloTag ligand. The fluorescence polarization of the ligand was continuously read to monitor labeling. Lysates were added at 3 minutes in the graph, and the baseline fluorescence polarization of the ligand and the subsequent increase in binding upon addition of lysates were measured. [Figure 56]Labeling activity at the endpoint of the HaloTag[22-297](M2F+D53G)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis mutant in E. coli lysates after complementation with synthetic peptide. The HaloTag[22-297](M2F+D53G)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis mutant was expressed in E. coli lysates and combined with 200 μM HaloTag[3-19] synthetic peptide. The reaction was incubated at room temperature for 3 hours and then combined with 100 nM JF646 HaloTag ligand. Fluorescence intensity was measured after 60 minutes of incubation with the ligand. [Figure 57] Stability and labeling reaction rate of purified HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis in E. coli lysates after complementation with synthetic peptide. 10 nM HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis was combined with 125 μM HaloTag[3-19] synthetic peptide and incubated at 4C or 32C for 10 minutes. After pre-incubation with the peptide at the indicated temperatures, 2.5 nM TMR HaloTag ligand was injected into the reaction mixture, and the labeling reaction rate was observed by monitoring fluorescence polarization over time. [Figure 58] Stability and labeling reaction rate of purified HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis after complementation with synthetic peptide. 10 nM HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis was combined with 125 μM HaloTag[3-19] synthetic peptide and incubated at 4°C or 32°C for 10 minutes. After pre-incubation with the peptide at the indicated temperatures, 2.5 nM TMR HaloTag® ligand was injected into the reaction mixture, and the labeling reaction rate was observed by monitoring fluorescence polarization over time. [Figure 59]Stability and labeling reaction rate of purified HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis after complementation with synthetic peptide. 10 nM HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis was combined with 125 μM HaloTag[3-19] synthetic peptide and incubated at 4°C or 32°C for 10 minutes. After pre-incubation with the peptide at the indicated temperatures, 2.5 nM TMR HaloTag® ligand was injected into the reaction mixture, and the labeling reaction rate was observed by monitoring fluorescence polarization over time. [Figure 60] Stability and labeling reaction rate of purified HaloTag[22-297](M2F+M49F+D53S+V177E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis after complementation with synthetic peptide. 10 nM HaloTag[22-297](M2F+D53S+V177E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis was combined with 125 μM HaloTag[3-19] synthetic peptide and incubated at 4°C or 37°C for 10 minutes. After pre-incubation with the peptide at the indicated temperatures, 2.5 nM TMR HaloTag ligand was injected into the reaction mixture, and the labeling reaction rate was observed by monitoring fluorescence polarization over time. [Figure 61] Stability and labeling reaction rate of purified HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis after complementation with synthetic peptide. 10 nM HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis was combined with 125 μM HaloTag[3-19] synthetic peptide and incubated at 4°C or 32°C for 10 minutes. After pre-incubation with the peptide at the indicated temperatures, 2.5 nM TMR HaloTag ligand was injected into the reaction mixture, and the labeling reaction rate was observed by monitoring fluorescence polarization over time. [Figure 62] Fluorescence intensity of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant co-expressing FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT in living mammalian cells. HeLa cells were transiently transfected with separate plasmids expressing the indicated HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused to FKBP. Approximately 48 hours after transfection, the cells were then labeled with 100 nM JF646 HaloTag ligand, and fluorescence detection was performed at the indicated time points. This figure illustrates the fluorescence intensity of JF646 HaloTag ligands over time in a live-cell assay, comparing the fluorescence activity of cells expressing both HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants with cells expressing only HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. HT-7 refers to the full-length HaloTag. A non-transfected cell (NTC) control was included for reference. Three technically replicated tests were measured for each sample. Error bars indicate the standard deviation (SD) of the mean of the data. [Figure 63]Fluorescence intensity of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant co-expressing FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT in living mammalian cells. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused to FKBP. Approximately 48 hours after transfection, the cells were then labeled with 100 nM JF646 HaloTag ligand, and fluorescence detection was performed at the indicated time points. This figure illustrates the fluorescence intensity of JF646 HaloTag ligands over time in a live-cell assay, comparing the fluorescence activity of cells expressing both HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants with cells expressing only HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. HT-7 refers to the full-length HaloTag. A non-transfected cell (NTC) control was included for reference. Three technically replicated tests were measured for each sample. Error bars indicate the standard deviation (SD) of the mean of the data. [Figure 64]This study compared the response factor of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant co-expressing FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT in living mammalian cells. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused to FKBP. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag ligand, and fluorescence was detected at the indicated time points. The response factor for each assay condition was calculated as the ratio of the fluorescence signal to cells expressing both the HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutants to the ratio to cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant. HT-7 refers to the full-length HaloTag. Non-transfected cells (NTCs) were included as a reference. Error bars indicate the standard deviation (SD) of the mean of the data. [Figure 65]Comparison of fluorescence intensity and fluorescence response magnification of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutants. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant and HaloTag[3-19]-5xGly / Ser-VS-HiBiT fused to FKBP. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag ligand. Fluorescence was detected after incubation with JF646 ligand for 5 hours. The response factor was calculated as the ratio of the fluorescence signal to cells expressing both the FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutants to the ratio to cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant. In this graph, M2F represents the template HaloTag[22-297](M2F)-12xGly / Ser-LgBiT construct. All dual mutants are added to the template, but for each construct, only the two added mutations are shown. Non-transfected cells (NTCs) were included as a reference. This plot shows a comparison of the response ratios to total fluorescence for each mutant, revealing that the performance of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant is improved in the presence of co-expressed FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT. [Figure 66]Fluorescence intensity of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant in live cell fluorescence plate assay. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant and HaloTag[3-19]-5xGly / Ser-VS-HiBiT fused to FKBP. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag ligand. Fluorescence intensity was detected after incubation with JF646 HaloTag ligand for 5 hours. This figure illustrates the time-course fluorescence intensity of JF646 HaloTag ligand in a live-cell assay, comparing the fluorescence activity of cells expressing both the HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutants with cells expressing only the HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutants. Three technical replicates were measured for each sample. Error bars indicate the standard deviation (SD) of the mean of the data. [Figure 67]Fluorescence response intensification of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant in a live cell fluorescence plate assay. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused to FKBP. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. Fluorescence activity was detected after 5 hours of incubation with JF646 ligand. The response factor for each assay condition was calculated as the ratio of the fluorescence signal to cells expressing both the HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutants to the ratio to cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant. This ratio was then normalized to the response factor of the template HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. Error bars indicate the standard deviation (SD) of the mean of the data. [Figure 68] Fluorescence activity of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant when co-expressed with FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT in living cells. HeLa cells were transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused to FKBP. Approximately 48 hours after transfection, cells were treated with a luminescent substrate for living cells (flimazine). The shown non-transfected cell (NTC) control was measured similarly except that it did not have the expression plasmid introduced. Three technical replicates were measured for each sample. Error bars indicate the standard deviation (SD) of the mean of the data. [Figure 69]Fluorescence activity of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant when co-expressed with FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT in living cells. HeLa cells were transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant fragment and HaloTag[3-19]-5xGly / Ser-VS-HiBiT fused to FKBP. Approximately 48 hours after transfection, cells were treated with a luminescent substrate for living cells (flimazine). Three technical replicates were measured for each sample. Error bars indicate the standard deviation (SD) of the mean of the data. [Figure 70] Expression of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant. HeLa cells were transiently transfected with a plasmid expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant. Approximately 48 hours after transfection, the luminescence signal intensity after cell lysis was measured by adding HiBiT peptide at a final concentration of 200 nM along with a luminescent substrate (flimazine). To prevent signal saturation and improve signal linearity, the initial cell sample was diluted 80-fold in Opti-MEM medium. An untransfected cell (NTC) control was included as a reference. Three technical replicates were measured for each sample. Error bars indicate the standard deviation (SD) of the mean of the data. [Figure 71]Comparison of response factor and expression of LgHT-LgBiT HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutants. To measure the response factor, HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant fragment and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused to FKBP. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. Fluorescence was detected after incubation with JF646 HaloTag® ligand for 5 hours. The response factor was calculated as the ratio of the fluorescence signal to cells expressing both the FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutants to the ratio to cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant. In this graph, M2F represents the template HaloTag[22-297](M2F)-12xGly / Ser-LgBiT construct. All dual mutants are added to the template, but for brevity, only the two added mutations are shown for each construct. Non-transfected cells (NTCs) were included as a reference. [Figure 72] Expression of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant. HeLa cells were transiently transfected with a plasmid expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant. Approximately 48 hours after transfection, the luminescence signal intensity after cell lysis was measured by adding HiBiT peptide at a final concentration of 200 nM along with a luminescent substrate (flimazine). To prevent signal saturation and improve signal linearity, the initial cell sample was diluted 80-fold in Opti-MEM medium. An untransfected cell (NTC) control was included as a reference. Three technical replicates were measured for each sample. Error bars indicate the standard deviation (SD) of the mean of the data. [Figure 73] Expression of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant. HeLa cells were transiently transfected with a plasmid expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant fragment. Approximately 48 hours after transfection, the luminescence signal intensity after cell lysis was measured by adding HiBiT peptide at a final concentration of 200 nM along with a luminescent substrate (flimazine). To prevent signal saturation and improve signal linearity, the initial cell sample was diluted 80-fold in Opti-MEM medium. Four technical replicates were measured for each sample. Error bars indicate the standard deviation (SD) of the mean of the data. [Figure 74] Expression variability of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant across transfection repeats. HeLa cells were transiently transfected with a plasmid expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant fragment. Approximately 48 hours after transfection, cells were lysed by adding a final concentration of 200 nM HiBiT peptide along with a lysing luminescent substrate (flimazine), and the luminescence signal intensity was subsequently measured. To prevent signal saturation and improve signal linearity, the initial cell sample was diluted 80-fold in Opti-MEM medium. Three technical repeats were measured for each sample. Error bars indicate the standard deviation (SD) of the mean of the data. [Figure 75]Expression variability of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant across transfection repeats. HeLa cells were transiently transfected with a plasmid expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. Approximately 48 hours after transfection, cells were lysed by adding a final concentration of 200 nM HiBiT peptide along with a lysing luminescent substrate (flimazine), and the luminescence signal intensity was subsequently measured. To prevent signal saturation and improve signal linearity, the initial cell sample was diluted 80-fold in Opti-MEM medium. Several technical repeats were measured for each transfection repeat. Error bars indicate the standard deviation (SD) of the mean of the data. This graph shows the observed variability of the lysed luminescence plate assay. The expression levels of the template across multiple transfection repeats allow for normalization of experimental results obtained from different repeats to the template. [Figure 76A-B]This is a density plot of untransfected cells in a cell cytometry assay for filtering dead cells, cell debris, doublets, and cell aggregates in a cell cytometry assay. Approximately 48 hours after seeding, untransfected HeLa cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with the ligand for 1 hour, the cells were washed in DPBS medium for 30 minutes (twice for 15 minutes each). The cells were then detached and their fluorescence activity was measured using a cell cytometry assay. The density plot of untransfected cells was used in a cell cytometry assay to remove dead cells, cell debris, doublets, and cell aggregates from a single population of living HeLa cells. A. The lateral scattering region (SSC-A) of untransfected cells was plotted against the forward scattering region (FSC-A), which effectively removes dead cells and cell debris. The same gating strategy was applied to every single sample in the cell cytometry assay to effectively remove dead cells, cell debris, and dilumas, ensuring accurate analysis of a single population of living HeLa cells. In all cytometry experiments listed in this document, the total number of events in the non-gated population was 10,000–12,000 cells. The numbers on the graphs indicate the proportion of the cell population within each gate depicted. B. This is a density plot showing how the forward scattering height (FSC-H) relative to the forward scattering area (FSC-A) of non-transfected cells effectively removes dilumas and cell aggregates. The same gating strategy was applied to every single sample in the cell cytometry assay to effectively remove dead cells, cell debris, and dilumas, ensuring accurate analysis of a single population of living HeLa cells. In all cytometry experiments listed in this document, the total number of events in the non-gated population was 10,000–12,000 cells. The numbers on the graphs indicate the proportion of the cell population within each gate depicted. [Figure 77]Gating strategy for untransfected HeLa cells for JF646-positive cell detection. Approximately 48 hours after seeding, untransfected HeLa cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with the ligand for 1 hour, the cells were washed in DPBS medium for 30 minutes (two 15-minute washes). The cells were then detached and their fluorescence activity was measured using a live cell cytometry assay. Untransfected samples were used for gating and activity measurement of transfected cells compared to untransfected or non-fluorescent cells. Histograms illustrate cell count (Y axis) against activity with JF646 ligand (X axis). A consistent gating strategy based on baseline activity detected in untransfected cells was applied to all transfected samples to accurately detect their fluorescence activity. Initial event numbers ranged from 10,000 to 12,000 cells. Specifically, in the case of JF646 activity analysis, the number of events analyzed is typically 2,000 to 4,000. The cells analyzed with the JF646+ gate depend on the transfection efficiency of the mutant and are based on the gate setting for JF646 activity. [Figure 78]Luminescence intensity of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant co-expressed with HaloTag[3-19]-5xGly / Ser-VS-HiBiT in the JF646+ gate, as measured by flow cytometry. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused to FKBP. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with ligand for 1 hour, cells were washed in DPBS medium for 30 minutes (twice for 15 minutes each). Subsequently, the cells were detached, and their fluorescence activity in the JF646+ gate was measured using a cell cytometry assay. Three technical replicates were measured for each sample. Error bars indicate the standard deviation (SD) of the mean of the data. [Figure 79]Comparison of fluorescence response intensities of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutants co-expressing FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT using flow cytometry. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant fragment and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused to FKBP. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with ligand for 1 hour, cells were washed in DPBS medium for 30 minutes (twice for 15 minutes each). Subsequently, the cells were detached, and their fluorescence activity in the JF646+ gate was measured using a cell cytometry assay. The response factor for each assay condition was calculated as the ratio of the mean fluorescence signal in the JF646+ gate for cells expressing both the FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants to the mean fluorescence intensity of cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant in the same JF646+ gate. Error bars indicate the standard deviation (SD) of the mean data. [Figure 80]Comparison of fluorescence intensity and response factor of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutants, measured by flow cytometry. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused to FKBP. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with ligand for 1 hour, cells were washed in DPBS medium for 30 minutes (twice for 15 minutes each). Subsequently, the cells were detached and their fluorescence activity at the JF646+ gate was measured using a cell cytometry assay. The response factor for each assay condition was calculated as the ratio of the mean fluorescence signal at the JF646+ gate for cells expressing both the FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutants to the mean fluorescence signal for cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant at the same JF646+ gate. In this graph, M2F represents the template HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. All dual mutants are added to the template, but for each construct, only the two mutations added to match the data are shown. Non-transfected cells (NTCs) were included as a reference. This plot shows a comparison of the response ratios to the total fluorescence activity of each mutant, revealing that the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant performs better when co-expressed with FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT. [Figure 81A-C]The fluorescence intensities of FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutants during co-expression, as measured by flow cytometry. (A) The histogram of mutants exhibiting a high response factor phenotype shows how the performance of the template HaloTag[22-297](M2F)-12xGly / Ser-LgBiT, as measured by flow cytometry, can change due to mutation. Cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant are shown in light gray, and cells co-expressing FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT are shown in dark gray. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused to FKBP. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with ligand for 1 hour, cells were washed in DPBS medium for 30 minutes (two 15-minute washes). Subsequently, cells were detached and their fluorescence activity was measured using a cell cytometry assay. In this graph, M2F represents the template HaloTag[22-297](M2F)-12xGly / Ser-LgBiT construct. All double mutants are added to the template, but only the two added mutants are shown to illustrate the data for each construct. The "Normalized to Mode" on the Y-axis scales the event count values ​​based on the most frequently occurring values, and in this graph, it shows the event count for the JF646 negative gate. This allows for standardized comparisons and reveals relative differences between populations. (B) Histograms of mutants representing low response magnification phenotypes show how the performance of the template HaloTag[22-297](M2F)-12xGly / Ser-LgBiT, measured using flow cytometry, can change with mutations.Cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant are shown in light gray, and cells co-expressing FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT are shown in dark gray. Cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant are shown in light gray, and cells co-expressing FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT are shown in dark gray. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused to FKBP. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with ligand for 1 hour, cells were washed in DPBS medium for 30 minutes (two 15-minute washes). Subsequently, cells were detached and their fluorescence activity was measured using a cell cytometry assay. In this graph, M2F represents the template HaloTag[22-297](M2F)-12xGly / Ser-LgBiT construct. All double mutants are added to the template, but only the two added mutants are shown to illustrate the data for each construct. The "Normalized to Mode" on the Y-axis scales the event count values ​​based on the most frequently occurring values, and in this graph, it shows the event count for the JF646 negative gate. This allows for standardized comparisons and reveals relative differences between groups. [Figure 82]Luminescence intensity of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant co-expressed with FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT, as measured by flow cytometry in a JF646+ gate. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant fragment and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused to FKBP. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with ligand for 1 hour, cells were washed in DPBS medium for 30 minutes (twice for 15 minutes each). Subsequently, the cells were detached, and their fluorescence activity in the JF646+ gate was measured using flow cytometry. Three technical replicates were measured for each sample. Error bars indicate the standard deviation (SD) of the mean of the data. [Figure 83]Comparison of fluorescence response intensities of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant co-expressed with FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT, measured by flow cytometry in living mammalian cells. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant fragment and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused to FKBP. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with ligand for 1 hour, cells were washed in DPBS medium for 30 minutes (twice for 15 minutes each). Subsequently, the cells were detached, and their fluorescence activity in the JF646+ gate was measured using a cell cytometry assay. The response factor for each assay condition was calculated as the ratio of the mean fluorescence signal in the JF646+ gate for cells expressing both the FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants to the mean fluorescence signal for cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant in the same JF646+ gate. Error bars indicate the standard deviation (SD) of the mean data. [Figure 84]Luminescence intensity of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant co-expressed with FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT in the JF646+ gate, measured by flow cytometry. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant fragment and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused to FKBP. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with ligand for 1 hour, cells were washed in DPBS medium for 30 minutes (twice for 15 minutes each). Subsequently, the cells were detached, and their fluorescence activity in the JF646+ gate was measured using a cell cytometry assay. Two technical replicates were measured for each sample. Error bars indicate the standard deviation (SD) of the mean of the data. [Figure 85]Comparison of fluorescence response intensities of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant co-expressed with FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT, measured by flow cytometry in living mammalian cells. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant fragment and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused to FKBP. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with ligand for 1 hour, cells were washed in DPBS medium for 30 minutes (twice for 15 minutes each). Subsequently, the cells were detached, and their fluorescence activity in the JF646+ gate was measured using a cell cytometry assay. The response factor for each assay condition was calculated as the ratio of the mean fluorescence signal in the JF646+ gate for cells expressing both the HaloTag[3-19]-5xGly / Ser-VS-HiBiT and FKBP-HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutants to the mean fluorescence signal for cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant in the same JF646+ gate. Error bars indicate the standard deviation (SD) of the mean data. [Figure 86]Luminescence intensity of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant co-expressed with FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT, measured by flow cytometry after prolonged washing in a JF646+ gate. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant fragment and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused to FKBP. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with ligand for 1 hour, cells were washed in DPBS medium for 2.5 hours (two 75-minute washes). Subsequently, the cells were detached, and their fluorescence activity in a JF646+ gate was measured using flow cytometry. Two technical replicates were measured for each sample. Error bars indicate the standard deviation (SD) of the mean of the data. [Figure 87]Comparison of fluorescence response intensities of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant co-expressed with the FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT mutant, measured by flow cytometry after prolonged washing in living mammalian cells. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused to FKBP. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with the ligand for 1 hour, cells were washed in DPBS medium for 2.5 hours (two 75-minute washes). Subsequently, the cells were detached, and their fluorescence activity in the JF646+ gate was measured using a cell cytometry assay. The response factor for each assay condition was calculated as the ratio of the mean fluorescence signal in the JF646+ gate for cells expressing both the HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants to the mean fluorescence signal for cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant in the same JF646+ gate. Error bars indicate the standard deviation (SD) of the mean data. [Figure 88]Luminescence activity of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant co-expressed with FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT was measured by flow cytometry in a JF646+ gate. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused to FKBP. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with ligand for 1 hour, cells were washed in DPBS medium for 30 minutes (twice for 15 minutes each). Subsequently, the cells were detached, and their fluorescence activity at the JF646+ gate was measured using a cell cytometry assay. Live cell cytometry assays revealed relatively consistent levels of fluorescence activity normalized to the template HaloTag[22-297](M2F)-12xGly / Ser-LgBiT between the first and second transfection repeats of the complementary FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants. [Figure 89]Fluorescence response intensities of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant co-expressed with FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT, measured by flow cytometry in living mammalian cells. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused to FKBP. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with ligand for 1 hour, cells were washed in DPBS medium for 1 hour (twice for 15 minutes each). Subsequently, the cells were detached, and their fluorescence activity at the JF646+ gate was measured using a live-cell cytometry assay. The response factor for each assay condition was calculated as the ratio of the mean fluorescence signal at the JF646+ gate for cells expressing both the FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutants to the mean fluorescence signal for cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant at the same JF646+ gate. Flow cytometry revealed a relatively consistent level of fluorescence factor response, normalized to the template HaloTag[22-297](M2F)-12xGly / Ser-LgBiT construct, between the first and second transfection repeats. [Figure 90]Variability of luminescence activity across transfection repeats of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutants co-expressed with FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT, as measured by flow cytometry in a JF646+ gate. HeLa cells were transiently transfected with separate plasmids expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragments fused to FKBP. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with ligand for 1 hour, cells were washed in DPBS medium for 30 minutes (twice for 15 minutes each). Subsequently, the cells were detached, and their fluorescence activity in the JF646+ gate was measured using flow cytometry. The expression levels of the template across multiple transfection repeats allow for normalization of experimental results obtained from different repeats to the template. [Figure 91]Comparison of fluorescence intensity and response factor of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant co-expressed with FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT using flow cytometry. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused to FKBP. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with ligand for 1 hour, cells were washed in DPBS medium for 30 minutes (twice for 15 minutes each). Subsequently, the cells were detached, and their fluorescence activity in the JF646+ gate was measured using flow cytometry. The response factor for each assay condition was calculated as the ratio of the mean fluorescence signal in the JF646+ gate for cells expressing both the FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutants to the mean fluorescence signal for cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant in the same JF646+ gate. The four transfection repeats for the template HaloTag[22-297](M2F)-12xGly / Ser-LgBiT construct are shown in darker gray. All single and double mutants were added to the template HaloTag[22-297](M2F)-12xGly / Ser-LgBiT construct, but only the added mutants are shown for each construct. This plot reveals a comparison of the response ratios to the total fluorescence activity of each mutant and highlights the improved performance of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant when co-expressed with FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT. [Figure 92]Gating of untransfected cells in flow cytometry for the separation of single and double-positive cells for JF646 and EGFP signaling. Untransfected HeLa cells were labeled with 100 nM JF646 HaloTag® ligand approximately 48 hours after seeding. After incubation with the ligand for 1 hour, the cells were washed in DPBS medium for 30 minutes (twice for 15 minutes each). The cells were then detached and their fluorescence activity was measured using a live cell cytometry assay. Using density plots of untransfected cells, gating regions were established: JF646-negative (Q4) and JF646-positive (Q3), as well as EGFP expression-negative (Q4) and EGFP expression-positive (Q1). As a result, double-negative cells were identified in Q4, and single-positive cells for JF646 and EGFP were observed in Q3 and Q1, respectively. Double-positive cells were identified in Q2. [Figure 93A-B]Comparison of cells expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT with cells expressing EGFP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT using flow cytometry. (A) HeLa cells were transiently transfected with separate plasmids expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT alone on separate plasmids. Approximately 48 hours after transfection, the cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with the ligand for 1 hour, the cells were washed in DPBS medium for 30 minutes (twice for 15 minutes each). Subsequently, the cells were detached and their fluorescence activity was measured using flow cytometry. As illustrated, when both the EGFP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag22-297-12xGly / Ser-LgBiT mutants are expressed, a large shift in cells from the Q3 quadrant to the Q2 quadrant is observed compared to when only HaloTag22-297-12xGly / Ser-LgBiT is expressed. This shift indicates the formation of a complex between the HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag22-297-12xGly / Ser-LgBiT mutants, or the co-expression of both EGFP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag22-297-12xGly / Ser-LgBiT components within the cell. (B) HeLa cells were transiently transfected with separate plasmids co-expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and EGFP-HaloTag[3-19]-5xGly / Ser-V or S-HiBiT on separate plasmids. Approximately 48 hours after transfection, the cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with the ligand for 1 hour, the cells were washed in DPBS medium for 30 minutes (twice for 15 minutes each). The cells were then detached and their fluorescence activity was measured using flow cytometry.As illustrated, when both the EGFP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag22-297-12xGly / Ser-LgBiT mutants are expressed, a large shift in cells from the Q3 quadrant to the Q2 quadrant is observed compared to when only HaloTag22-297-12xGly / Ser-LgBiT is expressed. This shift indicates the formation of a complex between the HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag22-297-12xGly / Ser-LgBiT mutants, or the co-expression of both EGFP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag22-297-12xGly / Ser-LgBiT components within the cell. [Figure 94] Luminescence intensity of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant co-expressed with EGFP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT, measured by flow cytometry. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused to EGFP. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with ligand for 1 hour, cells were washed in DPBS medium for 30 minutes (twice for 15 minutes each). Subsequently, the cells were detached and their fluorescence activity was measured by flow cytometry. Fluorescence intensity was determined based on the mean intensity in the second (double-positive) quadrant, while the fluorescence intensity of samples containing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant alone was measured based on the mean fluorescence intensity in the third (JF646+) quadrant. Two technical replicates were performed for each sample. Error bars indicate the standard deviation (SD) of the mean data. [Figure 95]Comparison of fluorescence response intensities of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant co-expressed with EGFP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT, as measured by low cytometry using JF646 HaloTag ligand in living mammalian cells. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused to EGFP. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with the ligand for 1 hour, cells were washed in DPBS medium for 30 minutes (twice for 15 minutes each). Subsequently, the cells were detached, and their fluorescence activity was measured using flow cytometry. Fluorescence activity within co-expression samples was determined based on the average intensity in the second (double-positive) quadrant, while the fluorescence activity of samples containing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mono-mutant was measured based on the average fluorescence intensity in the third (JF646+) quadrant. The response factor for each assay condition was calculated as the ratio of the average fluorescence signal in the second (double-positive) quadrant to the average fluorescence signal in the third (JF646+) quadrant. [Figure 96]Comparison of fluorescence response intensities of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant co-expressed with EGFP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT, measured by flow cytometry using JF635 HaloTag ligand in living mammalian cells. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant fragment and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused to EGFP. Approximately 48 hours after transfection, cells were labeled with 100 nM JF635 HaloTag® ligand. After incubation with the ligand for 1 hour, cells were washed in DPBS medium for 30 minutes (twice for 15 minutes each). Subsequently, the cells were detached, and their fluorescence activity was measured using flow cytometry. Fluorescence activity within co-expression samples was determined based on the average intensity in the second (double-positive) quadrant, while the fluorescence activity of samples containing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT monomutant was measured based on the average fluorescence intensity in the third (JF646+) quadrant. The response factor for each assay condition was calculated as the ratio of the average fluorescence signal in the second (double-positive) quadrant to the average fluorescence signal in the third (JF646+) quadrant. The results demonstrate that the response factor is enhanced when using the more fluorescence-generating ligand JF635 compared to the less fluorescence-generating ligand JF646. [Figure 97]Comparison of fluorescence intensity and response factor of selected HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutants, measured by flow cytometry. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused to EGFP. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with ligand for 1 hour, cells were washed in DPBS medium for 30 minutes (twice for 15 minutes each). Subsequently, the cells were detached and their fluorescence activity at the JF646+ gate was measured using flow cytometry. The response factor for each assay condition was calculated as the ratio of the mean fluorescence signal at the JF646+ gate for cells expressing both the EGFP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutants to the mean fluorescence signal for cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant at the same JF646+ gate. Two transfection repeats for the template HaloTag[22-297](M2F)-12xGly / Ser-LgBiT are shown in dark gray. This plot highlights the increased fluorescence intensity of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant when co-expressed with EGFP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT. [Figure 98A-C]Luminescence of HeLa cells co-expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant and EGFP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT was observed using confocal imaging. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused to EGFP. For comparison, cells were also transfected with the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant alone. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour incubation, cells were imaged using a confocal microscope in both the green channel (Ex. 488 nm) and the far-red channel (Ex. 640 nm). Laser and gain settings were optimized for each construct to minimize saturated pixels. Row (A) shows the co-expression signal and single-plasmid control signal for HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. Row (B) shows the co-expression signal and single-plasmid control signal for HaloTag[22-297](M2F+D53G+F148M)-12xGly / Ser-LgBiT. Row (C) shows the co-expression signal and single-plasmid control signal for HaloTag[22-297](M2F+D53G+V177E)-12xGly / Ser-LgBiT. [Figure 99]Quantified luminescence in HeLa cells co-expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant and EGFP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT was measured using confocal imaging. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused to EGFP. Furthermore, cells were transfected with only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant, and the signal from background labeling was measured. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After a 1-hour incubation, cells were imaged using a confocal microscope in both the green channel (Ex. 488 nm) and the far-red channel (Ex. 640 nm). Laser and gain settings were optimized for each construct to minimize saturated pixels. To quantify the confocal images, multiple fields of view were collected for each sample, and the intensity of individual cells was measured for both co-expression and background signals. The bars represent the average values ​​of the data. The response magnification shown for the samples above was calculated as the ratio of the specific signal for cells expressing both the FEGFP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants to the average signal intensity for cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant. This graph illustrates how the performance of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant is improved by mutating the template when EGFP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT is co-expressed in confocal imaging applications. [Figure 100]CRISPR knock-in efficiency at the endogenous PARP1 and CTNNB1 loci of the DLD-1 cell pool during the introduction period of the HaloTag[3-19]-VS-HiBiT mutant. The mutants included HaloTag[3-19]-VS-HiBiT without a linker and HaloTag[3-19]-VS-HiBiT mutant with a 4xGly / Ser linker (GGSG-HaloTag[3-19]-GGSG VS-HiBiT). Data were collected using droplet digital polymerase chain reaction. Knock-in efficiency was calculated as the ratio of PCR amplicon copies per volume containing tagged sequences to untagged sequences at the target locus. Error bars indicate the standard deviation (SD) of the mean of the data. [Figure 101] Expression of PARP1 and CTNNB1 tagged with the HaloTag[3-19]-VS-HiBiT mutant or HiBiT in a CRISPR pool. The luminescence signal intensity of a DLD-1 CRISPR cell pool (40,000 cells per pool repeat) was measured by adding LgBiT protein along with a luminescent substrate (flimazine) after cell lysis. Six technical replicates were tested for each construct's CRISPR pool. The results confirm the integration and functionality of each tag. Furthermore, the addition of a linker in the HaloTag[3-19]-GGSG-VS-HiBiT mutant improves detection. To ensure that luminescence activity is not affected by the knock-in efficiency of the inserts in the CRISPR pool, pools showing relatively similar knock-in efficiencies were selected for this experiment. Error bars indicate the standard deviation (SD) of the mean of the data. [Figure 102]In CRISPR clones, endogenous expression of PARP1 fused to the HaloTag[3-19]-VS-HiBiT mutant or HiBiT was investigated. The luminescence signal intensity of DLD-1 CRISPR cell clones (40,000 cells per clone for each replicate) was measured after cell lysis by adding LgBiT protein and a luminescent lysis substrate (flimazine). Multiple technical replicates were performed for each construct's CRISPR clone. These results successfully confirm the integration and functionality of each tag. Furthermore, the addition of a linker in the HaloTag[3-19]-GGSG-VS-HiBiT mutant improved detection at the CRISPR clone level. Error bars indicate the standard deviation (SD) of the mean of the data. [Figure 103A-C]Gating strategy for untransfected PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells against JF646-positive cells. Approximately 48 hours after seeding, DLD-1 cell lines expressing PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT were labeled with 100 nM JF646 HaloTag® ligand. After incubation with ligand for 1 hour, cells were washed in DPBS medium for 30 minutes (two 15-minute cycles). Subsequently, cells were cleaved and their fluorescence intensity was measured using flow cytometry. Density plots of untransfected cells were used in cell cytometry assays to remove dead cells, cell debris, doublets, and cell aggregates from a single population of DLD-1 cells. (A) The lateral scattering region (SSC-A) of untransfected cells is plotted against the forward scattering region (FSC-A), effectively removing dead cells and cell debris. (B) The height of the forward scattering (FSC-H) is plotted against the forward scattering (FSC-A) region, showing the density plot of untransfected cells, effectively removing diplos and cell aggregates. The same gating strategy was applied to every single sample in the cell cytometry assay to effectively remove dead cells, cell debris, and diplos, ensuring accurate analysis of a single population of living DLD-1 cells. (C) The histogram shows cell number on the Y axis and intensity with JF646 ligand on the X axis. A consistent gating strategy, established using baseline activity detected in untransfected cells, was applied to each transfected sample to accurately identify and quantify their fluorescence activity. Prior to this step, the gating strategy was consistent with previous cytometry experiments in excluding dead cells, cell debris, and cell aggregates. In all cytometry experiments listed in this document, the total number of events in the non-gated population is 10,000–12,000 cells. The numbers on the graphs indicate the proportion of cell populations within each gate depicted. Specifically, in the case of activity analysis of JF646, the number of events analyzed is typically 2,000–4,000.Cells analyzed with the JF646+ gate depend on the transfection efficiency of the mutant and are based on the gate setting for JF646 activity. [Figure 104] Comparison of fluorescence intensity of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT transiently expressed using different promoters in the PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cell line. DLD-1 cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT were transiently transfected with a plasmid expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT under the control of the CMV or TK promoter. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation for 1 hour, cells were washed in DPBS medium for 30 minutes (twice for 15 minutes each). Subsequently, the cells were cleaved and their fluorescence activity was measured using flow cytometry. The gating strategy used was consistent with previous cytometry experiments that excluded dead cells, cell debris, and cell aggregates. Furthermore, labeled CRISPR cells and untransfected CRISPR cells were used to distinguish between JF646-positive and JF646-negative populations. The x-axis values ​​correspond to the amount of plasmid DNA expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT used for transient transfection in each well of a 96-well plate. Error bars indicate the standard deviation (SD) of the mean of the data. This graph illustrates that, at all plasmid DNA concentrations, the fluorescence signal intensity at the JF646-positive gate is approximately 10 times higher when the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant is expressed under the CMV promoter compared to expression under the TK promoter. [Figure 105]Comparison of response factor and fluorescence intensity of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT transiently expressed using different promoters in the PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cell line. DLD-1 cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT were transiently transfected with a plasmid expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT under the control of the CMV or TK promoter. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation for 1 hour, cells were washed in DPBS medium for 30 minutes (twice for 15 minutes each). Subsequently, the cells were cleaved and their fluorescence activity was measured using flow cytometry. The response factor for each assay condition was calculated as the ratio of the mean fluorescence signal at the JF646+ gate for PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells transiently expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT to the mean fluorescence signal of parental DLD-1 cells transiently expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT at the same JF646+ gate. The x-axis value corresponds to the amount of plasmid DNA expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT used to transiently transfect cells in each well of a 96-well plate. Error bars indicate the standard deviation (SD) of the mean data. These results indicate that the response factor can be improved, particularly under promoters that express low levels of HaloTag22-297-12xGly / Ser-LgBiT (e.g., the TK promoter), by adjusting the concentration of plasmid DNA expressing HaloTag22-297-12xGly / Ser-LgBiT. [Figure 106A-C]Comparison of the promoter's effect on the proportion of cells labeled with HaloTag ligands in endogenously tagged PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells and parental DLD-1 cells transiently expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. (A) Parental DLD-1 cells and DLD-1 cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT were transiently transfected with different concentrations of plasmid expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT under the control of the CMV promoter. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation for 1 hour, the cells were washed in DPBS medium for 30 minutes (two 15-minute cycles). The cells were then cleaved and their fluorescence activity was measured using flow cytometry. The x-axis values ​​correspond to the amount of plasmid DNA encoding HaloTag[22-297](M2F)-12xGly / Ser-LgBiT used for transient transfection in each well of a 96-well plate. Error bars represent the standard deviation (SD) of the mean data. This graph illustrates that the proportion of DLD-1 cells overexpressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant under the CMV promoter in a JF646+ gate is generally higher than the corresponding proportion of CRISPR cells overexpressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant under the TK promoter. (B) Parental DLD-1 cells and DLD-1 cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT were transiently transfected with different concentrations of plasmid expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT under the control of the TK promoter. Approximately 48 hours after transfection, the cells were labeled with 100 nM JF646 HaloTag® ligand.After incubation for 1 hour, the cells were washed in DPBS medium for 30 minutes (two 15-minute cycles). The cells were then cleaved and their fluorescence activity was measured using flow cytometry. The x-axis values ​​correspond to the amount of plasmid DNA encoding HaloTag[22-297](M2F)-12xGly / Ser-LgBiT used for transient transfection in each well of a 96-well plate. Error bars represent the standard deviation (SD) of the mean data. This graph illustrates that the proportion of DLD-1 cells overexpressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant under the CMV promoter in a JF646+ gate is generally higher than the corresponding proportion of CRISPR cells overexpressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant under the TK promoter. (C) DLD-1 cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT were transiently transfected with different concentrations of plasmid expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT under the control of a CMV or TK promoter. Approximately 48 hours after transfection, the cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation for 1 hour, the cells were washed in DPBS medium for 30 minutes (twice for 15 minutes each). The cells were then cleaved and their fluorescence activity was measured using flow cytometry. The x-axis values ​​correspond to the amount of plasmid DNA encoding HaloTag[22-297](M2F)-12xGly / Ser-LgBiT used for transient transfection in each well of a 96-well plate. Error bars indicate the standard deviation (SD) of the mean of the data.This graph illustrates that the proportion of DLD-1 cells overexpressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant under the CMV promoter in the JF646+ gate is generally higher than the corresponding proportion of CRISPR cells overexpressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant under the TK promoter. [Figure 107]The multiplier difference in the proportion of cells labeled with HaloTag ligands in endogenously tagged PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells transiently expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT under different promoters. DLD-1 cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT were transiently transfected with a plasmid expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT under the control of either the CMV or TK promoter. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation for 1 hour, the cells were washed in DPBS medium for 30 minutes (two 15-minute cycles). The cells were then cleaved and their fluorescence activity was measured using flow cytometry. The response factor for each assay condition was calculated as the ratio of the parental frequency of PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT in the JF646+ gate to the parental frequency of parental DLD-1 cells transiently expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT in the same JF646+ gate. The x-axis values ​​correspond to the amount of plasmid DNA encoding HaloTag[22-297](M2F)-12xGly / Ser-LgBiT used for transient transfection in each well of a 96-well plate. Error bars indicate the standard deviation (SD) of the mean of the data. This graph illustrates that the overall fluorescence intensity with the TK promoter is approximately 1 log lower compared to expression with the CMV promoter, resulting in a lower proportion of cells in the JF646+ gate (lower frequency of parental values), and the fold difference of tagged CRISPR cells to parental cells in the JF646+ gate is higher with the TK promoter compared to the CMV promoter.The response rate is maximized when the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT plasmid DNA concentration used for expression with the TK promoter is approximately 10-40 ng / well. [Figure 108A-B]Comparison of fluorescence intensity between HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT in flow cytometry. (A) Overlay histogram of PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells transiently expressing either HaloTag[22-297](M2F)-12xGly / Ser-LgBiT (light gray) or its variant HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT (dark gray). The "normalized to mode" on the Y axis scales the event count value based on the most frequently occurring value, and in this graph, the event count is shown for the JF646 negative gate. This allows for standardized comparisons and reveals relative differences between populations. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour incubation, cells were washed in DPBS medium for 30 minutes (twice for 15 minutes each). Subsequently, cells were cleaved and their fluorescence activity was measured using flow cytometry. The gating strategy used to exclude dead cells, cell debris, and cell aggregates is consistent with previous cytometry experiments. This graph illustrates that the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant shows a higher mean fluorescence intensity within the gating applied to JF646 activity. Error bars indicate the standard deviation (SD) of the mean of the data. (B) Mean fluorescence intensity of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or mutants containing the K140E mutation in PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells within a JF646-positive gate. Cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT were transiently transfected with 20 ng / well of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or mutant plasmids in a 96-well plate.Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour incubation, cells were washed in DPBS medium for 30 minutes (twice for 15 minutes each). Subsequently, cells were cleaved and their fluorescence activity was measured using flow cytometry. Two technical replicates were tested for each construct. The gating strategy used to exclude dead cells, cell debris, and cell aggregates was consistent with previous cytometry experiments. This graph illustrates that the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant shows a higher mean fluorescence intensity within the gating applied for JF646 activity. Error bars indicate the standard deviation (SD) of the mean of the data. [Figure 109A-B]Comparison of fluorescence response magnifications at a JF646-positive gate between HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT mutants using flow cytometry. (A) Overlay histograms of PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells transiently expressing either HaloTag[22-297](M2F)-12xGly / Ser-LgBiT (left) or its variant HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT (right). For each mutant histogram, the light gray histograms are from CRIPSR cells transiently expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant, and the darker gray histograms are from parental DLD-1 cells transiently expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant. The "Normalized to Mode" on the Y-axis scales the number of events based on the most frequently occurring value, and in this graph, it shows the number of events for the JF646 negative gate. This allows for standardized comparisons and reveals relative differences between populations. (B) Response factor of HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT mutant in the JF646 positive gate. Cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT were transiently transfected in wells of a 96-well plate with either HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or the mutant plasmid (20 ng / well). Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation for 1 hour, the cells were washed with DPBS medium for 30 minutes (twice for 15 minutes each). The cells were then detached, and their fluorescence activity was measured using flow cytometry.The response factor for each assay condition was calculated as the ratio of the mean fluorescence signal at the JF646+ gate to PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells transiently expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT, compared to the mean fluorescence signal at the same JF646+ gate to parental DLD-1 cells expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. The gating strategies used to exclude dead cells, cell debris, and cell aggregates were consistent with previous cytometry experiments. Error bars indicate the standard deviation (SD) of the mean of the data. [Figure 110A-B]Effect of the K140E mutation on the retinometric difference of JF646-positive cells in endogenously tagged PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells transiently expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. (A) DLD-1 cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT were transiently transfected with 20 ng / well plasmids expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation for 1 hour, the cells were washed in DPBS medium for 30 minutes (twice for 15 minutes each). Subsequently, the cells were cleaved and their fluorescence activity was measured using flow cytometry. The response factor for each assay condition was calculated as the ratio of the parental frequency of PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant to the parental frequency of DLD-1 parent cells expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant in the same JF646+ gate. The gating strategies used to exclude dead cells, cell debris, and cell aggregates are consistent with previous cytometry experiments. Two technical replicates were tested for each construct. Error bars indicate the standard deviation (SD) of the mean of the data. We also demonstrate that parental frequency and response multipliers to parental frequency vary with different amounts of plasmid DNA expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT used to transfect the cells.(B) DLD-1 cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT were transiently transfected with 40 ng / well plasmids expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT. Approximately 48 hours after transfection, the cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation for 1 hour, the cells were washed in DPBS medium for 30 minutes (twice for 15 minutes each). Subsequently, the cells were cleaved and their fluorescence activity was measured using flow cytometry. The response factor for each assay condition was calculated as the ratio of the frequency of parental DLD-1 cells expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant in a JF646+ gate to the frequency of parental DLD-1 cells expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant in the same JF646+ gate. The gating strategies used to exclude dead cells, cell debris, and cell aggregates were consistent with previous cytometry experiments. Two technical replicates were tested for each construct. Error bars indicate the standard deviation (SD) of the mean of the data. Furthermore, we have demonstrated that the parental frequency and the response multiplier to the parental frequency change depending on the amount of plasmid DNA expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT used to transfect the cells. [Figure 111]Comparison of fluorescence intensity of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT transiently expressed using mRNA in parental DLD-1 cell lines and PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cell lines. DLD-1 cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT were transfected with mRNA encoding HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. Approximately 24 hours after transfection, the cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour of incubation, the cells underwent two 15-minute washes in DPBS medium for a total of 30 minutes. Subsequently, the cells were cleaved and their fluorescence activity was quantified using flow cytometry. The gating strategy used to exclude dead cells, cell debris, and cell aggregates was consistent with previous cytometry experiments. For each sample, the JF646-positive gate setting was optimized to maximize the distinction between tagged and untagged cells at different mRNA concentrations in order to compare optimal performance at each mRNA concentration. The values ​​on the x-axis correspond to the mRNA concentrations used for transfection in each well of the 96-well plate. Two technical replicates were performed for each construct. Error bars indicate the standard deviation (SD) of the mean of the data. Fluorescence intensity values ​​in the mRNA-free control are zero with the JF646-positive gate. [Figure 112]Comparison of response ratios of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT transiently expressed using mRNA in parental DLD-1 cell lines and PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cell lines. DLD-1 cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT were transfected with mRNA encoding HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. Approximately 24 hours after transfection, the cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour incubation, the cells underwent two 15-minute washes in DPBS medium for a total of 30 minutes. Subsequently, the cells were cleaved and their fluorescence activity was quantified using flow cytometry. The gating strategy used to exclude dead cells, cell debris, and cell aggregates was consistent with previous cytometry experiments. However, the gate settings for distinguishing between JF646-positive and negative populations differed for each mRNA concentration. This setting was optimized to maximize the separation between tagged cells and parental cells. The response factor for each assay condition was calculated as the ratio of the mean fluorescence signal at the JF646+ gate to the mean fluorescence intensity of parental DLD-1 cells expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT at the same JF646+ gate. The values ​​on the x-axis correspond to the mRNA concentration used for transfection in each well of the 96-well plate. This graph shows that the response factor increases as the concentration of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant mRNA decreases. The error bars indicate the standard deviation (SD) of the mean of the data.This observation suggests that in high concentrations of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant mRNA, the abundance of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutant exceeds that of endogenously expressed PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT, leading to the accumulation of nonspecific signals and a decrease in response magnification. [Figure 113A-B]Comparison of fluorescence intensity of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT transiently expressed using mRNA in PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells using flow cytometry. (A) Comparison of fluorescence intensity between DLD-1 cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT (right) and parental DLD-1 cells transiently transfected with various concentrations of mRNA expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT (left). A JF646 positive gate is shown for samples using 5 ng / well mRNA for transfection. The "Normalized to Mode" Y-axis scales the event count values ​​based on the most frequently occurring values, and in this graph, it shows the event count for JF646 negative gates. This allows for standardized comparisons and reveals relative differences between populations (B). PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells (light gray) were transiently transfected with mRNA encoding HaloTag[22-297](M2F)-12xGly / Ser-LgBiT (5 ng / well in a 96-well plate). As a control, parental DLD-1 cells (dark gray) were transfected with the same mRNA. Approximately 24 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour incubation, cells underwent two 15-minute washes in DPBS medium for a total of 30 minutes. Subsequently, the cells were cleaved and their fluorescence activity was measured using flow cytometry. The gating strategy used to exclude dead cells, cell debris, and cell aggregates was consistent with previous cytometry experiments. For samples using 5 ng / well mRNA transfection, JF646-positive gates are shown. The "Normalized to Mode" on the Y axis scales the number of events based on the most frequently occurring value, and in this graph, the number of events for JF646-negative gates is shown.This allows for standardized comparisons, revealing relative differences between groups. [Figure 114] Flow cytometry was used to determine the multiplier difference in the proportion of cells labeled with HaloTag ligand when transiently expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT using mRNA in PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells. DLD-1 cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT were transfected with the indicated amount of mRNA encoding HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. Approximately 24 hours after transfection, the cells were labeled with 100 nM of the HaloTag® ligand JF646 and TMR. After 1 hour of incubation, the cells were washed twice for 15 minutes each with DPBS medium, for a total of 30 minutes. Subsequently, the cells were cleaved, and their fluorescence activity was quantified using flow cytometry. The frequency of parental cells for each assay condition was measured using the exact same gate settings as those used for fluorescence intensity measurement. The response factor for each assay condition was calculated as the ratio of the frequency of parental cells in the JF646+ gate to the frequency of parental DLD-1 cells expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT in the same JF646+ gate compared to the frequency of parental DLD-1 cells expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT in the same JF646+ gate. The values ​​on the x-axis correspond to the mRNA concentration used for transfection in each well of the 96-well plate. Error bars indicate the standard deviation (SD) of the mean of the data. [Figure 115]Comparison of fluorescence intensity of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT transiently expressed using plasmid DNA or mRNA in PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells using flow cytometry. DLD-1 cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT were transfected with either 5 ng / well of plasmid DNA (dark gray) or mRNA (light gray) encoding HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. Approximately 24 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After a 1-hour incubation, the cells underwent two 15-minute washes with DPBS medium, for a total of 30 minutes. The cells were then cleaved and their fluorescence activity was measured using flow cytometry. The gating strategy used to exclude dead cells, cell debris, and cell aggregates was consistent with previous cytometry experiments. This graph shows the effect of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT expression methods on fluorescence intensity across the entire cell population. When expressed via mRNA, expression variability is reduced, resulting in a more pronounced peak in JF646-positive cells. In contrast, comparing the expression of the same amount of plasmid DNA reveals a broader peak, indicating a wider range of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT expression. "Normalized to Mode" on the Y-axis scales the event count values ​​based on the most frequently occurring values, and in this graph, it shows the event count for JF646-negative gates. This allows for standardized comparisons, revealing relative differences between groups. [Figure 116]Confocal imaging of live PARP1-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells transiently transfected with HaloTag[22-297](M2F)-12xGly / Ser-LgBiT plasmid DNA. DLD-1 cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT (upper panel) or parental DLD-1 cells (lower panel) were transfected with plasmid DNA encoding HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After a 1-hour incubation, cells were imaged using a confocal microscope in differential interference contrast (DIC) with a blue channel (Ex. 405 nm) and a far-red channel (Ex. 640 nm). Laser and gain settings were optimized to minimize saturated pixels in each channel. The PARP1 protein is originally localized to the cell nucleus, and images of PARP1-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells show localization of the far-red signal within the nucleus, along with clear co-localization of DAPI and JF646. These images provide evidence that tagging the endogenous target PARP1 with GGSG-HaloTag[3-19]-GGSG-VS-HiBiT does not affect the nuclear localization of the tagged protein. [Figure 117]Quantitative confocal imaging of live PARP1-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells transiently transfected with HaloTag[22-297](M2F)-12xGly / Ser-LgBiT plasmid DNA. DLD-1 cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT or parental DLD-1 cells were transfected with plasmid DNA encoding HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. Confocal microscopy was performed using a blue channel (Ex. 405 nm), a far-red channel (Ex. 640 nm), and DIC (differential interference contrast). The laser and gain settings were carefully optimized to minimize saturated pixels, thereby ensuring optimal image quality. These settings were consistently applied to collect both specific and background signals. For quantification, a single field of view (FOV) of PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells was analyzed to measure the nuclear localization fluorescence intensity of PARP1, while a separate FOV of parental cells was evaluated to quantify the background fluorescence intensity of all cells. The intensities of individual cells analyzed by this strategy were used to generate data points on the plot. The bars represent the mean of the data. The graph shows heterogeneity of expression using plasmid DNA, revealing outliers resulting from high expression that can affect the mean of the data. The imaging analysis correlates with the cytometry assay data analysis, as the fluorescence response factor in CRISPR cells does not differ significantly from the fluorescence intensity of parental cells. However, the distinguishing factor lies in the number of fluorescent cells within a defined gate in cytometry, or the number of fluorescent cells based on the threshold setting in imaging. [Figure 118]Confocal imaging of live PARP1-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells transiently transfected with HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mRNA. DLD-1 cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT (upper panel) or parental DLD-1 cells (lower panel) were transfected with 5 ng / well mRNA encoding HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. Approximately 24 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour of incubation, cells were imaged using a confocal microscope in differential interference contrast (DIC) with a blue channel (Ex. 405 nm) and a far-red channel (Ex. 640 nm). Laser and gain settings were optimized to minimize saturated pixels in each channel. The PARP1 protein is originally localized in the cell nucleus. Images of PARP1-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells show clear co-localization of DAPI and JF646, along with the localization of the far-red signal in the nucleus. The background signal from the non-complementary HaloTag[22-297](M2F)-12xGly / Ser-LgBiT label indicates the localization throughout the cell, as excessive HaloTag[22-297](M2F)-12xGly / Ser-LgBiT expression also occurs in the cytoplasm. A comparison of confocal images showing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT expression using plasmid DNA and mRNA reveals significantly more uniform expression in mRNA. These images also provide evidence that tagging the endogenous target PARP1 protein with GGSG-HaloTag[3-19]-GGSG-VS-HiBiT does not affect its localization. [Figure 119]Confocal imaging quantification of live PARP1-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells transiently transfected with HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mRNA. DLD-1 cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT or parental DLD-1 cells were transfected with 5 ng / well mRNA encoding HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. Approximately 24 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand and incubated for 1 hour. Confocal microscopy was performed using a blue channel (ex. 405 nm), a far-red channel (ex. 640 nm), and DIC (differential interference contrast). Laser and gain settings were carefully optimized to minimize saturated pixels, thereby ensuring optimal image quality. These settings were consistently applied to collect both specific and background signals. For quantification, the nuclear localization fluorescence intensity of PARP1 was measured by analyzing a single field of view (FOV) of PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells, while another FOV of parental cells was evaluated to quantify the background fluorescence intensity of all cells. The intensities of individual cells analyzed by this strategy were used to generate data points on the plot. Bars represent the mean of the data. The graph shows that mRNA expression exhibits higher uniformity, with only a few outliers. As a result, the mean (1215.003) and median (1192.750) of the data are similar. [Figure 120A-B]Dissociation rates of purified HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis or LgBiT after complementation with synthetic peptides. (A) Purified HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis (reporter) was mixed with 200 nM HaloTag[3-19](I2R)-4xGly / Ser-VS-HiBiT (tag) or HiBiT synthetic peptide and incubated at 4°C for 5 minutes. After pre-incubation with the peptides, the samples were diluted to 1 pM in either buffer alone or buffer containing 100 nM tag peptide. At various time points, the samples were transferred to a luminescent substrate (flimazine) and the dissociation of the complex was monitored. All measurements were performed in triplicate, and error bars indicate one standard deviation from the mean. (B) 100 nM LgBiT-6xHis was combined with 200 nM HaloTag[3-19](I2R)-4xGly / Ser-VS-HiBiT (tag) or HiBiT synthetic peptide and incubated at 4°C for 5 minutes. After pre-incubation with the peptide, the sample was diluted to 1 pM in either buffer alone or buffer containing the 100 nM tag peptide. At various time points, the sample was transferred to a luminescent substrate (flimazine) and the dissociation of the complex was monitored. All measurements were performed in triplicate, and error bars indicate one standard deviation from the mean. [Figure 121A-C]Bioluminescence resonance energy transfer (BRET) enables the measurement of ligand occupancy of purified HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis over a wide concentration range. 200 nM of purified HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis was mixed with 2 μM of HaloTag[3-19](I2R)-4xGly / Ser-VS-HiBiT or VS-HiBiT synthetic peptide. After pre-incubation with the peptide, the complex was serially diluted in buffer in semi-logarithmic increments to obtain concentrations from 0.1 pM to 10 nM, and then the buffer alone. These dilutions were added in a 1:1 ratio to flimazine substrates containing or not containing HaloTag® NanoBRET® 618 ligand at a final concentration of 100 nM. (A) Luminescence was measured for the luminescent donor signal using a 450 nm bandpass filter after incubation for approximately 1 minute. Measurements were performed in triplicate for all samples, and error bars indicate one standard deviation from the mean. (B) Luminescence was measured for the luminescent acceptor signal using a 600 nm longpass filter after incubation for approximately 1 minute. Measurements were performed in triplicate for all samples, and error bars indicate one standard deviation from the mean. (C) The BRET ratio was determined for each sample by dividing the acceptor signal by the donor signal. Measurements were performed in triplicate for all samples, and error bars indicate one standard deviation from the mean. [Figure 122A-C]Bioluminescence resonance energy transfer (BRET) enables the measurement of the stabilization of purified HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis. 200 nM of purified HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis (reporter) was mixed with 2 μM of HaloTag[3-19](I2R)-4xGly / Ser-VS-HiBiT (tag) or VS-HiBiT synthetic peptide. After pre-incubation with the peptide at 4°C, the solution was diluted 100-fold in buffer and incubated at room temperature. At each time point, the sample was added 1:1 to buffer containing or without HaloTag® NanoBRET® 618 ligand at a final concentration of 10 nM. For reporter-only conditions, the sample was added in a 1:1 ratio to a solution containing a VS-HiBiT peptide with a final concentration of 10 nM, either with or without 618 ligand, to generate a BRET donor by complementing LgBiT. (A) After the last time point, a flimazine substrate was added, followed by measurement of the fluorescence donor signal using a 450 nm bandpass filter. (B) After the last time point, a flimazine substrate was added, followed by measurement of the fluorescence acceptor signal using a 600 nm longpass filter. (C) The BRET ratio was determined for each sample by dividing the acceptor signal by the donor signal. [Figure 123A-B]Stabilization of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT activity in living cells in the presence of PARP1 endogenously tagged with HaloTag[3-19]-4xGly / Ser-HiBiT using flow cytometry. (A) Separate mRNAs encoding HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and EGFP were co-transfected into either parental DLD-1 cells (light gray) or PARP1-HaloTag[3-19]-4xGly / Ser-VS-HiBiT DLD-1 cells (dark gray). 48 hours after expression, cells were labeled with 100 nM JF646 HaloTag® ligand for 60 minutes and then analyzed by flow cytometry. Stabilization of HaloTag[22-297](M2F)-12xGly / Ser-VS-HiBiT in the presence of PARP1-HaloTag[3-19]-4xGly / Ser-VS-HiBiT results in increased JF646 HaloTag® ligand labeling in cells expressing both fragments compared to parental DLD-1 cells. Since the expression level and activity of HaloTag-LgBiT are unaffected by the presence or absence of DualTag, its HaloTag® activity exhibits excellent proportionality with co-transfected EGFP, while showing a very similar distribution in both cell lines. Therefore, the EGFP signal represents a good indicator of the expression level in each cell of the co-transfected reporter. (B) Separate mRNAs encoding HaloTag-LgBiT and EGFP were co-transfected into either parental DLD-1 cells (light gray) or PARP1-HaloTag[3-19]-4xGly / Ser-VS-HiBiT DLD-1 cells (dark gray). 48 hours after expression, cells were labeled with 100 nM JF646 HaloTag® ligand for 60 minutes and then analyzed by flow cytometry.Stabilization of HaloTag[22-297](M2F)-12xGly / Ser-VS-HiBiT in the presence of PARP1-HaloTag[3-19]-4xGly / Ser-VS-HiBiT results in increased JF646 HaloTag® ligand labeling in cells expressing both fragments compared to parental DLD-1 cells. Since the expression level and activity of HaloTag-LgBiT are unaffected by the presence or absence of DualTag, its HaloTag® activity exhibits excellent proportionality with co-transfected EGFP, while showing a very similar distribution in both cell lines. Therefore, the EGFP signal represents a good indicator of the expression level in each cell of the co-transfected reporter. [Figure 124] Stabilization of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT activity in the presence of PARP1 endogenously tagged with HaloTag[3-19]-4xGly / Ser-HiBiT using luminescence. mRNA encoding HaloTag[22-297](M2F)-12xGly / Ser-LgBiT was transfected into either parental DLD-1 cells or PARP1-HaloTag[3-19]-4xGly / Ser-VS-HiBiT DLD-1 cells. 24 hours after expression, cells were treated with 50 μg / ml cycloherepeat ximide (at hour 0), and the repeat samples were lysed at subsequent time points. To measure changes in total reporter levels, lysis reagents containing excess HiBiT peptide and flimazine substrate were added at various time points. For each condition, the ratio of luminescence signals of samples treated with + / - cycloheximide was calculated and then normalized to the initial ratio at 0 hours after cycloheximide treatment. The half-life was calculated using a one-phase exponential decay model. Five replicates were measured for each condition, and the error bars represent one standard deviation from the mean. [Figure 125]Gating settings were used to distinguish between JF646-positive and JF646-negative cell populations. The histogram shows cell number (Y-axis) and intensity due to JF646 ligand (X-axis). A consistent gating strategy, constructed using baseline activity from non-transfected cells, was applied to all transfected samples to accurately identify and quantify fluorescence activity. This strategy is consistent with conventional cytometry experiments, effectively excluding dead cells, cell debris, and aggregates. [Figure 126A-B]Comparison of fluorescence intensities of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT mutants transiently expressed using mRNA in the parental DLD-1 cell line and the PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cell line 24 and 48 hours after transfection. Parental DLD-1 cells and DLD-1 cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT were transfected with mRNA encoding HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT mutants. (A) Approximately 24 hours after transfection, the cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour incubation, the cells were washed twice in DPBS for 15 minutes each, for a total of 30 minutes. Subsequently, the cells were cleaved and their fluorescence activity was quantified using flow cytometry. A consistent gating strategy was used throughout all experiments to exclude dead cells, cell debris, and aggregates, and the same gate settings were used to distinguish between JF646-positive and negative populations. Values ​​on the x-axis correspond to the mRNA concentration used for transfection in each well of a 96-well plate. Two technical replicates were performed for each construct. Error bars indicate the standard deviation (SD) of the mean of the data. The absence of bars in any assay condition indicates the absence of a detectable signal in the JF646-positive gate of parental DLD-1 cells transfected with mRNA encoding the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT variants. Fluorescence intensity values ​​in the mRNA-free control are zero in the JF646-positive gate. These graphs show that the fluorescence intensity is approximately halved at 48 hours after transfection compared to 24 hours after transfection.(B) Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour incubation, cells were washed twice for 15 minutes each with DPBS medium, for a total of 30 minutes. Subsequently, the cells were cleaved and their fluorescence activity was quantified using flow cytometry. A consistent gating strategy was used throughout all experiments to exclude dead cells, cell debris, and aggregates, and the same gate settings were used to distinguish between JF646-positive and negative populations. Values ​​on the x-axis correspond to the mRNA concentration used for transfection in each well of a 96-well plate. Two technical replicates were performed for each construct. Error bars indicate the standard deviation (SD) of the mean of the data. The absence of bars in any assay condition indicates the absence of a detectable signal in the JF646-positive gate of parental DLD-1 cells transfected with mRNA encoding the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT variants. The fluorescence intensity value in the mRNA-free control is zero in the JF646-positive gate. These graphs show that the fluorescence intensity is approximately halved at 48 hours post-transfection compared to 24 hours post-transfection. [Figure 127A-B]Comparison of response ratios of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+d53g+K140E)-12xGly / Ser-LgBiT mutants transiently expressed using mRNA in the parental DLD-1 cell line and the PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cell line 24 and 48 hours after transfection. Parental DLD-1 cells and DLD-1 cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT were transfected with mRNA encoding HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT mutants. (A) Approximately 24 hours after transfection, the cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour incubation, the cells were washed twice in DPBS for 15 minutes each, for a total of 30 minutes. Subsequently, the cells were cleaved and their fluorescence activity was quantified using flow cytometry. A consistent gating strategy was used across all experiments to exclude dead cells, cell debris, and aggregates, and the same gate settings were used to distinguish between JF646-positive and JF646-negative populations. The response factor for each assay condition was calculated as the ratio of the average fluorescence signal at the JF646+ gate for PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT mutants to the average fluorescence intensity of DLD-1 parent cells expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT mutants at the same JF646+ gate. The values ​​on the x-axis correspond to the mRNA concentration used for transfection in each well of the 96-well plate.Error bars indicate the standard deviation (SD) of the mean of the data. The absence of bars in any assay condition indicates the absence of a detectable signal in the JF646-positive gate of parental DLD-1 cells transfected with mRNA encoding the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT variants. These results suggest no significant difference in response factor between assays performed at 24 and 48 hours post-transfection. Furthermore, a higher response factor is observed at lower mRNA concentrations. (B) Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour incubation, cells underwent two 15-minute washes in DPBS medium for a total of 30 minutes. Subsequently, the cells were cleaved and their fluorescence activity was quantified using flow cytometry. A consistent gating strategy was used throughout all experiments to exclude dead cells, cell debris, and aggregates, and the same gate settings were used to distinguish between JF646-positive and JF646-negative populations. The response factor for each assay condition was calculated as the ratio of the average fluorescence signal at the JF646+ gate for PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT mutants to the average fluorescence intensity of DLD-1 parent cells expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT mutants at the same JF646+ gate. The values ​​on the x-axis correspond to the mRNA concentration used for transfection in each well of the 96-well plate. The error bars indicate the standard deviation (SD) of the mean of the data.The absence of bars under any assay conditions indicates the absence of a detectable signal in the JF646-positive gate of parental DLD-1 cells transfected with mRNA encoding the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT variants. These results suggest no significant difference in response factor between assays performed 24 and 48 hours post-transfection. Furthermore, a higher response factor is observed at lower mRNA concentrations. [Figure 128A-B]Comparison of the proportion of cells labeled with HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT mutants after transient expression using mRNA in the parental DLD-1 cell line and the PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cell line 24 and 48 hours after transfection. Parental DLD-1 cells and DLD-1 cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT were transfected with mRNA encoding HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT mutants. (A) Approximately 24 hours after transfection, the cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour incubation, the cells were washed twice in DPBS for 15 minutes each, for a total of 30 minutes. Subsequently, the cells were cleaved and their fluorescence activity was quantified using flow cytometry. A consistent gating strategy was used throughout all experiments to exclude dead cells, cell debris, and aggregates, and the same gate settings were used to distinguish between JF646-positive and negative populations. The frequency of parental cells for each assay condition was measured using the exact same gate settings as those used for fluorescence intensity measurement. Values ​​on the x-axis correspond to the mRNA concentration used for transfection in each well of a 96-well plate. Two technical replicates were performed for each construct. Error bars indicate the standard deviation (SD) of the mean of the data. The absence of bars in any assay condition indicates the absence of detectably labeled cells in the JF646-positive gate of parental DLD-1 cells transfected with mRNA encoding the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT variants. The fluorescence intensity value in the mRNA-free control is zero when using the JF646-positive gate.These graphs show that while fluorescence intensity decreases by approximately 50% at 48 hours post-transfection compared to 24 hours, the proportion of PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells expressing the HaloTag® mutant and labeled is significantly increased at the JF646-positive gate compared to parental DLD-1 cells expressing the same mutant and labeled. At an mRNA concentration of 0.2 ng / well, labeled parental cells were undetectable, and at 2 ng / well, less than 1% were detected. In contrast, an average of 15% and 40% of PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells were labeled under these conditions, respectively. Therefore, for fluorescence sorting applications, it is recommended to perform the assay 48 hours post-transfection. These results demonstrate that both the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT mutants function similarly in cell cytometry assays. The latter mutant shows a slight improvement in the response factor of the percentage of fluorescent and labeled cells. (B) Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour incubation, cells underwent two 15-minute washes with DPBS medium for a total of 30 minutes. The cells were then cleaved and their fluorescence activity was quantified using flow cytometry. A consistent gating strategy was used throughout all experiments to exclude dead cells, cell debris, and aggregates, and the same gate settings were used to distinguish between JF646-positive and negative populations. The frequency of parental cells for each assay condition was measured using the exact same gate settings as those used for fluorescence intensity measurement. The values ​​on the x-axis correspond to the mRNA concentration used for transfection in each well of the 96-well plate. Two technical replicates were performed for each construct. Error bars indicate the standard deviation (SD) of the mean of the data.The absence of bars in any assay condition indicates that no detectable labeled cells exist in the JF646-positive gate of parental DLD-1 cells transfected with mRNA encoding the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT mutants. The fluorescence intensity value in the mRNA-free control is zero in the JF646-positive gate. These graphs show that while fluorescence intensity decreases by approximately 50% at 48 hours post-transfection compared to 24 hours, the proportion of PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells expressing the HaloTag® mutant and labeled is significantly increased in the JF646-positive gate compared to parental DLD-1 cells expressing the same mutant and labeled. At an mRNA concentration of 0.2 ng / well, labeled parental cells were not detected, and at 2 ng / well, less than 1% were detected. In contrast, an average of 15% and 40% of PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells were labeled under these conditions. Therefore, for fluorescence sorting applications, it is recommended to perform the assay 48 hours after transfection. These results demonstrate that both the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT mutants function similarly in cell cytometry assays. The latter mutant shows a slight improvement in the response factor of the percentage of fluorescent and labeled cells. [Figure 129A-B]Comparison of fluorescence intensities of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutants in a time-lapse ligand incubation cytometry assay in DLD-1 cell lines. Panel A shows the fluorescence intensities of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT, HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT, and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT mutants expressed in both the parent DLD-1 cell line and the PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cell line. These mutants were transiently expressed using mRNA in both the parental DLD-1 cell line and the PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cell line. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand and incubated for various time intervals (0.5, 2, 8, and 24 hours), followed by separation via flow cytometry and quantification of fluorescence. A consistent gating strategy was used throughout all experiments to exclude dead cells, cell debris, and aggregates, and the same gate settings were used to distinguish between JF646-positive and negative populations. Two technical replicates were performed for each construct. Error bars indicate the standard deviation (SD) of the mean of the data. Fluorescence intensity values ​​in the mRNA-free control are zero in the JF646-positive gate. These graphs show that the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT mutants exhibit similar fluorescence intensities, but slightly higher than that of the HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT mutant. Fluorescence intensity peaks approximately 2 hours after ligand addition.The maximum fluorescence magnification response is achieved approximately 2 hours after ligand incubation, but the magnification response of the HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT mutant peaks approximately 8 hours after ligand addition. Panel B illustrates the fluorescence magnification response of each mutant over these time points. The response magnification was calculated by comparing the mean fluorescence of the transfected PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cell line at the JF646+ gate with the mean fluorescence of the parental control at the same gate. Two technical replicates were performed for each construct. Error bars indicate the standard deviation (SD) of the mean of the data. Fluorescence intensity values ​​in the mRNA-free control are zero at the JF646-positive gate. These graphs show that the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT mutants exhibit similar fluorescence intensities, but slightly higher than that of the HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT mutant. Fluorescence intensity peaks around 2 hours after ligand addition. Maximum fluorescence fold rate response is achieved approximately 2 hours after ligand incubation, although the fold rate response for the HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT mutant peaks approximately 8 hours after ligand addition. [Figure 130A-B]Comparison of the proportion of labeled cells among HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mutants in a time-lapse ligand incubation cytometry assay in DLD-1 cell lines. Panel A shows the proportion of cells labeled with HaloTag[22-297](M2F)-12xGly / Ser-LgBiT, HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT, and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT mutants expressed in both the parental DLD-1 cell line and the PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cell line. These mutants were transiently expressed using mRNA in both the parental DLD-1 cell line and the PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cell line. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand and incubated for various times (0.5, 2, 8, and 24 hours), then separated via flow cytometry and fluorescence was quantified. A consistent gating strategy was used throughout all experiments to exclude dead cells, cell debris, and aggregates, and the same gate settings were used to distinguish between JF646-positive and negative populations. Two technical replicates were performed for each construct. Error bars indicate the standard deviation (SD) of the mean of the data. Fluorescence intensity values ​​in the mRNA-free control are zero in the JF646-positive gate. These graphs show that longer ligand incubation times result in better response multipliers for parental cell frequency. Across almost all time points, the HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT mutant exhibits a slightly higher response factor compared to the other two HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT mutants. Panel B illustrates the response factor (parental frequency) of the percentage of labeled cells for each mutant across these time points.The response factor for each assay condition was calculated as the ratio of the parental frequency in a JF646+ gate to the parental frequency of DLD-1 parent cells expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant in PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells, compared to the parental frequency of DLD-1 parent cells expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant in the same JF646+ gate. Two technical replicates were performed for each construct. Error bars indicate the standard deviation (SD) of the mean of the data. Fluorescence intensity values ​​in the mRNA-free control are zero in the JF646-positive gate. These graphs show that longer ligand incubation times result in better response factors for parental cell frequency. Across almost all time points, the HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT variant exhibits a slightly higher response factor compared to the other two HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants and the HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT variant. [Figure 131]Quantitative confocal imaging of live PARP1-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells transiently transfected with mRNA of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT, HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT, and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT mutants. Parental DLD-1 cells and DLD-1 cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT were transfected with 0.5 ng / well mRNA encoding HaloTag[22-297](M2F)-12xGly / Ser-LgBiT, HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT, and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT mutants. Approximately 24 hours after transfection, cells were labeled with 100 nM JF646 HaloTag ligand and incubated for 1 hour. Confocal microscopy was performed using a blue channel (ex. 405 nm), a far-red channel (ex. 640 nm), and DIC (differential interference contrast). Laser and gain settings were optimized to prevent pixel saturation and maintain uniform intensity across all mutants to capture specific signals and background signals. To quantify each mutant, a single field of view (FOV) of PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells was analyzed to measure the nuclear localization fluorescence intensity of PARP1, while another FOV of the parental cell was evaluated to quantify the background fluorescence intensity of all cells. The intensities of individual cells analyzed by this strategy were used to generate data points on the plot. Bars represent the mean values ​​of the data. The graph shows that mRNA expression exhibits higher uniformity and that there are only a few outliers. The results suggest similar fluorescence magnification responses across mutants. [Figure 132]Labeling reaction rates of HaloTag7 and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis with HaloTag ligand JF549. The labeling reaction rates of the JF549 HaloTag ligand with HaloTag standard protein or HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis were observed by monitoring fluorescence polarization over time. Experiments were completed in Tris-buffered saline + 0.01% CHAPS using 10 nM protein and 2.5 nM ligand. [Figure 133] Labeling reaction rates of HaloTag7 and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis with HaloTag ligand JF503. The labeling reaction rates of the JF503 HaloTag ligand with HaloTag standard protein or HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis were observed by monitoring fluorescence polarization over time. Experiments were completed in Tris-buffered saline + 0.01% CHAPS using 10 nM protein and 2.5 nM ligand. [Figure 134] Labeling reaction rates of HaloTag7 and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis with HaloTag ligand JFX554. The labeling reaction rates of the JFX554 HaloTag ligand with HaloTag standard protein or HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis were observed by monitoring fluorescence polarization over time. Experiments were completed in Tris-buffered saline + 0.01% CHAPS using 10 nM protein and 2.5 nM ligand. [Figure 135]Labeling reaction rates of HaloTag7 and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis with the HaloTag ligand Oregon Green. The labeling reaction rates of the Oregon Green HaloTag ligand with HaloTag standard protein or HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis were observed by monitoring fluorescence polarization over time. The experiment was completed in Tris-buffered saline + 0.01% CHAPS using 10 nM protein and 2.5 nM ligand. [Figure 136] Labeling reaction rates of HaloTag7 and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis with FAM HaloTag ligands. The labeling reaction rates of FAM HaloTag ligands with HaloTag standard protein or HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis were observed by monitoring fluorescence polarization over time. Experiments were completed in Tris-buffered saline + 0.01% CHAPS using 10 nM protein and 2.5 nM ligand. [Modes for carrying out the invention]

