Complementarity-based tags and reporter genes for bimodal labeling

Abstract

Provided herein are compositions and systems comprising complementary-based tags and reporter genes for labeling and detection of targets by luminescence and a second modality (e.g., fluorescence), as well as methods of use thereof. In particular, the present invention provides a tag comprising a fusion of a first component of a bioluminescent complex and a first component of a modified dehalogenase complex; providing a reporter gene comprising a second component of a bioluminescent complex and a second component of a modified dehalogenase complex; also provided are systems and methods comprising the tags and reporter genes herein for bimodal labeling and detection of targets.

Description

Cross-references to related applications

[0001] This invention claims priority to U.S. Provisional Patent Application 63 / 500,118, filed May 4, 2023, which is incorporated herein by reference in its entirety.

[0002] sequence list The text of the computer-readable sequence list, titled "PRMG_41898_601_SequenceListing.xml", created on May 3, 2024 and with a file size of 4,666,369 bytes, which was submitted along with the file, is hereby incorporated in its entirety by reference. Technical Field

[0003] This document provides compositions and systems comprising complementary tags and reporter genes for the labeling and detection of targets via luminescence and a second modality (e.g., fluorescence), as well as methods of using them. Specifically, the invention provides a tag comprising a fusion of a first component of a bioluminescent complex and a first component of a modified dehalogenase complex; a reporter gene comprising a second component of a bioluminescent complex and a second component of a modified dehalogenase complex; and systems and methods comprising the tags and reporter genes described herein for bimodal labeling and detection of targets. Background Technology

[0004] Reporter gene technology based on the complementation of split protein sequences has proven its applicability in a wide range of biomedical applications. In the most common configuration, these reporter genes function by initially producing little or no signal when their complementary sequences remain separated or interact unfavorably. When conditions are altered to enable interaction (such as through chemical induction, physical proximity, changes in concentration or affinity), these sequences interact and fold into an active protein complex that produces a measurable signal, for example, in the form of luminescence or fluorescence.

[0005] The practicality of split protein complementation systems as research tools has been demonstrated in applications such as biosensing, diagnostics, drug discovery, targeted molecular degradation, cellular and molecular imaging, and energy transfer detection (BRET, FRET). By fusing split protein sequences with intracellular proteins, these research tools can target specific molecular events, offering simple yet powerful versatility in method and application design. The advent of CRISPR genome editing technology has further enhanced the value of small (<35 amino acids) split protein sequences as fusion tags, as their small size allows for more efficient integration into the genome and makes them less likely to disrupt the function of target protein fusion chaperones.

[0006] Several examples of asymmetric cleavage protein reporter genes with small sequences have been reported, including fluorescent proteins (GFP, RFP), luciferases (NanoLuc), and self-labeled proteins (HaloTag, SNAP tags). These reporter genes have been introduced as fusions with endogenous genes using CRISPR-based genome editing, and this introduction has been shown to achieve many of the aforementioned functionalities. However, despite the practicality and versatility of these cleavage protein tags, users must choose a single detection method (e.g., fluorescence or luminescence) and subsequently work to generate assays or engineered cell lines limited by the advantages and disadvantages of the chosen detection type. For example, while luminescence-based detection (e.g., using NanoLuc binary technology (NANOBIT)) offers excellent sensitivity and linearity, it has not been widely used in cell imaging or flow cytometry due to the difficulty of performing luminescence detection on typical microscopic or cytometric instruments. Other methods preferred over bioluminescence imaging include: single-molecule tracking, localization, fixation, single-cell / subcellular resolution, and co-localization experiments using multiple colors. Conversely, while small split fluorescent protein tags can be used in fluorescence microscopy or cytometry, they are generally limited by their dynamic range and the emission wavelengths that can be used for measurement.