[0021] definition Any methods and substances similar to or equivalent to those described herein may be used in carrying out or testing the embodiments described herein, but several preferred methods, compositions, devices, and substances are described herein. However, before describing the substances and methods, it should be understood that the present invention is not limited to the specific molecules, compositions, methodologies, or protocols described herein, for these may be modified by customary experimentation and optimization. It should also be understood that the terminology used herein is for the sole purpose of describing specific versions or embodiments and is not intended to limit the scope of the embodiments described herein.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which the present invention pertains. However, in the event of any conflict, the specification of the present invention, including the definitions, shall prevail. Accordingly, the following definitions apply to the embodiments described herein.

[0023] As used herein and in the appended claims, the singular forms "a," "an," and "the" refer to multiple subjects unless otherwise explicitly required by the context. Thus, for example, a reference to "polypeptide" refers to one or more polypeptides and their equivalents known to those skilled in the art.

[0024] As used herein, the term "and / or" includes any combination of the listed items, including any one of the listed items. For example, "A, B, and / or C" includes A, B, C, AB, AC, BC, and ABC, each of which should be considered as being individually described by the statement "A, B, and / or C".

[0025] As used herein, the term “contains” and its linguistic variations mean that the detailed features, elements, method steps, etc., exist, and the existence of additional features, elements, method steps, etc., is not excluded. Conversely, the term “consist of” and its linguistic variations mean that the detailed features, elements, method steps, etc., exist, and any features, elements, method steps, etc., that are not detailed, except for impurities that are usually present. The phrase “essentially consisting of” means the detailed features, elements, method steps, etc., and any additional features, elements, method steps, etc., that do not substantially affect the fundamental nature of the composition, system, or method. Many embodiments herein are described using the non-restrictive term “contains.” Such embodiments encompass a plurality of closed "consisting of" and / or "consisting essentially of" embodiments, which, alternatively, may be claimed or described using such language.

[0026] As used herein, the term “substantially” means that the described properties, parameters, and / or values ​​do not need to be exactly achieved, and deviations or variations, including, for example, tolerances, measurement errors, measurement accuracy limits, and other factors known to those skilled in the art, may occur in an amount that does not preclude the effect that the feature is intended to provide. A substantially non-existent property or feature (e.g., substantially non-fluorescent) may be a property or feature that is within the range of noise, lower than the background, below the detection capability of the assay used, or is only a fraction of a significant property (e.g., fluorescence intensity of an active fluorophore) (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%).

[0027] When used herein, the phrase "corresponding to" refers to the relative position of an amino acid residue or amino acid segment with respect to the sequence being referred to, and does not necessarily imply the specific identity of the amino acid present at that position. For example, "peptide corresponding to positions 36-48 of SEQ ID NO: 1" may include less than 100% sequence identity (e.g., more than 70%) with positions 36-48 of SEQ ID NO: 1, but the context of the composition or system in which the peptide is described is relevant to those positions.

[0028] As used herein, the term “system” refers to a set of components (e.g., a device, a composition, etc.) used for a particular purpose. For example, two distinct biomolecules, whether or not they exist in the same composition, may constitute a system if they are useful together for a common purpose.

[0029] As used herein, the term “complementary” refers to the characteristic of two or more structural elements (e.g., peptides, polypeptides, nucleic acids, small molecules, etc.) that they can hybridize with each other, dimerize, or otherwise form complexes. For example, “complementary peptides and polypeptides” can assemble to form complexes. Complementary elements may require assistance (facilitation) to form complexes (e.g., from interacting elements), such as to give the elements a conformation suitable for complementarity, to position the elements proximal to complementarity, to colocalize the complementary elements, to reduce the interaction energy for complementarity, or to overcome insufficient affinity for each other.

[0030] As used herein, the term “complex” refers to a group or aggregate of molecules (e.g., peptides, polypeptides, etc.) that are in direct and / or indirect contact with one another. In one embodiment, “contact” or more specifically “direct contact” means that two or more molecules are in close proximity, and as a result, non-covalent attractive interactions, such as van der Waals forces, hydrogen bonds, ionic interactions, and hydrophobic interactions, occupy the interactions between the molecules. In such embodiments, the complex of molecules (e.g., peptides and polypeptides, etc.) is formed under assay conditions such that the complex is thermodynamically preferred (e.g., compared to the non-aggregated or non-complexed states of its constituent molecules). As used herein, the term “complex” refers to a group of two or more molecules (e.g., peptides, polypeptides, etc.) unless otherwise specified.

[0031] As used herein, the term “interacting element” refers to a moiety that assists or facilitates the assembly of two or more structural elements (e.g., peptides, polypeptides, etc.) to form a complex. In some embodiments, a pair of interacting elements (also known as an “interacting pair”) is bound to a pair of structural elements (e.g., peptides, polypeptides, etc.), and the attractive interaction between the two interacting elements facilitates the formation of a complex of the structural elements. Interacting elements may facilitate complex formation by any preferred mechanism (e.g., bringing structural elements into proximity, positioning structural elements in a three-dimensional structure suitable for stable interaction, reducing the activation energy for complex formation, or a combination thereof). Interacting elements may be proteins, polypeptides, peptides, small molecules, cofactors, nucleic acids, lipids, carbohydrates, antibodies, etc. An interacting pair may consist of two identical interacting elements (i.e., a homogeneous pair) or two different interacting elements (i.e., a heterogeneous pair). In the case of a heterogeneous pair, the interacting elements may be the same type of moiety (e.g., polypeptides) or two different types of moieties (e.g., a polypeptide and a small molecule). In some embodiments where complex formation by interaction pairs is studied, the interaction pair may be referred to as the “target pair” or “target pair,” and the individual interaction elements may be referred to as the “target element” (e.g., “target peptide,” “target polypeptide,” etc.) or “desired element” (e.g., “target peptide,” “desired polypeptide,” etc.).

[0032] As used herein, the term "low affinity" describes intermolecular interactions between two or more entities that are too weak to result in significant complex formation between entities, unless the concentration is considerably higher than physiological or assay conditions (e.g., 2x, 5x, 10x, 100x, 1000x or more) or facilitated by the formation of a second complex of attached elements (e.g., interacting elements).

[0033] As used herein, the term “high affinity” describes an intermolecular interaction between two or more (e.g., three) entities that is strong enough to cause detectable complex formation without the need for further enhancement by the formation of a second complex of the attached elements (e.g., interacting elements) under physiological or assay conditions.

[0034] As used herein, the term “existing protein” refers to an amino acid sequence that physically existed prior to a particular event or date. “Peptide that is not a fragment of an existing protein” is a short amino acid chain that is not a fragment or partial sequence of a protein (e.g., synthetic or naturally occurring) that physically existed prior to the design and / or synthesis of the peptide. As used herein, the term “fragment” refers to a peptide or polypeptide resulting from the cleavage or “fragmentation” of a larger entity (e.g., a protein, polypeptide, enzyme, etc.), or a peptide or polypeptide prepared to have the same sequence as such. Thus, a fragment is a partial sequence of an entity (e.g., a protein, polypeptide, enzyme, etc.) that is made and / or designed from the whole entity. 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 protein” is an amino acid chain that physically exists before the design and / or synthesis of the peptide or polypeptide and is not a partial sequence of a protein (e.g., native or synthetic). A fragment of a hydrolase or dehalogenase, as used herein, is a shorter sequence than the full-length sequence, but cannot form a substrate-binding site on its own and / or has substantially reduced substrate-binding activity, or has no substrate-binding activity, but exhibits substantially increased substrate-binding activity when adjacent to a second fragment of the hydrolase or dehalogenase. In one embodiment, the hydrolase or dehalogenase fragment is at least five, for example, at least 10, at least 20, at least 30, at least 40, or at least 50 consecutive residues of a wild-type hydrolase or mutant hydrolase, or a sequence having at least 70% sequence identity to them, and does not necessarily include the N-terminal or C-terminal residues, or the N-terminal or C-terminal sequence, of the corresponding full-length protein.

[0035] As used herein, the term “subsequence” refers to a peptide or polypeptide that has 100% sequence identity with a portion of another larger peptide or polypeptide. A subsequence is a complete sequence match with a portion of a longer amino acid chain.

[0036] The term "amino acid" refers to natural amino acids, unnatural amino acids, and amino acid analogs, all of which are D and L stereoisomers unless otherwise specified (where their structure allows for such stereoisomerization).

[0037] The term "protein-building amino acids" refers to the 20 amino acids encoded in the human genetic code, including 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). Selenocysteine ​​and pyrrolicin can also be considered protein-building amino acids.

[0038] The term "non-proteinogenic amino acid" refers to an amino acid that is not naturally encoded or found in the genetic code of any organism and is not biosynthetically incorporated into proteins during translation. Non-proteinogenic amino acids can be "non-natural amino acids" (amino acids that do not exist in nature) or "naturally occurring non-proteinogenic amino acids" (e.g., norvaline, ornithine, homocysteine). Non-protein constituent amino acids include azetidine carboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, naphthylalanine, 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, 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine, N-alkylglycine containing N-methylglycine, N-methylisoleucine, and N-alkylpentylglycine containing N-methylpentylglycine. Examples of non-protein constituent amino acids include, but are not limited to, N-methylvaline, naphthylalanine, norvaline, norleucine ("Norleu"), octylglycine, ornithine, pentylglycine, pipecolic acid, thioproline, homolysine, and homoarginine. Examples of non-protein constituent amino acids include the D-amino acid form of any of the amino acids herein, as well as the non-alpha amino acid form of any of the amino acids herein (beta-amino acids, gamma-amino acids, delta-amino acids, etc.), all of which are within the scope of this specification and may be included in the peptides herein.

[0039] The term "amino acid analog" refers to an amino acid (e.g., natural or unnatural, protein-based or non-protein-based) in which one or more of its C-terminal carboxyl group, N-terminal amino group, and side-chain bioactive groups are reversibly or irreversibly chemically blocked or otherwise modified with another bioactive group. For example, aspartate-(β-methyl ester) is an amino acid analog of aspartate, 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.

[0040] As used herein, unless otherwise specified, the terms “peptide” and “polypeptide” refer to polymer compounds of two or more amino acids linked together via a main chain by a peptide amide bond (--C(O)NH--). The term “peptide” usually refers to a short amino acid polymer (e.g., a chain with fewer than 30 amino acids), while the term “polypeptide” usually refers to a longer amino acid polymer (e.g., a chain with 30 or more amino acids).

[0041] As used herein, the terms “artificial” or “synthetic” refer to compositions and systems that are designed or prepared by humans and do not exist in nature. For example, artificial or synthetic peptides, peptoids, or nucleic acids are those that contain non-natural sequences (e.g., peptides that do not have 100% identity with naturally occurring proteins or their fragments).

[0042] As used herein, “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, such as size or charge. For the purposes of this disclosure, each of the following eight groups includes amino acids that are conservative substitutions with each other: 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).

[0043] Based on the properties of their common side chains, amino acid residues can be classified into the following classes, for example: polar positive charge (or basic) (e.g., histidine (H), lysine (K), and arginine (R)); polar negative charge (or acidic) (e.g., aspartic acid (D), glutamic acid (E)); polar neutral (e.g., serine (S), threonine (T), asparagine (N), glutamine (Q)); nonpolar aliphatic (e.g., alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M)); nonpolar aromatic (e.g., 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.

[0044] In some embodiments, unless otherwise specified, conserved or semi-conserved amino acid substitutions may include non-natural amino acid residues that have similar chemical properties to natural residues. These non-natural residues are typically incorporated by chemical peptide synthesis rather than synthesis in biological systems. These include, but are not limited to, peptide mimes and other inverted or reversed amino acid moieties. Embodiments described herein may, in some embodiments, be limited to natural amino acids, non-natural amino acids, and / or amino acid analogs.

[0045] Non-conservative substitutions can involve the exchange of a member of one class for a member of another class. 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 monomer subunit sequence composition. 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 similar biophysical characteristics and belong to families classified as, for example, acidic (e.g., aspartic acid, glutamic acid), basic (e.g., lysine, arginine, histidine), nonpolar (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). The "sequence identity percentage" (or "sequence similarity percentage") is calculated by (1) comparing two optimally aligned sequences across a comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, or a specified window); (2) determining the number of positions containing identical (similar) monomers (e.g., positions where the same amino acid is found in both sequences, or 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, or a specified window); and (4) multiplying the result by 100 to obtain the sequence identity percentage or sequence similarity percentage. For example, if peptides A and B are both 20 amino acid long and all amino acids except one position are identical, peptides A and B have 95% sequence identity. If amino acids at non-identical positions share the same biophysical properties (e.g., both are acidic), peptides A and B will have 100% sequence similarity.As another example, if peptide C is 20 amino acids long and peptide D is 15 amino acids long, and 14 of the 15 amino acids in peptide D are identical to some 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 purposes of calculating “sequence identity percentage” (or “sequence similarity percentage”) as used herein, any gaps in the aligned sequences are treated as mismatches at that position.

[0046] Any peptide / polypeptide described herein as having a specific percentage of sequence identity or similarity (e.g., at least 70%) to a reference sequence number may be described as having the maximum number of substitutions (or terminal deletions) relative to its reference sequence. For example, a sequence having at least Y% sequence identity (e.g., 90%) to sequence number Z (e.g., 100 amino acids) may have up to X substitutions (e.g., 10) compared to sequence number Z, and therefore may also be described as having X or fewer substitutions (e.g., 10) compared to sequence number Z.

[0047] As used herein, the term “wild-type” refers to a gene or gene product (e.g., a protein, polypeptide, peptide, etc.) that possesses the characteristics (e.g., sequence) of that gene or gene product isolated from a naturally occurring source and is most frequently observed in a population. In contrast, the term “variant” refers to a gene or gene product that exhibits a modification in sequence 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 exists naturally but has a modified sequence compared to the wild-type gene or gene product, and these are not the most frequently occurring sequences. An “artificial variant” or “synthetic variant” is a gene or gene product that has a modified sequence compared to the wild-type gene or gene product and does not exist naturally. A variant gene or gene product may be a naturally occurring sequence that exists in nature but is not the most commonly observed variant of the gene or gene product, or it may be a “synthetic” produced by human or experimental intervention.

[0048] As used herein, the term “physiological conditions” encompasses any conditions suitable for living cells, such as temperature, pH, salinity, and chemical composition that are primarily aqueous and suitable for living cells.

[0049] As used herein, the term “sample” is used in its broadest sense. In a sense, this term means specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and include liquids, solids, tissues, and gases. Biological samples include blood products such as plasma and serum. Samples may also refer to cell lysates, or purified forms of enzymes, peptides, and / or polypeptides as described herein. Cell lysates may include cells lysed with a solvent, or lysates such as rabbit reticulocytes or wheat germ lysates. Samples may also include cell-free expression systems. Environmental samples include environmental substances such as surface materials, soil, water, crystals, and industrial samples. However, such examples should not be construed as limiting the types of samples to which the present invention is applicable.

[0050] As used herein, the terms “fusion,” “fusion polypeptide,” and “fusion protein” refer to a chimeric protein having a first protein or polypeptide of interest linked to a second different peptide, polypeptide, or protein (e.g., an interacting element).

[0051] As used herein, the terms “conjugated” and “conjugation” refer to the covalent attachment of two molecular entities (e.g., after synthesis and / or during synthetic manufacturing). 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.

[0052] As used herein, the terms “polypeptide component” or “peptide component” are used synonymously with the terms “polypeptide component of the [mutant dehalogenase] complex” or “peptide component of the [mutant dehalogenase] complex.” Typically as used herein, the polypeptide component or peptide component can form a complex with a second component under appropriate conditions to obtain the desired complex.

[0053] As used herein, the term “dehalogenase” refers to an enzyme that catalyzes the removal of halogen atoms from a substrate. The term “haloalkane dehalogenase” refers to an enzyme that catalyzes the removal of halogens from haloalkane substrates to produce alcohols and halides. Dehalogenases and haloalkyl dehalogenases belong to the hydrolase enzyme family and may be referred to as such herein or elsewhere.

[0054] As used herein, the term "modified dehalogenase" refers to a dehalogenase variant (artificial variant) that has a mutation that prevents the release of the substrate from the protein after halogen removal, resulting in a covalent bond between the substrate and the modified dehalogenase. Because the modified dehalogenase does not release the substrate, it cannot undergo turnover and is not a classical enzyme. The HALOTAG system (Promega) is a commercially available modified dehalogenase and substrate system.

[0055] As used herein, the term "bioluminescence" refers to the generation and emission of light by chemical reactions catalyzed or enabled by enzymes, proteins, protein complexes, or other biomolecules (e.g., bioluminescent complexes). In a typical embodiment, a substrate of a bioluminescent entity (e.g., a bioluminescent protein or bioluminescent complex) is converted into an unstable form by the bioluminescent entity, after which the substrate emits light.

[0056] As used herein, the term “non-luminescent” refers to an entity (e.g., a peptide, polypeptide, complex, protein, etc.) that does not emit any detectable amount of visible spectral light (e.g., in the presence of a substrate). For example, an entity may be called non-luminescent if it does not exhibit detectable luminescence in a given assay. As used herein, the term “non-luminescent” is synonymous with “substantially non-luminescent.” In some embodiments, an entity is considered “non-luminescent” if any luminescence is small enough not to produce background that interferes with a particular assay.

[0057] As used herein, the terms "non-luminescent peptide" and "non-luminescent polypeptide" mean that, when compared to a significant signal (e.g., a bioluminescent complex) using typical instruments (e.g., a luminometer) under standard conditions (e.g., physiological conditions, assay conditions, etc.), they exhibit substantially no luminescence (e.g., in the presence of a substrate) or at a level lower than noise (e.g., 1 / 100, 1 / 200, 1 / 500, 1 × 10⁻¹⁶). 3 1 / 10 4 1 / 10 5 1 / 10 6 1 / 10 7 This refers to peptides and polypeptides exhibiting characteristics such as 1 / 1. In some embodiments, such non-luminescent peptides and polypeptides assemble according to the criteria described herein to form bioluminescent complexes.

[0058] As used herein, the term “Oplophorus luciferase” (“OgLuc”) refers to a luminescent polypeptide having remarkable sequence identity, structural conservation, and / or functional activity with the luciferase produced by or derived from the deep-sea shrimp Oplophorus gracilirostris. In particular, OgLuc polypeptide refers to a luminescent polypeptide having remarkable sequence identity, structural conservation, and / or functional activity with the mature 19kDa subunit of the Oplophorus luciferase protein complex (e.g., one without a signal sequence), such as Sequence ID No. 3034 (NanoLuc), which contains 10 β-strands (β1, β2, β3, β4, β5, β6, β7, β8, β9, β10), and generates luminescence using a substrate such as coelenterazine or a coelenterazine derivative or analog.

[0059] 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 thereof 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, resulting in enhanced luminescence of the complex 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).

[0060] 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 thereof 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, resulting in enhanced luminescence of the complex 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).

[0061] 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" can be named similarly (e.g., β 1-4 -like polypeptide, β 2-8 -like polypeptide, β 5-10 -like polypeptide, etc.).

[0062] As used herein, the term "NANOLUC" refers to an artificial luciferase or bioluminescent polypeptide commercially produced by Promega Corporation.

[0063] As used herein, the term "LgBiT" is used, for example, in the two-component complementation for forming a bioluminescent complex, corresponding to β3037. 1-9 This refers to a polypeptide equivalent to a specific polypeptide.

[0064] As used herein, the term "SmBiT" refers to a β that is useful, for example, in the two-component complementation for forming a bioluminescent complex, but has low affinity for LgBiT (e.g., requires enhancement for complex formation) and corresponds to SEQ ID NO: 3039. 10 This refers to peptides that correspond to similar peptides.

[0065] As used herein, the term "HiBiT" refers to a component that is useful, for example, in the two-component complementation for forming a bioluminescent complex, but has high affinity for LgBiT (e.g., requires promotion for complex formation), β 10 This refers to peptides that correspond to similar peptides. An exemplary HiBiT peptide corresponds to SEQ ID NO: 3038.

[0066] As used herein, the term "LgTrip" means β1-8 This refers to polypeptides equivalent to β9-like polypeptides. An example is LgTrip, which corresponds to SEQ ID NO: 3045, and is, for example, a β9-like and β-like polypeptide for forming a bioluminescent complex. 10 β for three-component complementation with various peptides, or for forming bioluminescent complexes. 9-10 It is useful in two-component complementation with various dipeptides.

[0067] As used herein, the term "SmTrip10" refers to, for example, a β component useful in the three-component complementation for forming a bioluminescent complex. 10 This refers to peptides that correspond to similar peptides.

[0068] As used herein, the term “SmTrip9” refers to a peptide equivalent to a β9-like peptide useful, for example, in the three-component complementation for forming a bioluminescent complex. As used herein, the term “split” ("sp") refers to a polypeptide that has been split into two fragments at an internal site of the original polypeptide. The fragments of an sp polypeptide can reconstitute the activity of the original polypeptide if they are structurally complementary and can form an active complex. The nomenclature used herein to refer to the split components of a polypeptide lists the positional numbers from the complete polypeptide corresponding to the last residue in the N-terminal component of the split polypeptide. For example, if the residue length of a polypeptide is 100 residues, the sp52 type of that polypeptide includes a first fragment corresponding to positions 1-52 of the parent polypeptide and a second fragment corresponding to positions 53-100 of the parent polypeptide. As another example, spHT(45) refers to a split variant of the commercially available HALOTAG protein in which the first fragment contains residues 1-45 of the HALOTAG polypeptide sequence and the second fragment contains residues 46-297 of the HALOTAG polypeptide sequence.

[0069] Alternatively, the components of a split polypeptide may be represented herein by reference to the name of the polypeptide from which it originates, the residues in the source polypeptide present in the component (in square brackets), and any subsequent substitutions in the component relative to the source polypeptide (in parentheses). For example, the split component of a commercially available HALOTAG protein corresponding to positions 22-297 of the HALOTAG sequence may be described as HaloTag[22-297]. If the second position of the component contains an M-to-F substitution, the component may be called HaloTag[22-297](M2F). Components may contain an N-terminal methionine residue that is not present in the source sequence. Such residues are counted when referring to the position of the substitution, but not when numbering the fragments in the source polypeptide.

[0070] As used herein, the term “gapded” refers to a split variant of a polypeptide that lacks a segment of the original polypeptide. For example, a “gapded sp polypeptide” is one in which a segment of the original sequence is missing at the split site. As used herein, the term “duplicate” refers to a split variant of a polypeptide that contains a duplication of a segment of the original polypeptide. For example, a “duplicate sp polypeptide” is one in which a segment of the original sequence adjacent to the split site is present (duplication) at the C-terminus of the first fragment and at the N-terminus of the second fragment.

[0071] The term "binding domain" refers to a domain that specifically binds to an antigen or epitope independently of different epitope or antigen-binding domains. Binding domains may include antibodies, antibody fragments, receptor domains that bind to target ligands, proteins that bind to immunoglobulins (e.g., protein A, protein G, protein A / G, protein L, protein M), binding domains of immunoglobulin-binding proteins (e.g., protein A, protein G, protein A / G, protein L, protein M), oligonucleotide probes, peptide nucleic acids, DARPin, aptamers, affimers, purified proteins (either the analyte itself or a protein that binds to the analyte), and analyte-binding domains of proteins. Table A provides a list of exemplary binding domains that may be used individually or in various combinations in the methods, systems, and assays (e.g., immunoassays) described herein. [Table 1]

[0072] Detailed explanation This specification provides compositions and systems comprising complementary tags and reporters for labeling and detecting targets by luminescence and a second mode (e.g., fluorescence), as well as methods for using them. In particular, a tag comprising a fusion of a first component of a bioluminescent complex and a first component of a modified dehalogenase complex is provided; a reporter comprising a second component of a bioluminescent complex and a second component of a modified dehalogenase complex is provided; and systems and methods comprising the tags and reporters described herein for dual-mode labeling and target detection are provided.

[0073] In some embodiments, a system comprising: (a) a tandem peptide tag comprising (i) a peptide component of a bioluminescent complex fused to (ii) a peptide component of a modified dehalogenase complex; and (b) a tandem polypeptide reporter comprising a polypeptide component of a bioluminescent complex fused to a polypeptide component of a modified dehalogenase complex; where the bioluminescent complex and the modified dehalogenase complex are formed by (facilitated or unfacilitated) interactions between the tandem peptide tag and the tandem polypeptide reporter; the bioluminescent complex can generate bioluminescence in the presence of a substrate (e.g., coelenterazine, flimazine, etc.); and the modified dehalogenase complex can bind to a haloalkyl ligand. In some embodiments, the formation of a complex (e.g., a component of the bioluminescent complex or a component of the modified dehalogenase complex) by a high-affinity interaction of a pair of components is sufficient to facilitate the formation of a second complex (e.g., a modified dehalogenase complex of the bioluminescent complex).

[0074] HaloTag In some embodiments, provided herein are compositions (e.g., fusion peptides and polypeptides) and systems (e.g., a plurality of complementary fusion peptides and polypeptides, substrates, ligands, etc.) comprising complementary peptide / polypeptide fragments that can interact (e.g., facilitated or unfavorable) to form an active modified dehalogenase complex capable of covalently bonding with a haloalkane ligand. In some embodiments, a first fusion is provided comprising a complementary peptide fragment of a modified dehalogenase, and a second fusion is provided comprising a complementary polypeptide fragment of a modified dehalogenase, wherein the complementary peptide and polypeptide interact (e.g., facilitated or unfavorable) to form an active modified dehalogenase complex capable of covalently bonding with a haloalkane ligand. In some embodiments, a first fusion is provided comprising a complementary peptide fragment of a modified dehalogenase, and a complementary polypeptide fragment of a modified dehalogenase (e.g., not the fusion) is provided, and the complementary peptide and polypeptide interact (e.g., if facilitated or not) to form an active modified dehalogenase complex that can form a covalent bond with a haloalkane ligand. In some embodiments, the complementary peptide and polypeptide are fragments of a split mutant dehalogenase. In alternative embodiments, both fragments may be polypeptides.

[0075] Split mutant dehalogenases, such as those derived from the commercially available HALOTAG protein (Promega), and / or mutant dehalogenases disclosed in U.S. Publication Application 20060024808, are provided herein as components of the compositions, systems, and methods described herein, the disclosures of which are incorporated herein by reference.

[0076] Although these mutant dehalogenases are not strictly enzymes (they lack substrate turnover), their stable binding of substrates depends on the appropriate protein structure. The results of reassociating split fragments of mutant dehalogenases differ from those of split enzyme systems. This is because the labeling function of the mutant dehalogenase is retained on one of the fragments even after separation from its partner, whereas split enzymes are active when they are together and do not retain artifacts of their previous activity after separation. In practice, the labeling reaction of split mutant dehalogenases provides molecular memory of protein interactions. In the case of fluorescent ligands, the label is retained on one of the fragments but may not be detectable after the complex dissociates (because the fluorescent activation contact with the protein may be disrupted / absent); therefore, the combination of split dehalogenases and fluorescent ligands results in a unique situation of permanent labeling, but with dynamic (on / off) fluorescence detection of the retained label.

[0077] Mutant dehalogenases enable efficient labeling within living cells or their lysates. This labeling is conditional only on the presence or expression of the protein and the presence of the labeled hydrolase substrate. In contrast, labeling with split mutant dehalogenases depends on specific protein interactions occurring within the cell and the presence of the labeled hydrolase substrate.

[0078] In some embodiments, the components of the compositions, systems, and methods of this specification are provided as split-modified dehalogenases. In some embodiments, a first fragment of the mutant dehalogenase is fused to a first fragment of a luminescent protein (e.g., and optionally, the protein or molecule of interest), and a second fragment of the mutant dehalogenase is fused to a second fragment of a luminescent protein (e.g., and optionally, the protein or molecule of interest). In some embodiments, at least one of the mutant dehalogenase fragments has a substitution that forms a bond with a haloalkane ligand, if present in a fully-length modified dehalogenase having a sequence of two fragments. In some embodiments, the first fragment of the mutant dehalogenase and the second fragment of the mutant dehalogenase can interact (e.g., if facilitated or not) to form an active modified dehalogenase complex.

[0079] HALOTAG is a 297-residue self-labeled polypeptide (33 kDa) derived from the bacterial hydrolase (dehalogenase) enzyme, modified to covalently bond to the haloalkane portion that serves as the ligand for this enzyme. The HALOTAG ligand can be ligated to a solid surface (e.g., beads) or a functional group (e.g., a fluorophore), and the HALOTAG polypeptide can be fused to a variety of proteins of interest, thus enabling covalent bonding to the solid surface or functional group of the target protein.

[0080] HALOTAG polypeptides are modified dehalogenases with genetically altered active sites that specifically bind to chloroalkane linkers, which are haloalkane ligands, resulting in enhanced and increased ligand binding rates (Pries et al. The Journal of Biological Chemistry. 270(18):10405-11; the entire article is incorporated by reference). The reaction that forms the bond between the protein tag and the chloroalkane linker is rapid under physiological conditions and is essentially irreversible (Waugh DS (June 2005). Trends in Biotechnology. 23(6):316-20; the entire article is incorporated by reference). In native hydrolase enzymes, nucleophilic attack of the chloroalkane-reactive linker leads to the substitution of the halogen with an amino acid residue, resulting in the formation of a covalent alkyl-enzyme intermediate. Subsequently, this intermediate is hydrolyzed by amino acid residues within the wild-type hydrolase (Chen et al. (February 2005) Current Opinion in Biotechnology. 16(1):35-40; the whole is incorporated by reference). This would lead to the regeneration of the enzyme after the reaction. However, in the modified haloalkane dehalogenase HALOTAG, the reaction intermediate cannot be hydrolyzed due to the enzyme mutation and therefore cannot proceed through the second reaction. As a result, the intermediate persists as a stable covalent adduct that does not undergo the reverse reaction (Marks et al. (August 2006) Nature Methods. 3(8):591-6; the whole is incorporated by reference).

[0081] HALOTAG fusion proteins can be expressed using standard recombinant protein expression techniques (Adams et al. (May 2002) Journal of the American Chemical Society. 124(21):6063-76; the entire article is incorporated by reference). HALOTAG polypeptides are relatively small proteins, and their reactions are exogenous to mammalian cells, thus avoiding interference from endogenous mammalian metabolic reactions (Naested et al. The Plant Journal. 18(5):571-6; the entire article is incorporated by reference). Once the fusion protein is expressed, a wide range of potential experimental areas exist, including enzyme assays, cell imaging, protein arrays, determination of intracellular localization, and many additional possibilities (Janssen DB (April 2004). Current Opinion in Chemical Biology. 8(2):150-9; the entire article is incorporated by reference).

[0082] Various HALOTAG ligands, functional groups, fusions, assays, modifications, uses, etc., are described in U.S. Patent Nos. 8,748,148; 9,593,316; 10,246,690; 8,742,086; 9,873,866; 10,604,745; U.S. Patent Application No. 2009 / 0253131; U.S. Patent Application No. 2010 / 0273186; 20130337539; U.S. Patent Application No. 2012 / 0258470; U.S. Patent Application No. 2012 / 0252048; U.S. Patent Application No. 2011 / 0201024; and U.S. 2014 / 0322794; each of these is incorporated in whole by reference.

[0083] In some embodiments, the modified dehalogenase fragments, complementary peptides, complementary polypeptides, etc. described herein constitute a HALOTAG-based complementary system. In some embodiments, the modified dehalogenase fragments, complementary peptides, complementary polypeptides, etc. described herein correspond to sequences within the HALOTAG protein (e.g., sequence identity, sequence similarity, 3D structure, etc.). In some embodiments, the modified dehalogenase complexes described herein, comprising two or more peptide or polypeptide components, correspond to the HALOTAG protein and can be similarly conjugated to a halcowalkyl ligand.

[0084] In some embodiments, as described in USProv.App.No.63 / 338,323 and USApp.No.18 / 312,117 (both incorporated herein by reference in their entirety), extensive experiments have been conducted to demonstrate the feasibility of generating fragments of HALOTAG (and its variants) that can interact to form modified dehalogenase complexes capable of binding to haloalkyl ligands, and further experiments have been conducted to optimize the variants of the HALOTAG fragments to obtain desired properties. As described herein, embodiments are not limited to the HALOTAG sequence. In some embodiments, the split-modified dehalogenases provided herein have a sequence different from HALOTAG (SEQ ID NO: 1).

[0085] In some embodiments, compositions and systems are provided that include components of a split-modified dehalogenase, such as a split HaloTag ("spHT") or a variant thereof. In some embodiments, the systems and compositions herein include spHT peptides and polypeptides (for example, as part of a fusion described herein).

[0086] In some embodiments, compositions (e.g., fusions) and systems (e.g., multiple fusions having suitable ligands and substrates) are provided, comprising polypeptides, peptides, fragments, and combinations thereof derived from the modified dehalogenase sequence of Sequence ID No. 1 (HALOTAG): MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETF QAFRTTDVGRKLIIDQNVFIEGTLMGVVRPLTEEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISG.

[0087] In some embodiments, the spHT components of this specification lack mutations (multiple) (e.g., 272 and / or 106) that result in covalent bonding to a haloalkane substrate. Such sp dehalogenases are true enzymes capable of substrate turnover, but otherwise include the sequences and features of the embodiments described herein.

[0088] In some embodiments, the spHT peptides and polypeptides described herein (e.g., as part of a fusion described herein, as an independent reporter or tag, etc.) include at least 70% sequence identity with a portion of SEQ ID NO: 1 (e.g., greater than 70% sequence identity, greater than 75% sequence identity, greater than 80% sequence identity, greater than 85% sequence identity, greater than 90% sequence identity, greater than 95% sequence identity, greater than 96% sequence identity, greater than 97% sequence identity, greater than 98% sequence identity, greater than 99% sequence identity). In some embodiments, the spHT peptides and polypeptides described herein (e.g., as part of a fusion described herein, as an independent reporter or tag, etc.) include 100% sequence identity with all or a portion of SEQ ID NO: 1. In some embodiments, the spHT peptides and polypeptides described herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) have at least 70% sequence similarity to all or part of SEQ ID NO: 1 (e.g., greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%). In some embodiments, the spHT peptides and polypeptides described herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) have 100% sequence similarity to all or part of SEQ ID NO: 1.

[0089] In some embodiments, the spHT peptide or polypeptide used herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) contains A at the position corresponding to position 2 of SEQ ID NO: 1. In other embodiments, the spHT peptide or polypeptide used herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) contains S at the position corresponding to position 2 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide used herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) contains V at the position corresponding to position 47 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide used herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) contains T at the position corresponding to position 58 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide used herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) contains G at the position corresponding to position 78 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide used herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) contains F at the position corresponding to position 88 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) contains M at the position corresponding to position 89 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) contains F at the position corresponding to position 128 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) contains T at the position corresponding to position 155 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) contains K at the position corresponding to position 160 of SEQ ID NO: 1.In some embodiments, the spHT peptide or polypeptide used herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) contains V at the position corresponding to position 167 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide used herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) contains T at the position corresponding to position 172 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide used herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) contains M at the position corresponding to position 175 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide used herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) contains G at the position corresponding to position 176 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide used herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) contains N at the position corresponding to position 195 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) contains E at the position corresponding to position 224 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) contains D at the position corresponding to position 227 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) contains K at the position corresponding to position 257 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) contains A at the position corresponding to position 264 of SEQ ID NO: 1. In some embodiments, the peptide or polypeptide as specified herein contains N at the position corresponding to position 272 of SEQ ID NO: 1.In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) contains L at the position corresponding to position 273 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) contains S at the position corresponding to position 291 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) contains T at the position corresponding to position 292 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) contains E at the position corresponding to position 294 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) contains I at the position corresponding to position 295 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as described herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) contains S at the position corresponding to position 296 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as described herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) contains G at the position corresponding to position 297 of SEQ ID NO: 1.

[0090] In some embodiments, the spHT peptide or polypeptide used herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) does not have an S at the position corresponding to position 2 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide used herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) does not have an L at the position corresponding to position 47 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide used herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) does not have an S at the position corresponding to position 58 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide used herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) does not have a D at the position corresponding to position 78 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide used herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) does not have a Y at the position corresponding to position 88 of SEQ ID NO: 1. In some embodiments, the spHT peptides or polypeptides described herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) do not have L at the position corresponding to position 89 of SEQ ID NO: 1. In some embodiments, the spHT peptides or polypeptides described herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) do not have C at the position corresponding to position 128 of SEQ ID NO: 1. In some embodiments, the spHT peptides or polypeptides described herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) do not have A at the position corresponding to position 155 of SEQ ID NO: 1. In some embodiments, the spHT peptides or polypeptides described herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) do not have E at the position corresponding to position 160 of SEQ ID NO: 1.In some embodiments, the spHT peptide or polypeptide used herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) does not have an A at the position corresponding to position 167 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide used herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) does not have an A at the position corresponding to position 172 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide used herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) does not have a K at the position corresponding to position 175 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide used herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) does not have a C at the position corresponding to position 176 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide used herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) does not have a K at the position corresponding to position 195 of SEQ ID NO: 1. In some embodiments, the spHT peptides or polypeptides described herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) do not have an A at the position corresponding to position 224 of SEQ ID NO: 1. In some embodiments, the spHT peptides or polypeptides described herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) do not have an N at the position corresponding to position 227 of SEQ ID NO: 1. In some embodiments, the spHT peptides or polypeptides described herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) do not have an E at the position corresponding to position 257 of SEQ ID NO: 1. In some embodiments, the spHT peptides or polypeptides described herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) do not have a T at the position corresponding to position 264 of SEQ ID NO: 1.In some embodiments, the spHT peptides or polypeptides described herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) do not have H at the position corresponding to position 272 of SEQ ID NO: 1. In some embodiments, the spHT peptides or polypeptides described herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) do not have Y at the position corresponding to position 273 of SEQ ID NO: 1. In some embodiments, the spHT peptides or polypeptides described herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) do not have P at the position corresponding to position 291 of SEQ ID NO: 1. In some embodiments, the spHT peptides or polypeptides described herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) do not have A at the position corresponding to position 292 of SEQ ID NO: 1. In some embodiments, the spHT peptides or polypeptides described herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) do not have an amino acid at the position corresponding to position 294 of SEQ ID NO: 1. In some embodiments, the spHT peptides or polypeptides described herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) do not have an amino acid at the position corresponding to position 295 of SEQ ID NO: 1. In some embodiments, the spHT peptides or polypeptides described herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) do not have an amino acid at the position corresponding to position 296 of SEQ ID NO: 1. In some embodiments, the spHT peptides or polypeptides described herein (e.g., as part of a fusion, as an independent reporter or tag, etc.) do not have an amino acid at the position corresponding to position 297 of SEQ ID NO: 1.

[0091] In some embodiments, the sp dehalogenase (e.g., spHT) comprises two peptide and / or polypeptide components that, as a whole, have at least 70% sequence similarity or identity with all or part of Sequence ID No. 1 (e.g., more than 70% sequence similarity or identity, more than 75% sequence similarity or identity, more than 80% sequence similarity or identity, more than 85% sequence similarity or identity, more than 90% sequence similarity or identity, more than 95% sequence similarity or identity, more than 96% sequence similarity or identity, more than 97% sequence similarity or identity, more than 98% sequence similarity or identity, more than 99% sequence similarity or identity). For example, the first peptide / polypeptide component of the sp polypeptide corresponds to the first part of SEQ ID NO: 1 (e.g., at least 70% sequence similarity or sequence identity with respect to the first part), and the second peptide / polypeptide component of the sp polypeptide corresponds to the second part of SEQ ID NO: 1 (e.g., at least 70% sequence similarity or sequence identity with respect to the second part). In some embodiments, the sp dehalogenase (e.g., spHT) comprises two fragments having 100% sequence similarity or sequence identity with all or part of SEQ ID NO: 1 as a whole. For example, the first fragment of the sp polypeptide has 100% sequence similarity or sequence identity with respect to the first part of SEQ ID NO: 1, and the second fragment of the sp polypeptide has 100% sequence similarity or sequence identity with respect to the second part of SEQ ID NO: 1.