[0007] What is needed is a single-splitting protein technology that can simultaneously use luminescence and fluorescence to detect complementarity between the tag and complementary reporter gene sequences. Such a system would allow users to measure the functional dynamics of the target protein under minimal interference conditions, while simultaneously achieving the benefits of both detection methods. Summary of the Invention

[0008] This document provides compositions and systems comprising a complementary tag and a reporter gene for the labeling and detection of targets by luminescence and a second modality (e.g., fluorescence), and methods of using them. Specifically, the invention provides a tag comprising a fusion of a first component of a bioluminescent complex and a first component of a modified dehalogenase complex; a reporter gene comprising a second component of a bioluminescent complex and a second component of a modified dehalogenase complex; and systems and methods comprising the tag and reporter gene described herein for bimodal labeling and detection of targets. In some embodiments, this document provides a dual luminescence-fluorescence detection technique based on a fusion of split NanoLuc® (LgBit and SmBiT / HiBiT) and split HaloTag® (LgHT and SmHT) sequences. Embodiments of such a configuration retain as: (1) a single small-sized tag comprising a fusion between SmHT and SmBiT / HiBiT sequences; and (2) a complementary reporter gene peptide comprising a fusion between LgHT and LgBiT sequences. The advantages offered by this implementation scheme are initially realized by introducing a small-sized fusion tag (e.g., the SmHT-HiBiT tag) into the target protein. Once tagged, the protein target is detected through a high-affinity, spontaneous interaction between the protein target and the LgHT-LgBiT reporter peptide, the detection reconstructed from a “detection complex” consisting of a reconstructed active NanoLuc® and HaloTag® complex. In a non-complementary / non-complexed state, the dipeptide tag cannot generate a signal, providing a zero-background state unattainable with full-length fluorescent proteins. The high binding affinity between LgBiT and HiBiT drives the interaction between LgHT and SmHT through induced proximity. The introduction of a luciferase substrate and a fluorescent HaloTag ligand, respectively, generates separate luminescent and fluorescent signals that can be configured to achieve a variety of functional measurements or assay types. The high-affinity complementarity with the LgHT-LgBiT detection peptide provides the advantage of selecting the most favorable detection modality in a given situation without altering the tag fused to the protein target.

[0009] The choice of detection modality exemplifies the simplicity and versatility of this technology. Another advantage is the ability to measure two signals sequentially or simultaneously within the same reaction or the same cell (enclosed in a single test tube or microplate well), as fluorescence and luminescence signals can be detected separately without interference. For example, the expression level of a tagged target protein can be sensitively quantified using the luminescence of the active cleavage NanoLuc® component of the complex, while its subcellular localization can be measured using fluorescence microscopy of the active cleavage HaloTag component of the complex. These measurements can be configured for multiplex, homogenized, live-cell assays without the need for lysis, washing, or purification steps. In these examples, LgBiT or LgHT, or a fusion of both, is generated within the cells detecting the tag or dual tag. However, the system can also be used in in vitro biochemical or cytosolic forms, where the dipeptide tag is generated as a fusion with the target gene and detected in cytosolic form by recombinant LgBiT, LgHT, or LgBiT-LgHT fusions.

[0010] The various implementations described herein include different detection complex substrates, particularly HaloTag® ligands, among which many modified chloroalkanes with dyes that fluoresce (in fluorescent or non-fluorescent modes) in the visible and near-IR spectral wavelength ranges have been described, as well as non-fluorescent ligands with multiple functionalities (e.g., mechanosensors and biosensors, chemically induced proximity, targeted degradation and post-translational modification, energy transfer, etc.). This offers a significant advantage over similar competing technologies with small-tag, split fluorescent proteins (GFP, RFP), since split FP emits only at one wavelength and does not provide the non-fluorescent labeling functionality of HaloTag. The combination of the advantages of dual luminescence and fluorescence detection using small fusion tags and a single complementary detection peptide, along with the versatility of its substrates, endows the technologies with potentially broad advantages while providing new functionalities that can only be achieved by combining them.

[0011] In some embodiments, this document provides a dual reporter gene 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 gene comprising (i) a polypeptide component of a bioluminescent complex and (ii) a polypeptide component of a modified dehalogenase complex; wherein the peptide and polypeptide components of the bioluminescent complex are capable of interacting to form a bioluminescent complex, and wherein the bioluminescent complex is capable of generating bioluminescence in the presence of a substrate of the bioluminescent complex; and wherein the peptide and polypeptide components of the modified dehalogenase complex are capable of interacting to form a modified dehalogenase complex, and wherein the modified dehalogenase complex is capable of forming a covalent bond with a haloalkyl ligand. In some embodiments, the system further comprises a substrate of the bioluminescent complex. In some embodiments, the system further comprises a haloalkyl ligand. In some embodiments, the haloalkyl ligand comprises a haloalkyl moiety attached to a fluorophore. In some embodiments, the tandem peptide tag is attached to a target element (e.g., a cellular target, protein, peptide, etc.). In some implementations, the tandem peptide tag and target element are expressed as a fusion within the cell.

[0012] In some embodiments, this document provides a tandem peptide tag comprising a peptide component of a bioluminescent complex fused with a peptide component of a modified dehalogenase complex; wherein the peptide component of the bioluminescent complex is capable of interacting with the polypeptide component of the bioluminescent complex to form a bioluminescent complex, and wherein the bioluminescent complex is capable of generating bioluminescence in the presence of a substrate of the bioluminescent complex; and wherein the peptide component of the modified dehalogenase complex is capable of interacting with the polypeptide component of the modified dehalogenase complex to form a modified dehalogenase complex, and wherein the modified dehalogenase complex is capable of forming a covalent bond with a haloalkyl ligand.

[0013] In some embodiments, this document provides a system comprising a polypeptide component of the tandem peptide tag and the bioluminescent complex described herein. In some embodiments, the system further comprises a substrate of the bioluminescent complex. In some embodiments, the system further comprises a polypeptide component of a modified dehalogenase complex. In some embodiments, the system further comprises a haloalkyl ligand. In some embodiments, the polypeptide component of the modified dehalogenase complex and the polypeptide component of the bioluminescent complex are present in the system as independent reporter peptides. In some embodiments, the polypeptide component of the modified dehalogenase complex and the polypeptide component of the bioluminescent complex are present in the system as a tandem peptide reporter gene.

[0014] In some embodiments, this document provides a system comprising a tandem peptide tag of this document and a polypeptide component of a modified dehalogenase complex. In some embodiments, the system also comprises a substrate of a bioluminescent complex.

[0015] In some embodiments, this document provides a tandem polypeptide reporter gene comprising a polypeptide component of a bioluminescent complex and a polypeptide component of a modified dehalogenase complex; wherein the polypeptide component of the bioluminescent complex is capable of interacting with the peptide component of the bioluminescent complex to form a bioluminescent complex, and wherein the bioluminescent complex is capable of generating bioluminescence in the presence of a substrate of the bioluminescent complex; and wherein the polypeptide component of the modified dehalogenase complex is capable of interacting with the peptide component of the modified dehalogenase complex to form a modified dehalogenase complex, and wherein the modified dehalogenase complex is capable of forming a covalent bond with a haloalkyl ligand.

[0016] In some embodiments, this document provides a system comprising the peptide components of the tandem peptide reporter gene and the bioluminescent complex described herein. In some embodiments, the system further comprises a substrate of the bioluminescent complex. In some embodiments, the system further comprises a peptide component of a 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 bioluminescent complex are present in the system as independent reporter peptides. In some embodiments, the peptide components of the modified dehalogenase complex and the bioluminescent complex are present in the system as a tandem peptide reporter gene.

[0017] In some embodiments, this document provides a system comprising the tandem polypeptide reporter gene of this document and a peptide component of a modified dehalogenase complex. In some embodiments, the system also comprises a substrate of a bioluminescent complex.