[0092] In some embodiments, the sp dehalogenase (e.g., existing as part of a fusion as described herein, or as an independent reporter or tag) includes an sp site. The sp site is an internal position in the parent sequence that defines the C-terminus of a first component or fragment of the sp dehalogenase and the N-terminus of a second component or fragment. For example, if a theoretical 100-amino acid polypeptide is split at an sp site between residues 57 and 58 of the parent polypeptide (referred to herein as the sp site at 57), the first constituent polypeptide corresponds to positions 1-57, and the second constituent polypeptide corresponds to positions 58-100. In some embodiments described herein, the sp site in SEQ ID NO: 1 can occur at any position from position 5 to position 290 of SEQ ID NO: 1. In some embodiments, SEQ ID NOs: 2-577 are exemplary components of an spHT polypeptide having 100% sequence identity with respect to SEQ ID NO: 1. In some embodiments, the active spHT complex is formed between two fragments that collectively contain the amino acids corresponding to each position in SEQ ID NO: 1. For example, a polypeptide having the sequence of SEQ ID NO: 26 and a peptide having the sequence of SEQ ID NO: 27 both contain, as a whole, the amino acids corresponding to each position in SEQ ID NO: 1. Any pair of peptides and polypeptides (or two polypeptides) corresponding to two of SEQ ID NOs: 2-577, and together containing the amino acids corresponding to each position in SEQ ID NO: 1 (with or without positional deletions or duplications), are used in the embodiments herein. In some embodiments, spHT dehalogenase (for example, for use in the fusions herein, or as a standalone reporter or tag) includes any of the following fragment pairs: SEQ ID NOs: 2 and 3, 4 and 5, 6 and 7, 8 and 9, 10 and 11, 12 and 13, 14 and 15, 16 and 17, 18 and 19, 20 and 21, 22 and 23, 24 and 25 , 26 and 27, 28 and 29, 30 and 31, 32 and 33, 34 and 35, 36 and 37, 38 and 39, 40 and 41, 42 and 43, 44 and 45, 46 and 47, 48 and 49, 50 and 51, 52 and 53, 54 and 55, 56 and 57, 58 and 59, 60 and 61, 62 and 63, 64 and 65, 66 and 67, 68 and 69, 70 and 71, 72 and 73,74 and 75, 76 and 77, 78 and 79, 80 and 81, 82 and 83, 84 and 85, 86 and 87, 88 and 89, 90 and 91, 92 and 93, 94 and 95, 96 and 97, 98 and 99, 100 and 101, 102 and 103, 104 and 105, 106 and 107, 108 and 109, 110 and 111, 112 and 113, 114 and 115, 116 and 117, 118 and 119, 120 and 121, 121, 122 and 123, 124 and 125, 126 and 127, 128 and 129, 130 and 131, 132 and 133, 134 and 135, 136 and 137, 138 and 139, 140 and 141, 142 and 143, 144 and 145, 146 and 147, 148 and 149, 150 and 151, 152 and 153, 154 and 155, 156 and 157, 158 and 159, 160 and 161, 172 and 173, 174 and 175, 176 and 177, 178 and 179, 180 and 181, 182 and 183, 184 and 185, 186 and 187, 188 and 189, 190 and 191, 192 and 193, 194 and 195, 196 and 197, 198 and 1 99, 200 and 201, 202 and 203, 204 and 205, 206 and 207, 208 and 209, 190 and 211, 212 and 213, 214 and 215, 216 and 217, 218 and 219, 220 and 221, 222 and 223, 224 and 225, 226 and 227, 228 and 229, 300 and 301, 302 and 303, 304 and 305, 306 and 307, 308 and 309, 310 and 311, 312 and 313, 314 and 315, 316 and 317, 318 and 319, 320 and 321, 322 and 323, 324 and 325, 326 and 327, 328 and 329, 330 and 331, 332 and 333, 334 and 335, 336 and 337, 338 and 339, 340 and 341, 342 and 343, 344 and 345, 346 and 347, 348 and 349, 350 and 351, 352 and 353, 354 and 355, 356 and 357, 358 and 359, 360 and 361, 362 and 363, 364 and 365, 366 and 367, 368 and 369, 370 and 371, 372 and 373, 374 and 375, 376 and 377, 378 and 379,380 and 381, 382 and 383, 384 and 385, 386 and 387, 388 and 389, 390 and 391, 392 and 393, 394 and 395, 396 and 397, 398 and 399, 400 and 401, 402 and 403, 404 and 405, 406 and 407, 408 and 409, 410 and 411, 412 and 413, 414 and 415, 416 and 417, 418 and 419, 420 and 421, 422 and 423, 424 and 425, 426 and 427, 428 and 42 9, 430 and 431, 432 and 433, 434 and 435, 436 and 437, 438 and 439, 440 and 441, 442 and 443, 444 and 445, 446 and 447, 448 and 449, 450 and 451, 452 and 453, 454 and 455, 456 and 457, 458 and 459, 460 and 461, 462 and 463, 464 and 465, 466 and 467, 468 and 469, 470 and 471, 472 and 473, 474 and 475, 476 and 477, 478 and 4 79, 480 and 481, 482 and 483, 484 and 485, 486 and 487, 488 and 489, 490 and 491, 492 and 493, 494 and 495, 496 and 497, 498 and 499, 500 and 501, 502 and 503, 504 and 505, 506 and 507, 508 and 509, 510 and 511, 512 and 513, 514 and 515, 516 and 517, 518 and 519, 520 and 521, 522 and 523, 524 and 525, 526 and 527, 528 and 529, 530 and 531, 532 and 533, 534 and 535, 536 and 537, 538 and 539, 540 and 541, 542 and 543, 544 and 545, 546 and 547, 548 and 549, 550 and 551, 552 and 553, 554 and 555, 556 and 557, 558 and 559, 560 and 561, 562 and 563, 564 and 565, 566 and 567, 568 and 569, 570 and 571, 572 and 573, 574 and 575, and 576 and 577.

[0093] In some embodiments, an spHT comprises a pair of peptides and polypeptides (or two polypeptides) corresponding to two of SEQ ID NOs: 2-577, which together contain amino acids corresponding to each position in SEQ ID NO: 1, but have a deletion of up to 40 amino acids in length at the C-terminus or N-terminus of one or both fragments (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or in between). For example, the pair corresponding to SEQ ID NOs: 7 and 28, when combined, corresponds to the positions in SEQ ID NO: 1, but has a deletion of 11 residues. In some embodiments, any pair of SEQ ID NOs: 2-577, which, when combined, corresponds to the sequence of SEQ ID NO: 1, but has a deletion of up to 40 amino acids, is within the scope of spHT as defined herein. In some embodiments, the deletion is adjacent to the splitting site. In some embodiments, the deletion corresponds to the N-terminus or C-terminus of SEQ ID NO: 1.

[0094] In some embodiments, spHT comprises a pair of peptides and polypeptides (or two polypeptides) corresponding to two of SEQ ID NOs: 2-577, which together contain amino acids corresponding to each position in SEQ ID NO: 1, but have overlaps of up to 40 amino acids in length at the C-terminus or N-terminus of one or both fragments (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or in between). For example, the pair corresponding to SEQ ID NOs: 6 and 29, when combined, correspond to each position in SEQ ID NO: 1, but have an overlap of 11 residues. In some embodiments, any pair of SEQ ID NOs: 2-577, which, when combined, correspond to the sequence of SEQ ID NO: 1, but have an overlap of up to 40 amino acids, is within the scope of spHT as defined herein. In some embodiments, the overlap is adjacent to the splitting site. In some embodiments, the overlap corresponds to the N-terminus or C-terminus of SEQ ID NO: 1.

[0095] For example, a fragment utilizing any sp site corresponding to positions 5 through 290 of sequence number 1 is readily conceivable and falls within the scope of this specification.

[0096] In some embodiments, spHT is at positions 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 31, 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 313, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, – 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, – 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219 of SEQ ID NO: 1.220th, 221st, 222nd, 223rd, 224th, 225th, 226th, 227th, 228th, 229th, 230th, 231st, 232nd, 233rd, 234th, 235th, 236th, 237th, 238th , 239th, 240th, 241st, 242nd, 243rd, 244th, 245th, 246th, 247th, 248th, 249th, 250th, 251st, 252nd, 253rd, 254th, 255th, 256th, 257th It is provided having sp parts corresponding to the 258th, 259th, 260th, 261st, 262nd, 263rd, 264th, 265th, 266th, 267th, 268th, 269th, 270th, 271st, 272nd, 273rd, 274th, 275th, 276th, 277th, 278th, 279th, 280th, 281st, 282nd, 283rd, 284th, 285th, 286th, 287th, 288th, 289th, or 290th rank.

[0097] In some embodiments, spHT is provided having sp portions corresponding to positions between positions 5 and 13, 36 and 51, 63 and 72, 84 and 92, 104 and 130, 142 and 148, 160 and 174, 186 and 189, 311 and 313, 221 and 229, or 269 and 290 of SEQ ID NO: 1.

[0098] In some embodiments, the spHT peptides and polypeptides herein (e.g., as fusions or independent reporters or tags) have one or more substitutions or deletions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19m, 20, 25, 30, 35, 40, 50, 75, or more) for one of SEQ ID NOs. In some embodiments, the sp peptides and polypeptides are provided having 70% to 100% sequence identity (e.g., greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%) to one of Sequence IDs 2 to 557 (e.g., greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%). In some embodiments, sp peptides and polypeptides are provided having a sequence similarity of 70% to 100% to one of SEQ ID NOs: 2 to 557 (for example, sequence similarity of over 70%, over 75%, over 80%, over 85%, over 90%, over 95%, over 96%, over 97%, over 98%, and over 99%).

[0099] In some embodiments, pairs of sp peptides and / or polypeptides capable of forming an active sp dehalogenase complex (active spHT complex) are provided (for example, in the fusion body herein, or as independent reporters or tags). In some embodiments, such pairs have at least 70% sequence identity or similarity to two of SEQ ID NOs. 2-557, and together they contain residues corresponding to the position (100%) of SEQ ID NO. 1, and allow up to 40 deletions or duplications at the C-terminus or N-terminus of the peptide / polypeptide.

[0100] In some embodiments, the first fragment of the spHT complementary pair is located at positions 1 through 5 of SEQ ID NO: 1, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 31, 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 , 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, 74th, 7 5th, 76th, 77th, 78th, 79th, 80th, 81st, 82nd, 83rd, 84th, 85th, 86th, 87th, 88th, 89th, 90th, 91st, 92nd, 93rd, 94th, 95th, 96th, 97th, 98th, 99th, 100th, 101st, 102nd, 313th, 104th, 1 05th, 106th, 107th, 108th, 109th, 110th, 111th, 112th, 113th, 114th, 115th, 116th, 117th , 118th, 119th, 120th, 121st, 122nd, 123rd, 124th, 125th, 126th, 127th, 128th, 129th, 1 30th, 131st, 132nd, 133rd, 134th, 135th, 136th, 137th, 138th, 139th, 140th, 141st, 142nd , 143rd, 144th, 145th, 146th, 147th, 148th, 149th, 150th, 151st, 152nd, 153rd, 154th, 1 55th, 156th, 157th, 158th, 159th, 160th, 161st, 162nd, 163rd, 164th, 165th, 166th, 167th , 168th, 169th, 170th, 171st, 172nd, 173rd, 174th, 175th, 176th, 177th, 178th, 179th, 1 80th, 181st, 182nd, 183rd, 184th, 185th, 186th, 187th, 188th, 189th, 190th, 191st, 192nd , 193rd, 194th, 195th, 196th, 197th, 198th, 199th, 310th, 311th, 312th, 313th, 314th, 31st 5th place, 316th place, 317th place, 318th place, 319th place, 210th place, 211th place, 212th place, 213rd place, 214th place, 215th place, 216th place, 217th place,218th, 219th, 220th, 221st, 222nd, 223rd, 224th, 225th, 226th, 227th, 228th, 229th, 230th, 231st, 232nd, 233rd, 234th, 235th, 236th 237th, 238th, 239th, 240th, 241st, 242nd, 243rd, 244th, 245th, 246th, 247th, 248th, 249th, 250th, 251st, 252nd, 253rd, 254th, 25th It supports positions 5, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, or up to 290.

[0101] In some embodiments, the second fragment of the spHT complementary pair is located at positions 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 31, 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 6 2nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, 74th, 75th, 76th, 77th , 78th, 79th, 80th, 81st, 82nd, 83rd, 84th, 85th, 86th, 87th, 88th, 89th, 90th, 91st, 92nd, 93 No. 94, No. 95, No. 96, No. 97, No. 98, No. 99, No. 100, No. 101, No. 102, No. 313, No. 104, No. 105, No. 106, 1 07th, 108th, 109th, 110th, 111th, 112th, 113th, 114th, 115th, 116th, 117th, 118th, 119th , 120th, 121st, 122nd, 123rd, 124th, 125th, 126th, 127th, 128th, 129th, 130th, 131st, 1 32nd, 133rd, 134th, 135th, 136th, 137th, 138th, 139th, 140th, 141st, 142nd, 143rd, 144th , 145th, 146th, 147th, 148th, 149th, 150th, 151st, 152nd, 153rd, 154th, 155th, 156th, 1 57th, 158th, 159th, 160th, 161st, 162nd, 163rd, 164th, 165th, 166th, 167th, 168th, 169th , 170th, 171st, 172nd, 173rd, 174th, 175th, 176th, 177th, 178th, 179th, 180th, 181st, 1 82nd, 183rd, 184th, 185th, 186th, 187th, 188th, 189th, 190th, 191st, 192nd, 193rd, 194th , 195th, 196th, 197th, 198th, 199th, 310th, 311th, 312th, 313th, 314th, 315th, 316th, 31st 7th, 318th, 319th, 210th, 211th, 212nd, 213rd, 214th, 215th, 216th, 217th, 218th, 219th,220th, 221st, 222nd, 223rd, 224th, 225th, 226th, 227th, 228th, 229th, 230th, 231st, 232nd, 233rd, 234th, 235th, 236th, 237th, 23 8th, 239th, 240th, 241st, 242nd, 243rd, 244th, 245th, 246th, 247th, 248th, 249th, 250th, 251st, 252nd, 253rd, 254th, 255th, 256th This corresponds to ranks 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, or ranks 290-294.

[0102] In some embodiments, the overlap portion of the spHT complement is 1 to 40 amino acid lengths (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 31, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or a range between those).

[0103] In some embodiments, the deletion portion of the spHT complement is 1 to 40 amino acids long (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 31, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or a range between these).

[0104] The exemplary spHT fragment sequences of SEQ ID NOs: 2-577 contain 100% sequence identity with respect to the portion of SEQ ID NO: 1, and these sequences contain no portions that do not match SEQ ID NO: 1 with 100% sequence identity. However, as described herein, spHT peptides and polypeptides may have less than 100% sequence identity with SEQ ID NO: 1 (e.g., greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, but less than 100% sequence identity). Therefore, peptides and polypeptides having less than 100% sequence identity with respect to one or more SEQ ID NOs: 2-577 (e.g., greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, but less than 100% sequence identity) are provided herein for use in complementary pairs and complexes herein.

[0105] In some embodiments, the spHT complements described herein include a peptide corresponding to SEQ ID NO: 578 and a polypeptide corresponding to SEQ ID NO: 1188. SEQ ID NOs: 578 and 1188 are fragments of SEQ ID NO: 1 and have 100% sequence identity to the portion of SEQ ID NO: 1. In some embodiments, the spHT complements include a peptide having 100% sequence identity to SEQ ID NO: 578, and such peptides are referred to herein as "SmHT". In some embodiments, the spHT complements include a polypeptide having 100% sequence identity to SEQ ID NO: 1188, and such polypeptides are referred to herein as "LgHT". During the development of the embodiments described herein, extensive experiments were conducted to analyze variants of SmHT and LgHT. SEQ ID NOs: 579–1187 correspond to peptide variants of SEQ ID NO: 588 in which at least one to up to all positions are substituted. Each peptide of SEQ ID NOs: 578–1187 was synthesized and tested for various properties, including the ability to form an active complex with complementary LgHT variant polypeptides. Sequence IDs 1189-3033 correspond to polypeptide variants having one or more substitutions compared to Sequence ID 1188. Each peptide from Sequence IDs 1188-3033 was synthesized and tested for various properties, including its ability to form an active complex with complementary SmHT variant polypeptides.

[0106] In some embodiments, mutant SmHT peptides having one or more substitutions to a reference SmHT peptide sequence, such as the peptides of SEQ ID NOs. 3061, 3064-3066, and 3079-3091, are provided herein.

[0107] In some embodiments, provided herein (e.g., in a fusion of the Specified, or as a standalone reporter or tag, etc.) are SmHT peptides or SmHT variant peptides having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) sequence similarity (e.g., conserved or semi-conserved similarity) to one of SEQ ID NOs. 578-1187, 3061, 3064-3066, and 3079-3091. In some embodiments, the peptide (e.g., in a fusion of the Specified, or as a standalone reporter or tag, etc.) corresponds to SmHT (SEQ ID NO: 578), but has one or more substitutions of SEQ ID NOs. 588-1187 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or a range between these) to SEQ ID NO: 578. In some embodiments, the SmHT variant (e.g., within the fusion as herein, or as a standalone reporter or tag, etc.) has 1 to 8 non-conservative substitutions for one of SEQ ID NOs. 578 to 1187 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or a range between them).

[0108] In some embodiments, the peptide component of the modified dehalogenase (e.g., in the fusion as described herein, or as an independent reporter or tag) is a variant of HT[3-19] (SEQ ID NO: 3061) having one or more substitutions in one or more sequences of SEQ ID NOs: 3061, 3064-3066, and 3079-3091 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or a range between them). In some embodiments, the HT[3-19] variant (e.g., in the fusion as described herein, or as an independent reporter or tag) has 1 to 8 non-conservative substitutions in one of SEQ ID NOs: 3061, 3064-3066, and 3079-3091 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or a range between them).

[0109] In some embodiments, what is provided herein (for example, within the fusion body herein, or as a standalone reporter or tag, etc.) is a SmHT peptide or SmHT variant peptide comprising: X1X2X3X4X5(F / W / Y / M / H)X7(F / W / Y / D / R)X9X 10 X 11 (F / W / Y / M / H / R) (V / I / L / M / A / C)X 14 (V / I / L / A / C / MI / L / F / W)X 16 X 17 (SEQ ID NO:3034); and / or X1X2X3X4X5(F / W / Y)X7(F / W / Y)X9X 10 X 11 (F / W / Y) (V / I / L / M)X 14 (V / I / L)X 16 X 17 (Sequence ID 3035); Here, each X is an arbitrary amino acid (for example, a protein-forming amino acid).

[0110] In some embodiments, provided herein are LgHT polypeptides or LgHT variant polypeptides having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or in between) sequence similarity (e.g., conserved or semi-conserved similarity) to one of SEQ ID NOs. 1188-3033 (e.g., within the fusion body herein, or as an independent reporter or tag). In some embodiments, the polypeptide (e.g., within the fusion body herein, or as an independent reporter or tag) corresponds to LgHT (SEQ ID NOs. 1188) but has one or more substitutions of SEQ ID NOs. 1188 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or more, or in between) In some embodiments, the LgHT variant (e.g., in the fusion as herein, or as a standalone reporter or tag, etc.) has at least 70% sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) with one of sequence numbers 1188-3033. In some embodiments, the LgHT polypeptide of the LgHT variant polypeptide includes a substitution for the reference LgHT sequence (sequence number 1188), which corresponds to a substitution present in the LgHT portion of one of sequence numbers 3110-4064 constructs.

[0111] In some embodiments, provided herein are spHT complementary pairs (for example, each component of the pair exists in a separate fusion body), the complementary pair comprising: (a) an SmHT peptide or SmHT mutant peptide, (1) one of SEQ ID NOs: 578-1187, 3061, 3064-3066, and 3079-3091, and at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%) (2) Sequence similarity of %, 100%, or a range between them (e.g., conservative or semi-conservative similarity), (2) one or more substitutions for sequence number 578 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or a range between them), and / or (3) one to eight substitutions for one of sequence numbers 578-1187, 3061, 3064-3066, and 3079-3091 (e.g., (b) LgHT polypeptides or LgHT variant polypeptides having non-conservative substitutions (1, 2, 3, 4, 5, 6, 7, 8, or in the range between thereof); and (b) LgHT polypeptides or LgHT variant polypeptides having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or in the range between thereof) sequence similarity (e.g., conserved or semi-conservative similarity) to one of SEQ ID NOs: 1188-3033; (2) SEQ ID NOs: 1 One or more substitutions for 188 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or more, or a range in between) and / or (3) having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range in between) sequence identity with one of sequence numbers 1188 to 3033.

[0112] The formation of an spHT complex from two complementary fragments may be reversible or irreversible. In some embodiments, the spHT complex can be denatured, restored, and its activity reconstituted. In some embodiments, such spHTs are used in a method that involves exposing the spHT-containing sample to denaturing conditions (e.g., manufacturing conditions, storage conditions, etc.) before substrate binding.

[0113] NanoLuc In some embodiments, provided herein are compositions (e.g., fusion peptides and polypeptides) and systems (e.g., multiple complementary fusion peptides and polypeptides, substrates, ligands, etc.) comprising peptide / polypeptide fragments that can interact (e.g., facilitated or intangible) to form an active luminescent protein capable of generating luminescence using a suitable substrate.

[0114] In some embodiments, provided herein are compositions (e.g., fusion peptides and polypeptides) and systems (e.g., multiple complementary fusion peptides and polypeptides, substrates, ligands, etc.) comprising complementary peptide / polypeptide fragments that can interact (e.g., facilitated or unfavorable) to form an active bioluminescent complex capable of generating luminescence upon interaction with a suitable luminescent substrate. In some embodiments, a first fusion comprising a complementary peptide fragment of a luminescent protein is provided, and a second fusion comprising a complementary polypeptide fragment of a luminescent protein is also provided, and upon interaction (e.g., facilitated or unfavorable), the complementary peptide and polypeptide form an active bioluminescent complex capable of generating luminescence upon interaction with a suitable luminescent substrate. In some embodiments, the complementary peptide and polypeptide are fragments of a split luminescent protein (e.g., luciferase). In alternative embodiments, both fragments may be polypeptides.

[0115] This specification provides two- or multi-component bioluminescent complexes, such as commercially available NANOLUC protein (Promega) and / or those derived from NANOBIT (Promega) or the NANOTRIP structural complementation system, as components of the compositions, systems, and methods described herein.