[0018] In some embodiments, this document provides a method for detecting a target component in a system, the method comprising: (a) attaching the target component to a tandem peptide tag, the tandem peptide tag comprising a peptide component of a bioluminescent complex fused with a peptide component of a modified dehalogenase complex, wherein the peptide component of the bioluminescent complex is capable of interacting with a polypeptide component of the bioluminescent complex to form a bioluminescent complex, wherein the bioluminescent complex is capable of generating bioluminescence in the presence of a substrate of the bioluminescent complex, wherein the peptide component of the modified dehalogenase complex is capable of interacting with a polypeptide component of the modified dehalogenase complex to form a modified dehalogenase complex, and wherein the modified dehalogenase complex is capable of forming a covalent bond with a haloalkyl ligand; (b) contacting the target component attached to the tandem peptide tag with the polypeptide component of the modified dehalogenase complex under conditions that cause the formation of the modified dehalogenase complex; (c) contacting the modified dehalogenase complex with a haloalkyl ligand comprising a haloalkane attached to a fluorophore; and (d) detecting fluorescence intensity.

[0019] In some embodiments, this document provides a method for detecting a target component in a system, the method comprising: (a) attaching the target component to a tandem peptide tag, the tandem peptide tag comprising a peptide component of a bioluminescent complex fused with a peptide component of a modified dehalogenase complex, wherein the peptide component of the bioluminescent complex is capable of interacting with a polypeptide component of the bioluminescent complex to form a bioluminescent complex, wherein the bioluminescent complex is capable of generating bioluminescence in the presence of a substrate of the bioluminescent complex, wherein the peptide component of the modified dehalogenase complex is capable of interacting with a polypeptide component of the modified dehalogenase complex to form a modified dehalogenase complex, and wherein the modified dehalogenase complex is capable of forming a covalent bond with a haloalkyl ligand; (b) contacting the target component attached to the tandem peptide tag with the polypeptide component of the bioluminescent complex under conditions that enable the formation of the bioluminescent complex; (c) contacting the bioluminescent complex with a substrate of the bioluminescent complex; and (d) detecting the bioluminescence intensity.

[0020] In some embodiments, this document provides a method for detecting a target component in a system, the method comprising: (a) linking the target component to a tandem peptide tag, the tandem peptide tag comprising a peptide component of a bioluminescent complex fused with a peptide component of a modified dehalogenase complex, wherein the peptide component of the bioluminescent complex is capable of interacting with a polypeptide component of the bioluminescent complex to form a bioluminescent complex, wherein the bioluminescent complex is capable of generating bioluminescence in the presence of a substrate of the bioluminescent complex, wherein the peptide component of the modified dehalogenase complex is capable of interacting with a polypeptide component of the modified dehalogenase complex to form a modified dehalogenase complex. (a) Modifying the dehalogenase complex, wherein the modified dehalogenase complex is capable of forming a covalent bond with a haloalkyl ligand; (b) under conditions that cause the formation of the bioluminescent complex and the modified dehalogenase complex, contacting a target component linked to a tandem peptide tag with a tandem polypeptide reporter gene, the tandem polypeptide reporter gene comprising (i) a polypeptide component of the bioluminescent complex and (ii) a polypeptide component of the modified dehalogenase complex; (c) contacting the bioluminescent complex with a substrate of the bioluminescent complex and / or a haloalkyl ligand comprising a haloalkane linked to a fluorophore; and (d) detecting bioluminescence and / or fluorescence.

[0021] In some embodiments, this document provides a method for detecting a target component in a system, the method comprising: (a) linking the target component to a tandem peptide tag, the tandem peptide tag comprising a peptide component of a bioluminescent complex fused with a peptide component of a modified dehalogenase complex, wherein the peptide component of the bioluminescent complex is capable of interacting with a polypeptide component of the bioluminescent complex to form a bioluminescent complex, wherein the bioluminescent complex is capable of generating bioluminescence in the presence of a substrate of the bioluminescent complex, wherein the peptide component of the modified dehalogenase complex is capable of interacting with a polypeptide component of the modified dehalogenase complex to form a bioluminescent complex. (a) forming a modified dehalogenase complex, wherein the modified dehalogenase complex is capable of forming a covalent bond with a haloalkyl ligand; (b) under conditions that cause the formation of the bioluminescent complex and the modified dehalogenase complex, contacting a target component linked to a tandem peptide tag with an independent polypeptide comprising (i) a polypeptide component of the bioluminescent complex and (ii) a polypeptide component of the modified dehalogenase complex; (c) contacting the bioluminescent complex with a substrate of the bioluminescent complex and / or a haloalkyl ligand comprising a haloalkane linked to a fluorophore; and (d) detecting bioluminescence and / or fluorescence. Attached Figure Description

[0022] Figure 1 A sketch description of an exemplary implementation of the dual-label / reporter gene technology described herein.

[0023] Figure 2The complement of HaloTag was split when using the SmHT-HiBiT fusion. HaloTag[23-297]-FRB expression was then used. E. coli Lysates were combined with lysates of FKBP-SmHT expressing FKBP-SmHT with specified modifications (+ / - HiBiT tags) and incubated for 2 hours with or without rapamycin. Fluorescence activity of each combination was measured after adding JF646 HaloTag ligand to the mixture and incubating for 1 hour. SmHT refers to the HaloTag[3-19] fragment.

[0024] Figure 3 The complementary components of HaloTag were split when using the SmHT-HiBiT fusion. HaloTag[22-297](Q145H+P154)-FRB were expressed. E. coli Lysates were combined with lysates of FKBP-SmHT expressing FKBP-SmHT with specified modifications (+ / - HiBiT tags) and incubated for 2 hours with or without rapamycin. Fluorescence activity of each combination was measured after adding JF646 HaloTag ligand to the mixture and incubating for 1 hour. SmHT refers to the HaloTag[3-19] fragment.

[0025] Figure 4 The complement of HaloTag was split when using the SmHT-HiBiT fusion. HaloTag[23-297]-FRB expression was then used. E. coli Lysates were combined with lysates of FKBP-SmHT expressing a specified modification (+ / - HiBiT tag) and incubated for 2 hours with or without rapamycin. Fluorescence activity of each combination was measured after adding JF646 HaloTag ligand to the mixture and incubating for 1 hour. Fold-up response was calculated by dividing fluorescence with rapamycin by fluorescence without rapamycin. SmHT refers to the HaloTag[3-19] fragment.

[0026] Figure 5 The complementary components of HaloTag were split when using the SmHT-HiBiT fusion. HaloTag[22-297](Q145H+P154)-FRB were expressed. E. coli Lysates were combined with lysates of FKBP-SmHT expressing a specified modification (+ / - HiBiT tag) and incubated for 2 hours with or without rapamycin. Fluorescence activity of each combination was measured after adding JF646 HaloTag ligand to the mixture and incubating for 1 hour. Fold-up response was calculated by subtracting the fluorescence without rapamycin from the fluorescence with rapamycin. SmHT refers to the HaloTag[3-19] fragment.

[0027] Figure 6 The luminescent activity of the SmHT-HiBiT fusion tag when complementary to LgBiT. Purified LgBiT protein and furimazine substrate were added to FKBP-SmHT expression. E. coli The lysate was modified as shown (+ / - HiBiT tag) and then incubated for 2 hours with or without rapamycin. The luminescent activity of each combination was measured after 20 minutes of incubation. SmHT refers to the HaloTag[3-19] fragment.