[0116] Natural Oplophorus gracilirostris luciferase (OgLuc) and commercially available NANOLUC luciferase (Promega Corporation) each contain 10 beta chains (β1, β2, β3, β4, β5, β6, β7, β8, β9, β10). U.S. Patent No. 9,797,889 (which is incorporated herein by reference in its entirety) describes the development and use of a complementary system containing β1-β9-like polypeptides and a β10-like peptide (certain OgLuc / NANOLUC-based polypeptide and peptide sequences in U.S. Patent No. 9,797,889 differ from the corresponding sequences in NANOLUC and wild-type natural OgLuc). Similarly, U.S. Patent Application No. 16 / 439,565 (which is incorporated herein by reference in its entirety) describes the development and use of a complementary system comprising two or more OgLuc / NANOLUC peptides and / or polypeptides (certain OgLuc / NANOLUC-based polypeptide and peptide sequences in U.S. Patent No. 16 / 439,565 differ from the corresponding sequences in NANOLUC and wild-type natural OgLuc).

[0117] In some embodiments, provided herein are peptide components of a two-component bioluminescent complex (e.g., within the fusion body as herein, or as an independent reporter or tag) having more than 40% sequence identity (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%, greater than 98%, greater than 99%, greater than 100%) of SEQ ID NO: 3036, wherein a detectable bioluminescent signal is generated (e.g., higher luminescence than the components of the complex in the presence of the substrate) when the peptide component of the two-component bioluminescent complex comes into contact with a polypeptide comprising SEQ ID NO: 3037 (e.g., within the fusion body as herein, or as an independent reporter or tag) in the presence of a substrate of the bioluminescent complex. In some embodiments, the peptide has less than 100% sequence identity with SEQ ID NO: 3036. In some embodiments, a detectable bioluminescent signal is generated when the peptide component of a two-component bioluminescent complex comes into contact with the polypeptide component of a two-component bioluminescent complex having 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%, 100%) sequence identity with SEQ ID NO: 3037. In certain embodiments, a detectable bioluminescent signal is generated or substantially increased when the peptide associates with a polypeptide containing or comprising SEQ ID NO: 3037. In preferred embodiments, the peptide exhibits changes (e.g., enhancements) in one or more properties compared to the peptide of SEQ ID NO: 3038 or 3039, where the properties are selected from: affinity for the polypeptide comprising SEQ ID NO: 3037, expression, intracellular solubility, intracellular stability, and bioluminescent activity when combined with the polypeptide comprising SEQ ID NO: 3037.

[0118] Exemplary sequences of the peptide components of the two-component bioluminescent complexes used in the embodiments herein are described, for example, in U.S. Patent No. 9,797,889 (which is incorporated in its entirety by reference). The peptide components of the two-component bioluminescent complexes herein are not limited to these sequences, but in some embodiments, the peptide components of the two-component bioluminescent complexes herein may be selected from the amino acid sequences described in SEQ ID NOs. 3-438 and 2162-2365 of U.S. Patent No. 9,797,889 (which is incorporated in its entirety by reference).

[0119] In some embodiments, provided herein are peptide components of a two-component bioluminescent complex (e.g., within the fusion body as herein, or as an independent reporter or tag) having more than 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%, greater than 98%, greater than 99%, greater than 100%) sequence identity with SEQ ID NO: 3038, wherein a detectable bioluminescent signal is generated (e.g., higher luminescence than the components of the complex in the presence of the substrate) when the peptide component of the two-component bioluminescent complex comes into contact with a polypeptide comprising SEQ ID NO: 3037 (e.g., within the fusion body as herein, or as an independent reporter or tag) in the presence of a substrate of the bioluminescent complex. In some embodiments, the peptide has less than 100% sequence identity with SEQ ID NO: 3036. In some embodiments, a detectable bioluminescent signal is generated when the peptide component of a two-component bioluminescent complex comes into contact with the polypeptide component of a two-component bioluminescent complex having more than 40% sequence identity with SEQ ID NO: 3037 (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%, 100%). In certain embodiments, a detectable bioluminescent signal is generated or substantially increased when the peptide associates with a polypeptide containing or consisting of SEQ ID NO: 3037. In some embodiments, the peptide exhibits high affinity for the polypeptide of SEQ ID NO: 3037 and can form a bioluminescent complex without facilitating it. In preferred embodiments, the peptide exhibits changes (e.g., enhancements) to one or more properties compared to the peptide of SEQ ID NO: 3036, where the property is selected from: affinity for the polypeptide comprising SEQ ID NO: 3037, expression, intracellular solubility, intracellular stability, and bioluminescent activity when combined with the polypeptide comprising SEQ ID NO: 3037.

[0120] In some embodiments, provided herein are peptide components of a two-component bioluminescent complex (e.g., within the fusion body as herein, or as an independent reporter or tag) having more than 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%, greater than 98%, greater than 99%, greater than 100%) sequence identity with SEQ ID NO: 3039, wherein a detectable bioluminescent signal is generated (e.g., higher luminescence than the components of the complex in the presence of the substrate) when the peptide component of the two-component bioluminescent complex comes into contact with a polypeptide comprising SEQ ID NO: 3037 (e.g., within the fusion body as herein, or as an independent reporter or tag) in the presence of a substrate of the bioluminescent complex. In some embodiments, the peptide has less than 100% sequence identity with SEQ ID NO: 3036. In some embodiments, a detectable bioluminescent signal is generated when the peptide component of a two-component bioluminescent complex comes into contact with the polypeptide component of a two-component bioluminescent complex having 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%, 100%) sequence identity with SEQ ID NO: 3037. In certain embodiments, a detectable bioluminescent signal is generated or substantially increased when the peptide associates with a polypeptide containing or consisting of SEQ ID NO: 3037. In some embodiments, the peptide exhibits low affinity for the polypeptide of SEQ ID NO: 3037 and does not form a stable bioluminescent complex without facilitation. In preferred embodiments, the peptide exhibits changes (e.g., enhancements) to one or more properties compared to the peptide of SEQ ID NO: 3036, where the property is selected from: affinity (or low affinity) to the polypeptide comprising SEQ ID NO: 3037, expression, intracellular solubility, intracellular stability, and bioluminescent activity when combined with the polypeptide comprising SEQ ID NO: 3037.In some embodiments, provided herein are polypeptide components of a two-component bioluminescent complex (e.g., within the fusion as defined herein, or as an independent reporter or tag) having more than 40% sequence identity (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%, greater than 98%, greater than 99%, greater than 100%) of SEQ ID NO: 3037, wherein a detectable bioluminescent signal is generated (e.g., higher luminescence than the components of the complex in the presence of the substrate) when the polypeptide comes into contact with the polypeptide comprising SEQ ID NO: 3036 (e.g., within the fusion as defined herein, or as an independent reporter or tag) in the presence of the substrate of the bioluminescent complex. In some embodiments, the polypeptide components of the two-component bioluminescent complex (e.g., within the fusion as defined herein, or as an independent reporter or tag) have less than 100% sequence identity with SEQ ID NO: 3037. In some embodiments, a detectable bioluminescent signal is generated when the polypeptide comes into contact with a peptide having more than 40% sequence identity with SEQ ID NO: 3036 (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%, 100%). In some embodiments, the polypeptide exhibits changes (e.g., enhancements) in one or more properties compared to the peptide of SEQ ID NO: 3037, where the properties are selected from: affinity for the polypeptide consisting of SEQ ID NO: 3036 or 3038, expression, intracellular solubility, intracellular stability, and bioluminescent activity when combined with the polypeptide consisting of SEQ ID NO: 3036, 3038, or 3039.

[0121] Exemplary sequences of polypeptide components of the two-component bioluminescent complexes used in the embodiments herein are described, for example, in U.S. Patent No. 9,797,889 (which is incorporated in its entirety by reference). The peptide components of the two-component bioluminescent complexes herein are not limited to these sequences, but in some embodiments, the polypeptide components of the two-component bioluminescent complexes herein may be selected from the amino acid sequences described in SEQ ID NOs. 441-2156 of U.S. Patent No. 9,797,889 (which is incorporated in its entirety by reference).

[0122] In certain embodiments, the present invention provides bioluminescent complexes (formed between fusions described herein) comprising: (a) a peptide comprising a peptide amino acid sequence having more than 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%, greater than 98%, greater than 99%, greater than 100%) sequence identity with SEQ ID NO: 3036, 3038, or 3039; and (b) a polypeptide comprising a polypeptide amino acid sequence having more than 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%, greater than 98%, greater than 99%, greater than 100%) sequence identity with SEQ ID NO: 3037. Here, the bioluminescent complex exhibits detectable luminescence in the presence of a substrate (for example, higher luminescence than the components of the complex in the presence of the substrate).

[0123] In some embodiments, provided herein are polypeptide components of a two-component bioluminescent complex (e.g., within the fusion body as herein, or as an independent reporter or tag) having more than 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%, greater than 98%, greater than 99%, greater than 100%) sequence identity with SEQ ID NO: 3037, wherein a detectable bioluminescent signal is generated (e.g., higher luminescence than the components of the complex in the presence of the substrate) when the polypeptide comes into contact with a peptide comprising SEQ ID NO: 3036, 3038, or 3039 (e.g., within the fusion body as herein, or as an independent reporter or tag) in the presence of a substrate of the bioluminescent complex.

[0124] In some embodiments, provided herein (alone and / or within a fusion described herein) are components of a bioluminescent complex comprising: a first component having sequence identity of 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%), or within these ranges) to a first fragment of SEQ ID NO: 3040 or SEQ ID NO: 3041; and a complementary portion of SEQ ID NO: 3040 or SEQ ID NO: 3041 One or more complementary fragments collectively containing 40% or more sequence identity (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%), or within these ranges); where the bioluminescent signal produced by a bioluminescent complex assembled from these components in the presence of a coelenterazine or coelenterazine derivative substrate is substantially amplified compared to the bioluminescent signal produced from the coelenterazine substrate and the individual components alone. In some embodiments, the first component includes 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%) or within these ranges) sequence identity to sequence number 3042, and one or more complementary components collectively include 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%) or within these ranges) sequence identity to sequence number 3046. In some embodiments, the first component includes 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%) or within these ranges) sequence identity to sequence number 3043, and one or more complementary components collectively include 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%) or within these ranges) sequence identity to sequence number 3047.In some embodiments, the first component includes 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%) or within these ranges) sequence identity to sequence number 3044, and one or more complementary components collectively include 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%) or within these ranges) sequence identity to sequence number 3048. In some embodiments, the first component includes 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%) or within these ranges) sequence identity to sequence number 3045, and one or more complementary components collectively include 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%) or within these ranges) sequence identity to sequence number 3049. In some embodiments, the first component includes 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%) or within these ranges) sequence identity to sequence number 3042, and one or more complementary components collectively include 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%) or within these ranges) sequence identity to sequence number 3050.In some embodiments, the first component includes 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%) or within these ranges) sequence identity to sequence number 3043, and one or more complementary components collectively include 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%) or within these ranges) sequence identity to sequence number 3051. In some embodiments, the first component includes 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%) or within these ranges) sequence identity to sequence number 3044, and one or more complementary components collectively include 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%) or within these ranges) sequence identity to sequence number 3052. In some embodiments, the first component includes 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%) or within these ranges) sequence identity to SEQ ID NO: 3045, and one or more complementary components collectively include 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%) or within these ranges) sequence identity to SEQ ID NO: 3053. In some embodiments, the bioluminescent signal is substantially increased when the first component associates with one or more complementary components.

[0125] Exemplary sequences of peptide and polypeptide components of two-component or multi-component bioluminescent complexes used in the embodiments herein are described, for example, in U.S. Patent Application No. 16 / 439,565 (which is incorporated in its entirety by reference). The peptide and polypeptide components of two-component or multi-component bioluminescent complexes herein are not limited to these sequences, but in some embodiments, the peptide and polypeptide components of two-component or multi-component bioluminescent complexes herein may be selected from the amino acid sequences of SEQ ID NOs. 1 to 804 of U.S. Patent Application No. 16 / 439,565 (which is incorporated in its entirety by reference).

[0126] In some embodiments, what is provided herein is a polypeptide (e.g., within a fusion body described herein) containing 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%) or within these ranges) sequence identity with any one of SEQ ID NOs. 790, 791, 792, or 793.

[0127] In some embodiments, what is provided herein is a peptide polypeptide comprising SEQ ID NOs. 3054-3060 (e.g., in a fusion body described herein). In some embodiments, what is provided herein is a peptide comprising any one of SEQ ID NOs. 3054-3060 with sequence identity of 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%), or within these ranges) (e.g., in a fusion body described herein).

[0128] In some embodiments, what is provided herein is a β6-7-like peptide comprising SEQ ID NOs. 3054 and 3055 (e.g., in the fusion described herein). In some embodiments, what is provided herein is a β6-7-like peptide having 40% or more sequence identity with SEQ ID NOs. 3054 and 3055 (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%), or within these ranges).

[0129] In some embodiments, what is provided herein is a β7-8-like peptide (e.g., in a fusion body described herein) comprising SEQ ID NOs. 3055 and 3056. In some embodiments, what is provided herein is a β7-8-like peptide (e.g., in a fusion body described herein) having 40% or more sequence identity (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%) or within these ranges) sequence identity with SEQ ID NOs. 3055 and 3056.

[0130] In some embodiments, what is provided herein is a β8-9-like peptide (e.g., in a fusion body described herein) comprising SEQ ID NO: 3056 / 3059 or 3056 / 3060. In some embodiments, what is provided herein is a β8-9-like peptide (e.g., in a fusion body described herein) having 40% or more sequence identity (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%) or within these ranges) with SEQ ID NO: 3056 / 3059 or 3056 / 3060.

[0131] In some embodiments, what is provided herein is a β9-10-like peptide (e.g., in a fusion described herein) comprising SEQ ID NOs. 3059 / 3057, 3059 / 3058, 3060 / 3057, or 3060 / 3058. In some embodiments, what is provided herein is a β8-9-like peptide (e.g., in a fusion described herein) having 40% or more sequence identity (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%) or within these ranges) of SEQ ID NOs. 3059 / 3057, 3059 / 3058, 3060 / 3057, or 3060 / 3058.

[0132] In some embodiments, what is provided herein is a β6-8-like peptide or polypeptide (e.g., in the fusion described herein) comprising SEQ ID NOs. 3054-3056. In some embodiments, what is provided herein is a β6-8-like peptide or polypeptide (e.g., in the fusion described herein) having 40% or more sequence identity (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%) or within these ranges) sequence identity with SEQ ID NOs. 3054-3056.

[0133] In some embodiments, what is provided herein is a β7-9-like peptide or polypeptide (e.g., in the fusion described herein) comprising SEQ ID NOs. 3055 / 3056 / 3059 or 3055 / 3056 / 3060. In some embodiments, what is provided herein is a β7-9-like peptide or polypeptide (e.g., in the fusion described herein) having 40% or more sequence identity (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%) or within these ranges) sequence identity with SEQ ID NOs. 3055 / 3056 / 3059 or 3055 / 3056 / 3060.

[0134] In some embodiments, what is provided herein is a β8-10-like peptide or polypeptide (e.g., in a fusion body described herein) comprising SEQ ID NOs: 3056 / 3059 / 3057, 3056 / 3059 / 3058, 3056 / 3060 / 3057, or 3056 / 3060 / 3058. In some embodiments, what is provided herein is a β7-9-like peptide peptide or polypeptide (e.g., within the fusion body described herein) having 40% or more sequence identity (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%) or within these ranges) with SEQ ID NOs. 3056 / 3059 / 3057, 3056 / 3059 / 3058.

[0135] Tandem peptide tag In some embodiments, provided herein is a tandem peptide tag comprising (i) a peptide component of a bioluminescent complex fused to (ii) a peptide component of a modified dehalogenase complex. In some embodiments, the peptide component of the bioluminescent complex can form a bioluminescent complex with the polypeptide component of the bioluminescent complex upon interaction between them (e.g., facilitated or unfavorable). In some embodiments, the peptide component of the modified dehalogenase complex can form a modified dehalogenase complex with the polypeptide component of the modified dehalogenase complex upon interaction between them (e.g., facilitated or unfavorable). In some embodiments, the bioluminescent complex and the modified dehalogenase complex are formed upon interaction (facilitated or unfavorable) between the tandem peptide tag and a tandem polypeptide reporter (e.g., including the polypeptide component of the bioluminescent complex fused to the polypeptide component of the modified dehalogenase complex). In some embodiments, the bioluminescent complex can generate bioluminescence in the presence of a substrate (e.g., coelenterazine, flimazine, etc.), and the modified dehalogenase complex can bind to a haloalkane ligand.

[0136] In some embodiments, the tandem peptide tag herein comprises a first component which is a component of a bioluminescent complex, as described herein and throughout U.S. Patent No. 9,797,889 and U.S. Patent Application No. 16 / 439,565, all of which are incorporated herein by reference in their entirety. Any component of a bioluminescent complex described herein or incorporated by reference may be used in the tandem peptide tag herein. In certain embodiments, the tandem peptide tag herein comprises a peptide component of a bioluminescent complex.

[0137] In some embodiments, the tandem peptide tag herein includes a second component which is a component of a modified dehalogenase complex, as described herein and throughout U.S. Provisional Patent Application No. 63 / 338,323 and U.S. Patent Application No. 18 / 312,117, all of which are incorporated herein by reference in their entirety. Any component of the modified dehalogenase complex described herein or incorporated by reference may be used in the tandem peptide tag herein. In certain embodiments, the tandem peptide tag herein includes a peptide component of a modified dehalogenase complex.

[0138] In some embodiments, a tandem peptide tag containing components of a bioluminescent complex and components of a modified dehalogenase complex is fused to or otherwise linked to the peptide or protein of interest. In some embodiments, the tandem peptide tag is expressed as a fusion with the peptide or protein of interest. In some embodiments, the peptide or protein of interest is a cellular target to be detected, quantified, or otherwise characterized by using the systems and methods described herein.

[0139] In some embodiments, the tandem peptide tags herein include at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between them) sequence identity with respect to one or more of SEQ ID NOs: 3062-3063, 3067-3078, 3092, 3094-3109, and 4177-4181. In some embodiments, the peptide component of the modified dehalogenase moiety (e.g., SmHT) of the tandem peptide tag herein includes one or more substitutions with respect to the reference SmHT peptide and / or one or more SmHT moieties of any of the SmHT variant peptides herein and / or SEQ ID NOs: 3062-3063, 3067-3078, 3092, and 3094-3109. In some embodiments, the peptide component of the bioluminescent complex portion (e.g., HiBiT) of the tandem peptide tag herein comprises one or more substitutions to the reference HiBiT peptide of any of the HiBiT mutant peptides herein and / or to the reference HiBiT peptides of one or more HiBiT portions of SEQ ID NOs. 3062-3063, 3067-3078, 3092, 3094-3109, and 4177-4181.

[0140] Tandem Polypeptide Reporter In some embodiments, provided herein is a tandem polypeptide reporter comprising a polypeptide component of a bioluminescent complex fused to a polypeptide component of a modified dehalogenase complex. In some embodiments, the polypeptide component of the bioluminescent complex can form a bioluminescent complex with the peptide component of the bioluminescent complex upon interaction between them (e.g., facilitated or unfavorable). In some embodiments, the polypeptide component of the modified dehalogenase complex can form a modified dehalogenase complex with the peptide component of the modified dehalogenase complex upon interaction between them (e.g., facilitated or unfavorable). In some embodiments, the bioluminescent complex and the modified dehalogenase complex are formed upon interaction (e.g., facilitated or unfavorable) between the tandem polypeptide reporter and the tandem peptide tag (e.g., including the peptide component of the bioluminescent complex fused to the peptide component of the modified dehalogenase complex). In some embodiments, the bioluminescent complex can generate bioluminescence in the presence of a substrate (e.g., coelenterazine, flimazine, etc.), and the modified dehalogenase complex can bind to a haloalkane ligand.

[0141] In some embodiments, a first component which is a component of a bioluminescent complex is included, as described herein and throughout U.S. Patent No. 9,797,889 and U.S. Patent Application No. 16 / 439,565, all of which are incorporated herein by reference in their entirety. Any of the components of a bioluminescent complex described herein or incorporated by reference may be used in the tandem peptide reporter herein. In certain embodiments, the tandem peptide reporter herein includes a peptide component of a bioluminescent complex.

[0142] In some embodiments, the tandem peptide reporters herein include a second component which is a component of a modified dehalogenase complex, as described herein and throughout U.S. Provisional Patent Application No. 63 / 338,323 and U.S. Patent Application No. 18 / 312,117, all of which are incorporated herein by reference in their entirety. Any component of the modified dehalogenase complex described herein or incorporated by reference may be used in the tandem peptide reporters herein. In certain embodiments, the tandem peptide reporters herein include a peptide component of a modified dehalogenase complex.

[0143] In some embodiments, the tandem peptide reporters herein contain at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between them) sequence identity with respect to one or more of SEQ ID NOs: 3110-4064. In some embodiments, the polypeptide component of the modified dehalogenase moiety (e.g., LgHT) of the tandem polypeptide reporter herein contains one or more substitutions with respect to the reference LgHT peptide of any of the LgHT mutant peptides herein and / or one or more LgHT moieties of SEQ ID NOs: 3110-4064. In some embodiments, the peptide component of the bioluminescence complex moiety (e.g., HiBiT) of the tandem peptide reporter herein contains one or more substitutions with respect to the reference HiBiT peptide of any of the HiBiT mutant peptides herein and / or one or more HiBiT moieties of SEQ ID NOs: 3110-4064.

[0144] Other tandem tags and reporters Embodiments of this specification are primarily described as tags containing a fusion peptide component of a bioluminescent and modified dehalogenase complex, and reporters containing a fusion polypeptide component of the complex. However, embodiments within the scope of this specification also include tags (for example, for linking / fusion to a target) that include: a polypeptide component of a bioluminescent and modified dehalogenase complex, a polypeptide component of a bioluminescent complex and a peptide component of a modified dehalogenase complex, or a peptide component of a bioluminescent complex and a polypeptide component of a modified dehalogenase complex; and reporters that include: a peptide component of a bioluminescent and modified dehalogenase complex, a polypeptide component of a bioluminescent complex and a peptide component of a modified dehalogenase complex, or a peptide component of a bioluminescent complex and a polypeptide component of a modified dehalogenase complex. Any suitable arrangement of the components described herein is within the scope of this specification.

[0145] Linker Various fusions are described herein. In some embodiments, the components of the fusion are directly linked (e.g., C-terminus to N-terminus). In other embodiments, the fusions herein include a peptide or polypeptide linker between the components. Such a linker can be any suitable sequence and is up to 100 amino acids long (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 445, 50, 60, 70, 80, 90, 100, or a range in between). In some embodiments, the tandem peptide tag herein includes a linker sequence between the peptide component of the modified dehalogenase complex and the peptide component of the bioluminescent complex. In some embodiments, the tandem peptide tag is linked to a target element by a suitable linker sequence. In some embodiments, the tandem detector herein includes a linker sequence between the polypeptide component of the modified dehalogenase complex and the polypeptide component of the bioluminescent complex. Exemplary linker sequences are provided in the tandem tags and tandem detectors exemplified herein, but other linker sequences are within the scope of this specification. Any peptide / polypeptide sequences capable of linking the components of the constructs herein are within the scope of this specification, including, but not limited to, those specifically exemplified herein in terms of both length and amino acid composition.