[0028] Figure 7 The detection of SmHT-HiBiT fusion tags in peptides was performed by promoting complementarity and LgHT-LgBiT detection. Peptides expressing HaloTag[22-297(M2F)]-FRB or HaloTag[22-297(M2F)]-LgBiT were then tested. E. coli Lysates were combined with lysates expressing FKBP-SmHT (with specified modifications, + / - HiBiT tags) and incubated for 2 hours with or without rapamycin. Fluorescence activity of each combination was measured after adding the JF646 HaloTag ligand to the mixture and incubating for 1 hour. SmHT refers to the HaloTag[3-19] fragment.

[0029] Figure 8 The detection of SmHT-HiBiT fusion tags of peptides by promoting complementarity and LgHT-LgBiT detection. Peptides expressing HaloTag[22-297(Q145H+P154R)]-FRB or HaloTag[22-297](Q145H+P154R)-LgBiT were used. the large intestine bacilli Lysates were combined with lysates of FKBP-SmHT expressing FKBP-SmHT with specified modifications (+ / - HiBiT tags) and incubated for 2 hours with or without rapamycin. Fluorescence activity of each combination was measured after adding JF646 HaloTag ligand to the mixture and incubating for 1 hour. SmHT refers to the HaloTag[3-19] fragment.

[0030] Figure 9 The SmHT-HiBiT fusion label was detected using promoted complementary luminescence and the LgHT-LgBiT detection peptide. The LgHT-FRB or LgHT-LgBiT fusion expressing the indicated peptide was then tested. E. coliThe lysates were combined with lysates expressing FKBP-SmHT (with specified modifications, + / - HiBiT tags) and incubated for 2 hours with or without rapamycin. Fluorescence activity was measured after adding the JF646 HaloTag ligand to the mixture and incubating for 1 hour, followed by dilution of the mixture 1:10, addition of furimazine, and measurement of luminescence activity. SmHT refers to the HaloTag[3-19] fragment.

[0031] Figure 10 The synthesis of peptides was detected using LgHT-LgBiT and SmHT-HiBiT peptides. Peptides expressing HaloTag[22-297(M2F)]-LgBiT or HaloTag[22-297(Q145H+P174R)]-LgBiT were analyzed. E. coli The lysates were combined with different concentrations of synthetic SmHT-HiBiT peptides and incubated 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. For each combination, the lowest fluorescence intensity was used as a baseline for subtraction from all data points to enable nonlinear curve fitting and affinity calculation. SmHT refers to the HaloTag[3-19] fragment.

[0032] Figure 11 The synthesis of peptides was detected using LgHT-LgBiT and SmHT-HiBiT peptides. Peptides expressing HaloTag[22-297(M2F)]-LgBiT or HaloTag[22-297(Q145H+P174R)]-LgBiT were analyzed. E. coli The lysates were combined with different concentrations of synthetic SmHT-HiBiT peptides and incubated for 2 hours. After adding furimazine to the mixture and incubating for 20 minutes, the luminescent activity of each combination was measured. SmHT refers to the HaloTag[3-19] fragment.

[0033] Figure 12 Different HaloTag[3-19]-HiBiT internal connector lengths as C-terminal fusion pairs of FKBP Escherichia coli bacteria The effect of luminescence activity in the lysate. Constructs with different internal linker lengths separating the HaloTag[3-19] and HiBiT sequences were subjected to... E. coli The lysate was expressed as a C-terminal FKBP fusion. The lysate expressing the FKBP fusion was then compared with those expressing LgBiT, HaloTag[22-297](M2F)-12xGly / Ser-LgBiT, or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). E. coliLysis composition. The reaction was incubated at room temperature for 30 minutes. Fluorofurimazine substrate was added to the reaction system 5 minutes before luminescence detection.

[0034] Figure 13 Different HaloTag[3-19]-HiBiT internal connector lengths E. coli The effect of fluorescence activity in the lysate. Constructs with different internal linker lengths separating the HaloTag[3-19] and HiBiT sequences were used in... E. coli The lysate was expressed as a C-terminal FKBP fusion. The lysate expressing the FKBP fusion was then compared with lysates expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). E. coli Lysis mixture. 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 another 2 hours, followed by fluorescence detection.

[0035] Figure 14 Different HaloTag[3-19]-HiBiT internal connector lengths E. coli The effect of fluorescence activity fold response in the lysate. Constructs with different internal linker lengths separating the HaloTag[3-19] and HiBiT sequences were used in... big Enterobacteriaceae The lysate was expressed as a C-terminal FKBP fusion. The lysate expressing the FKBP fusion was then compared with lysates expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). E. coli Lysis mixture. 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 an additional 2 hours, followed by fluorescence detection. Fold-over-fold response was calculated by dividing the raw fluorescence reading of each reaction containing the FKBP fusion and the peptide reporter lysate by the fluorescence reading of the peptide reporter lysate alone.

[0036] Figure 15 The different internal HaloTag[3-19]-HiBiT connector lengths of the N-terminal tag E. coli The effect of luminescence activity in the lysate. Constructs with different internal linker lengths separating the HaloTag[3-19] and HiBiT sequences were subjected to... E. coliThe lysate was expressed as an N-terminal FKBP fusion. The lysate expressing the FKBP fusion was then compared with lysates expressing LgBiT, HaloTag[22-297](M2F)-12xGly / Ser-LgBiT, or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). E. coli Lysis composition. The reaction was incubated at room temperature for 30 minutes. The reaction was diluted 10-fold in buffer, and the fluorofurimazine substrate was added to the diluted reaction 5 minutes before luminescence detection.

[0037] Figure 16 The different internal HaloTag[3-19]-HiBiT connector lengths of the N-terminal fusion with FKBP Escherichia coli bacteria The effect of fluorescence activity in the lysate. Constructs with different internal linker lengths separating the HaloTag[3-19] and HiBiT sequences were used in... E. coli The lysate was expressed as an N-terminal FKBP fusion. The lysate expressing the FKBP fusion was then compared with that expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). E. coli Lysis mixture. 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 another 2 hours, followed by fluorescence detection.

[0038] Figure 17 The different internal HaloTag[3-19]-HiBiT connector lengths of the N-terminal fusion with FKBP Escherichia coli bacteria The effect of fold-reaction on fluorescence activity in the lysate. Constructs with different internal linker lengths separating the HaloTag[3-19] and HiBiT sequences were subjected to... E. coli The lysate was expressed as an N-terminal FKBP fusion. The lysate expressing the FKBP fusion was then compared with that expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). E. coli Lysis mixture. 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 an additional 2 hours, followed by fluorescence detection. Fold-over-fold response was calculated by dividing the raw fluorescence reading of each reaction containing the FKBP fusion and the peptide reporter lysate by the fluorescence reading of the peptide reporter lysate alone.

[0039] Figure 18 Different HaloTag[3-19]-HiBiT internal connector lengths and compositions E. coli The effect of luminescent activity in the lysate. Constructs with different internal linker lengths and compositions separating the HaloTag[3-19] and HiBiT sequences were subjected to... E. coli The lysate was expressed as a C-terminal FKBP fusion. The lysate expressing the FKBP fusion was then compared with those expressing LgBiT, HaloTag[22-297](M2F)-12xGly / Ser-LgBiT, or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). E. coli Lysis composition. The reaction was incubated at room temperature for 30 minutes. Fluorofurimazine substrate was added to the reaction system 5 minutes before luminescence detection.

[0040] Figure 19 Different HaloTag[3-19]-HiBiT internal connector lengths and compositions E. coli The effect of fluorescence activity in the lysate. Constructs with different internal linker lengths separating the HaloTag[3-19] and HiBiT sequences were used in... the large intestine bacilli The lysate was expressed as a C-terminal FKBP fusion. The lysate expressing the FKBP fusion was then compared with lysates expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). E. coli Lysis mixture. 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 another 2 hours, followed by fluorescence detection.