[0146] Haloalkyl ligand As described herein, spHT systems (e.g., those in the fusions herein) utilize haloalkane ligands. In some embodiments, the ligand is of formula (I):R-linker-AX, where R is a solid surface, one or more functional groups, or absent; the linker is a polyatomic linear or branched chain containing C, N, S, or O, or a group containing one or more rings (e.g., saturated or unsaturated rings, e.g., one or more aryl rings, heteroaryl rings, or any combination thereof); and AX is a dehalogenase, hydrolase, HALOTAG, or ligand for the spHT system herein (e.g., where A is (CH2) 4-20(I) is a halogen (e.g., Cl or Br). Suitable ligands are described, for example, in U.S. Patent Nos. 11,072,812; 11,028,424; 10,618,907; and 10,101,332, which are incorporated in their entirety by reference. In certain embodiments, X in formula (I) is not a halogen but a methylsulfonamide or trifluoromethylsulfonamide, and such embodiments result in interchangeable ligands that reversibly bind to modified dehalogenases (e.g., HALOTAG). Such ligands are described, for example, in Kompa et al. J.Am.Chem.Soc.2023,145,5,3075-3083, which are incorporated in their entirety by reference.

[0147] In some embodiments, R is one or more functional groups (e.g., fluorophores, biotins, luminescent groups, or fluorescent or luminescent molecules). Exemplary functional groups for use in the present invention include amino acids, proteins (e.g., enzymes, antibodies or other immunogenic proteins), radionuclides, nucleic acid molecules, drugs, lipids, biotin, avidin, streptavidin, magnetic beads, solid supports, electron-impermeable molecules, chromophores, MRI contrast agents, dyes (e.g., xanthene dyes), calcium-sensitive dyes (e.g., 1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxy-9-xanthenyl)-phenoxy]-2-(2'-amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid (Fluo-3)), sodium-sensitive dyes (e.g., 1,3-benzenedicarboxylic acid, 4,4'-[1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diirbis(5-methoxy- Examples include, but are not limited to, 6,2-benzofranzyl)bis(PBFI), No-sensitive dyes (e.g., 4-amino-5-methylamino-2',7'-difluorescein), or other fluorophores. In one embodiment, the functional group is conjugated by an immunogenic molecule, i.e., an antibody specific to that molecule.

[0148] In some embodiments, the ligands of the present invention are permeable to the cell plasma membrane (i.e., they can pass from outside the cell (e.g., eukaryotic cells, prokaryotic cells) into the cell without chemically, enzymatically, or mechanically disrupting the cell membrane).

[0149] In some embodiments, the ligands herein include cleavable linkers, such as those described in U.S. Patent No. 10,618,907, which are incorporated in their entirety by reference.

[0150] In some embodiments, the ligand includes a fluorescent functional group (R).Suitable fluorescent functional groups include, but are not limited to, the following: stilbazolium derivatives (Marquesa et al. Mechanism-Based Strategy for Optimizing HaloTag Protein Labeling. ChemRxiv. Cambridge: Cambridge Open Engage;2021;the whole is incorporated by reference), xanthene derivatives (e.g., fluorescein, rhodamine, Oregon Green, eosin, Texas Red, etc.), cyanine derivatives (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, etc.), naphthalene derivatives (e.g., dansyl and prodan derivatives), oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole, benzooxadiazole, etc.), pyrene derivatives (e.g., Cascade Blue), oxazine derivatives (e.g., Nile Red, Nile Blue, Cresyl Violet, Oxazine 170, etc.), acridine derivatives (e.g., proflavin, acridine orange, acridine yellow, etc.), allylmethine derivatives (e.g., auramine, crystal violet, malachite green, etc.), tetrapyrrole derivatives (e.g., porfin, phthalocyanine, bilirubin, etc.), CF dyes (Biotium), BODIPY (Invitrogen), ALEXA FLOUR (Invitrogen), DYLIGHT FLUOR (Thermo Scientific, Pierce), ATTO and TRACY (Sigma Aldrich), FluoProbes (Interchim), DY and MEGASTOKES (Dyomics), SULFO CY dye (CYANDYE, LLC), SETAU and SQUARE dyes (SETA BioMedicals), QUASAR and CAL FLUOR dyes (Biosearch Technologies), SURELIGHT dyes (APC, RPE, PerCP, phycobilisome) (Columbia Biosciences), APC, APCXL, RPE, BPE (Phyco-Biotech), autofluorescent proteins (e.g., YFP, RFP, mCherry, mKate), quantum dot nanocrystals, etc.

[0151] In some embodiments, the ligand includes a fluorescing functional group (R). The fluorescing functional group generates and enhances a fluorescent signal upon binding of the ligand to a target (e.g., binding of a haloalkane to a modified dehalogenase). The significant increase in fluorescence upon binding to the target (e.g., 10-fold, 31-fold, 50-fold, 100-fold, 310-fold, 500-fold, 1000-fold, or more) mitigates the problem of background signaling. Exemplary fluorescing dyes for use in the embodiments herein include fluorophores of the JANELIA FLUOR family, as listed below: [ka] [ka] [ka] [ka] (See, for example, U.S. Patents 9,933,417, 10,018,624, 10,161,932, and 10,495,632; each of these is incorporated in whole by reference). In some embodiments, exemplary conjugates of JANELIA FLUOR 549 and JANELIA FLUOR 646 with haloalkane ligands for modified dehalogenases (e.g., HALOTAG) are commercially available (Promega Corp.). The use and design of fluorescent functional groups, dyes, probes, and ligands are described, for example, in Grimm et al. Nat Methods. 3117 Oct;14(10):987-994.; and in Wang et al. Nat Chem. 3120 Feb;12(2):165-172, which are incorporated in whole by reference.

[0152] In some embodiments, the fluorophores or fluorescent dyes of this specification are rhodamine and / or rhodol dyes.

[0153] In some embodiments, the fluorophore (R) has the following structure: [ka] In the formula, Y is C, O, or Si, and if Y is C or Si, it is substituted with two CH3 groups, R 1 , R 2 , R 3 , R 4 , and R 5 Each of them is independently H or F (for example, R 1 -R 5 All are H; R 1 -R 5 All are F; R 1 and R 2 F is R 3 H is; R 1 -R 3 F is R 4 -R 5は H and R 1 and R 2 F is R 3 -R 5 (where is H, etc.). In some embodiments, azetidine is further substituted with one or two non-hydrogen substituents at the 3-position (e.g., CO 2 (H, CH3, F, etc.). In some embodiments, the exemplary compounds described herein have the following structures: [ka] In the formula, R 1-5 (and Y are defined as above, and Y and azetidine are optionally substituted as above, and alternative linkers and AX groups are also within range).

[0154] In some embodiments, the fluorophore (R) has the following structure: [ka] In the formula, Y is C, O, or Si; when Y is C or Si, it is substituted with two CH3 groups, and R 1 , R 2 , R 3 , R 4 , and R 5 each independently is H or F (for example, R 1 -R 5 are all H; R 1 -R 5 are all F; R 1 and R 2 are F, and R[[ID=2 \theta]] 3 is H; R 1 -R 3 is F, and R 4 -R 5は is H, and R 1 and R 2 are F, and R 3 -R 5 is H, etc.); also in the formula, each

Chemical formula

Chemical formula

Chemical formula

[0155] In some embodiments, the linker is a polyatomic linear or branched chain containing C, N, S, or O, or a group containing one or more rings (e.g., saturated or unsaturated rings, e.g., one or more aryl rings, heteroaryl rings, or any combination thereof). In some embodiments, the linker includes combinations of -O(CH2)2-, -(CH2)O-, -CH2-, -NHC(O)O-, -OC(O)NH-, NHC(O)-, and -C(O)NH-. In some embodiments, the linker length is 5 to 50 atoms (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or in between). In some embodiments, the linker length for tethering an alkyl halide to R allows for optimization of proximity and geometry (e.g., for the binding of a modified dehalogenase complex to an alkyl halide, for the function of a functional group (e.g., fluorescence), etc.). The scope of embodiments herein is not limited by the types of linkers available. The fluorophores and AX may be directly linked (e.g., the linker consists of a single covalent bond) or linked via a suitable linker. Embodiments are not limited to any particular linker group. Various linker groups are intended, and suitable linkers 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, and dendritic polymers (WO93 / 06868 and Tomalia et al. in Angew). This may include Chem.Int.Ed.Engl.29:138-175(1990), (these are incorporated herein by reference in their entirety), PEG-chelating polymers (W94 / 08629, WO94 / 09056, and WO96 / 26754 (these 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., enzymatic (e.g., TEV protease site), chemical, photoinducible, etc.). In some embodiments, the cleavable linker contains an allyl-heteroatom group or a propargyl-heteroatom group, such as, for example, the linker described in U.S. Patent Application No. 16 / 813,295, which is incorporated herein by reference in its entirety.

[0156] The haloalkyl ligands herein contain a haloalkyl group, a linker, and a functional group. Exemplary compounds containing a rhodamine dye linked to a haloalkane include the following:

Chemical Structure

Chemical Structure

Chemical Structure

[0157] Bioluminescent substrate In some embodiments, the systems herein (including the bioluminescent complex and / or its components) produce bioluminescence using an imidazopyrazine lumophore substrate. In some embodiments, the substrate is coelenterazine:

Chemical Structure

[0158] In some embodiments, the substrates are coelenterazine derivatives (e.g., flimazine, flimazine analogs (e.g., fluoroflimazine), coelenterazine-n, coelenterazine-f, coelenterazine-h, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, bis-deoxycoelenterazine ("coelenterazine-hh"), coelenterazine-i, coelenterazine-icp, coelenterazine-v, and 2-methylcoelenterazine) in addition to those disclosed in WO2003 / 040100, U.S. Patent Application No. 12 / 056,073 (paragraph

[0086] ), U.S. Patent No. 8,669,103, and U.S. Provisional Patent Application No. 63 / 379,573), and these disclosures are incorporated herein by reference in their entirety.

[0159] In some embodiments, the substrate is frimazine: [ka]

[0160] In some embodiments, the substrate is fluoroflimazine: [ka]

[0161] Additional reporter peptides In some embodiments, the bioluminescent and / or modified dehalogenase complex is a three-component or multi-component complex (e.g., comprising three or more peptide / polypeptide components). In such embodiments, the first component of the complex may be located within a tandem peptide tag, the second component may be located within a tandem polypeptide reporter, and the third (or fourth or more) component may be located as an additional reporter. In some embodiments, the interaction of the additional reporter, the tandem polypeptide reporter, and the tandem peptide tag (if facilitated or not) results in the formation of the modified dehalogenase and bioluminescent complex.

[0162] The additional reporter may exist as a fusion with one or more addit...

Claims

1. A dual reporter system, including the following: (a) a tandem peptide tag containing (i) a peptide component of a bioluminescent complex fused to (ii) a peptide component of a modified dehalogenase complex; and (b) A tandem peptide reporter comprising (i) the peptide component of the bioluminescent complex and (ii) the polypeptide component of the modified dehalogenase complex; The peptide component and the polypeptide component of the bioluminescent complex can interact to form the bioluminescent complex, and the bioluminescent complex can produce bioluminescence in the presence of a substrate for the bioluminescent complex; The dual reporter system wherein the peptide component and the polypeptide component of the modified dehalogenase complex can interact to form the modified dehalogenase complex, and the modified dehalogenase complex can form a covalent bond with a haloalkyl ligand.

2. The system according to claim 1, wherein the tandem polypeptide reporter exhibits high affinity for the tandem peptide tag.

3. The system according to claim 2, wherein the peptide component of the bioluminescent complex exhibits high affinity for the polypeptide component of the bioluminescent complex.

4. The aforementioned high affinity is less than 1 μM of K d The system according to claim 3.

5. The aforementioned high affinity is K less than 1 nM. d The system according to claim 4.

6. The system according to claim 1, wherein the peptide component of the bioluminescent complex has at least 70% identity with SEQ ID NO: 3038.

7. The system according to claim 6, wherein the peptide component of the bioluminescent complex has at least 100% identity with SEQ ID NO: 3038.

8. The system according to claim 1, wherein the peptide component of the bioluminescent complex has at least 70% identity with SEQ ID NO: 3037.

9. The system according to claim 8, wherein the peptide component of the bioluminescent complex has at least 100% identity with SEQ ID NO: 3037.

10. The system according to claim 1, wherein the peptide component of the modified dehalogenase complex has at least 70% identity with SEQ ID NO: 3034.

11. The system according to claim 10, wherein the peptide component of the modified dehalogenase complex has at least 100% identity with SEQ ID NO: 3034.

12. The system according to claim 1, wherein the polypeptide component of the modified dehalogenase complex has at least 70% identity with SEQ ID NO: 1188.

13. The system according to claim 12, wherein the polypeptide component of the modified dehalogenase complex has at least 100% identity with SEQ ID NO: 1188.

14. The system according to claim 1, wherein the tandem peptide tag has at least 70% identity with one of sequence numbers 3062-3063, 3067-3078, 3092, 3094-3109, or 4177-4181.

15. The system according to claim 14, wherein the tandem peptide tag has at least 100% identity with one of sequence numbers 3062-3063, 3067-3078, 3092, 3094-3109, or 4177-4181.

16. The system according to claim 1, wherein the tandem polypeptide reporter has at least 70% identity with one of sequence numbers 3110 to 4064.

17. The system according to claim 16, wherein the tandem polypeptide reporter has at least 100% identity with one of sequence numbers 3110 to 4064.

18. The system according to claim 1, further comprising the substrate for the bioluminescent complex.

19. The system according to claim 1, further comprising the haloalkyl ligand.

20. The system according to claim 19, wherein the haloalkyl ligand includes a haloalkane moiety linked to a fluorophore.

21. The system according to claim 20, wherein the fluorophore has an excitation spectrum that overlaps with the emission spectra of the bioluminescent complex and the substrate.

22. The system according to claim 1, wherein the tandem peptide tag is linked to a target element.

23. The system according to claim 22, wherein the tandem peptide tag and the target element are expressed in a cell as a fusion.

24. A tandem peptide tag comprising a peptide component of a bioluminescent complex fused to a peptide component of a modified dehalogenase complex; The peptide component of the bioluminescent complex can interact with the polypeptide component of the bioluminescent complex to form the bioluminescent complex, and the bioluminescent complex can produce bioluminescence in the presence of a substrate for the bioluminescent complex; The tandem peptide tag wherein the peptide component of the modified dehalogenase complex can interact with the polypeptide component of the modified dehalogenase complex to form the modified dehalogenase complex, and the modified dehalogenase complex can form a covalent bond with a haloalkyl ligand.

25. The tandem peptide tag according to claim 24, wherein the tandem peptide tag has at least 70% identity with one of sequence numbers 3062-3063, 3067-3078, 3092, 3094-3109, or 4177-4181.

26. The tandem peptide tag according to claim 24, wherein the tandem peptide tag has 100% identity with one of sequence numbers 3062-3063, 3067-3078, 3092, 3094-3109, or 4177-4181.

27. The tandem peptide tag according to claim 24, wherein the peptide component of the bioluminescent complex has at least 70% identity with SEQ ID NO: 3038.

28. The tandem peptide tag according to claim 27, wherein the peptide component of the bioluminescent complex has 100% identity with SEQ ID NO: 3038.

29. The tandem peptide tag according to claim 24, wherein the peptide component of the modified dehalogenase complex has at least 70% identity with SEQ ID NO: 3034.

30. The tandem peptide tag according to claim 29, wherein the peptide component of the modified dehalogenase complex has 100% identity with SEQ ID NO: 3034.

31. A fusion polypeptide comprising a target polypeptide linked to a tandem peptide tag according to one of claims 24 to 30.

32. The fusion polypeptide according to claim 31, wherein the tandem peptide tag is linked to the C-terminus, N-terminus, or internally of the target polypeptide.

33. A polynucleotide encoding the fusion polypeptide according to claim 30 or 31.

34. A vector comprising the polynucleotide described in claim 33.

35. A system comprising a tandem peptide tag according to one of claims 24 to 30, or a fusion polypeptide according to claim 30 or 31, and the polypeptide component of the bioluminescent complex.

36. The system according to claim 35, further comprising the substrate for the bioluminescent complex.

37. The system according to claim 35, further comprising the polypeptide component of the modified dehalogenase complex.

38. The system according to claim 37, further comprising the haloalkyl ligand.

39. The system according to claim 37, wherein the polypeptide component of the modified dehalogenase complex and the polypeptide component of the bioluminescent complex are present in the system as independent reporter polypeptides.

40. The system according to claim 37, wherein the polypeptide component of the modified dehalogenase complex and the polypeptide component of the bioluminescent complex are present in the system as tandem polypeptide reporters.

41. A system comprising the tandem peptide tag and the polypeptide component of the modified dehalogenase complex according to claim 24.

42. The system according to claim 41, further comprising the substrate for the bioluminescent complex.

43. The system according to claim 37, further comprising the haloalkyl ligand.

44. A tandem peptide tag comprising the polypeptide component of the bioluminescent complex and the polypeptide component of the modified dehalogenase complex; The polypeptide components of the bioluminescent complex can interact with other polypeptide components of the bioluminescent complex to form the bioluminescent complex, and the bioluminescent complex can produce bioluminescence in the presence of a substrate for the bioluminescent complex; The tandem peptide tag wherein the polypeptide component of the modified dehalogenase complex can interact with the peptide component of the modified dehalogenase complex to form the modified dehalogenase complex, and the modified dehalogenase complex can form a covalent bond with a haloalkyl ligand.

45. The tandem polypeptide reporter according to claim 44, wherein the tandem polypeptide reporter has at least 70% identity with one of sequence numbers 3110 to 4064.

46. The tandem polypeptide reporter according to claim 46, wherein the tandem polypeptide reporter has at least 100% identity with one of sequence numbers 3110 to 4064.

47. The tandem polypeptide reporter according to claim 44, wherein the polypeptide component of the modified dehalogenase complex has at least 70% identity with SEQ ID NO: 1188.

48. The tandem polypeptide reporter according to claim 47, wherein the polypeptide component of the modified dehalogenase complex has 100% identity with SEQ ID NO: 1188.

49. The tandem polypeptide reporter according to claim 44, wherein the polypeptide component of the bioluminescent complex has at least 70% identity with SEQ ID NO: 3037.

50. The tandem polypeptide reporter according to claim 49, wherein the polypeptide component of the bioluminescent complex has 100% identity with SEQ ID NO: 3037.

51. A system comprising the tandem peptide reporter according to claim 44, and the peptide component of the bioluminescent complex.

52. The system according to claim 51, further comprising the substrate for the bioluminescent complex.

53. The system according to claim 51, further comprising the peptide component of the modified dehalogenase complex.

54. The system according to claim 53, further comprising the haloalkyl ligand.

55. The system according to claim 53, wherein the peptide component of the modified dehalogenase complex and the peptide component of the bioluminescent complex are present in the system as independent reporter polypeptides.

56. The system according to claim 53, wherein the peptide component of the modified dehalogenase complex and the peptide component of the bioluminescent complex are present in the system as tandem polypeptide reporters.

57. A system comprising the tandem polypeptide reporter according to claim 44 and the peptide component of the modified dehalogenase complex.

58. The system according to claim 57, further comprising the substrate for the bioluminescent complex.

59. A polynucleotide encoding the tandem polypeptide reporter according to claim 44.

60. A vector comprising the polynucleotide described in claim 59.

61. A method for detecting a target component within a system, (a) Linking the target component to a tandem peptide tag containing a peptide component of a bioluminescent complex fused to a peptide component of a modified dehalogenase complex, wherein the peptide component of the bioluminescent complex interacts with the polypeptide component of the bioluminescent complex to form the bioluminescent complex, the bioluminescent complex can produce bioluminescence in the presence of a substrate for the bioluminescent complex, the peptide component of the modified dehalogenase complex interacts with the polypeptide component of the modified dehalogenase complex to form the modified dehalogenase complex, and the modified dehalogenase complex can form a covalent bond with a haloalkyl ligand; (b) Contacting the target component linked to the tandem peptide tag with the polypeptide component of the modified dehalogenase complex under conditions that the modified dehalogenase complex is formed; (c) Contacting the modified dehalogenase complex with a haloalkyl ligand containing a haloalkane linked to a fluorophore; and (d) The method comprising detecting fluorescence.

62. A method for detecting a target component within a system, (a) Linking the target component to a tandem peptide tag containing a peptide component of a bioluminescent complex fused to a peptide component of a modified dehalogenase complex, wherein the peptide component of the bioluminescent complex interacts with the polypeptide component of the bioluminescent complex to form the bioluminescent complex, the bioluminescent complex can produce bioluminescence in the presence of a substrate for the bioluminescent complex, the peptide component of the modified dehalogenase complex interacts with the polypeptide component of the modified dehalogenase complex to form the modified dehalogenase complex, and the modified dehalogenase complex can form a covalent bond with a haloalkyl ligand; (b) Contacting the component of the interest linked to the tandem peptide tag with the polypeptide component of the bioluminescent complex under conditions such that the bioluminescent complex is formed; (c) Contacting the bioluminescent complex with a substrate for the bioluminescent complex; and (d) The method comprising detecting bioluminescence.

63. A method for detecting a target component within a system, (a) Linking the target component to a tandem peptide tag containing a peptide component of a bioluminescent complex fused to a peptide component of a modified dehalogenase complex, wherein the peptide component of the bioluminescent complex interacts with the polypeptide component of the bioluminescent complex to form the bioluminescent complex, the bioluminescent complex can produce bioluminescence in the presence of a substrate for the bioluminescent complex, the peptide component of the modified dehalogenase complex interacts with the polypeptide component of the modified dehalogenase complex to form the modified dehalogenase complex, and the modified dehalogenase complex can form a covalent bond with a haloalkyl ligand; (b) Contacting the target component linked to the tandem peptide tag with a tandem polypeptide reporter containing (i) the polypeptide component of the bioluminescent complex and (ii) the polypeptide component of the modified dehalogenase complex, under conditions in which the bioluminescent complex and the modified dehalogenase complex are formed; (c) Contacting the bioluminescent complex with a substrate for a haloalkyl ligand containing a haloalkane linked to the bioluminescent complex and / or a fluorophore; and (d) The method comprising detecting bioluminescence and / or fluorescence.

64. A method for detecting a target component within a system, (a) Linking the target component to a tandem peptide tag containing a peptide component of a bioluminescent complex fused to a peptide component of a modified dehalogenase complex, wherein the peptide component of the bioluminescent complex interacts with the polypeptide component of the bioluminescent complex to form the bioluminescent complex, the bioluminescent complex can produce bioluminescence in the presence of a substrate for the bioluminescent complex, the peptide component of the modified dehalogenase complex interacts with the polypeptide component of the modified dehalogenase complex to form the modified dehalogenase complex, and the modified dehalogenase complex can form a covalent bond with a haloalkyl ligand; (b) Contacting the target component linked to the tandem peptide tag with (i) the polypeptide component of the bioluminescent complex and (ii) an independent polypeptide comprising the polypeptide component of the modified dehalogenase complex, under conditions in which the bioluminescent complex and the modified dehalogenase complex are formed; (c) Contacting the bioluminescent complex with a substrate for a haloalkyl ligand containing a haloalkane linked to the bioluminescent complex and / or a fluorophore; and (d) The method comprising detecting bioluminescence and / or fluorescence.

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