[0041] Figure 20 The different connector lengths between FKBP and HaloTag[3-19]-HiBiT C-terminal fusions are important for... E. coli The effect of luminescence activity in the pyrolysis products. Constructs with different linker lengths separating FKBP and HaloTag[3-19]-HiBiT were subjected to... E. coli Expression in lysates. Lysates expressing the FKBP fusion were mixed with those expressing LgBiT, HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). E. coliLysis composition. The reaction was incubated at room temperature for 30 minutes. Fluorofurimazine substrate was added to the reaction system 5 minutes before luminescence detection.

[0042] Figure 21 The different connector lengths between FKBP and HaloTag[3-19]-HiBiT C-terminal fusions are important for... E. coli The effect of fluorescence activity in the lysate. Constructs with different linker lengths separating FKBP and HaloTag[3-19]-HiBiT were subjected to... E. coli Expression in lysates. Lysates expressing the FKBP fusion were mixed with those expressing LgBiT, HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). E. coli Lysis mixture. 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 another 2 hours, followed by fluorescence detection.

[0043] Figure 22 The pair of different connector lengths between the N-terminal HaloTag[3-19]-HiBiT fusion with FKBP E. coli The effect of luminescence activity in the pyrolysis products. Constructs with different linker lengths separating FKBP and HaloTag[3-19]-HiBiT were subjected to... E. coli Expression in lysates. Lysates expressing the FKBP fusion were mixed with those expressing LgBiT, HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). E. coli Lysis composition. The reaction was incubated at room temperature for 30 minutes. The reaction was diluted 10-fold in buffer, and the fluorofurimazine substrate was added to the diluted reaction 5 minutes before luminescence detection.

[0044] Figure 23 The pair of different connector lengths between the N-terminal HaloTag[3-19]-HiBiT fusion with FKBP E. coli The effect of fluorescence activity in the lysate. Constructs with different linker lengths separating FKBP and HaloTag[3-19]-HiBiT were subjected to... E. coliExpression in lysates. Lysates expressing the FKBP fusion were mixed with those expressing LgBiT, HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). E. coli Lysis mixture. 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 another 2 hours, followed by fluorescence detection.

[0045] Figure 24 . HaloTag[3-19] truncates pairs E. coli The effect of pyrolysis product luminescence activity. The HaloTag[3-19]-HiBiT construct containing the truncated HaloTag[3-19] in... E. coli The lysate was expressed as a C-terminal fusion with FKBP. The lysate expressing the FKBP fusion was then compared with lysates expressing LgBiT, HaloTag[22-297](M2F)-12xGly / Ser-LgBiT, or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). E. coli Lysis composition. The reaction was incubated at room temperature for 30 minutes. Fluorofurimazine substrate was added to the reaction system 5 minutes before luminescence detection.

[0046] Figure 25 . HaloTag[3-19] truncates pairs E. coli The effect of HaloTag[3-19]-HiBiT on the fluorescence activity of the lysate. The HaloTag[3-19]-HiBiT construct containing the truncated HaloTag[3-19] in... E. coli The lysate was expressed as a C-terminal fusion with FKBP. The lysate expressing the FKBP fusion was then compared with that expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). E. coli Lysis mixture. 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 another 2 hours, followed by fluorescence detection.

[0047] Figure 26 HiBiT mutations E. coli The effect of HaloTag[3-19]-HiBiT on the luminescent activity of the lysate. The HaloTag[3-19]-HiBiT construct containing the HiBiT mutation in... E. coli The lysate was expressed as an FKBP fusion. The lysate expressing the FKBP fusion was then compared with lysates expressing LgBiT, HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). E. coli Lysis composition. The reaction was incubated at room temperature for 30 minutes. Fluorofurimazine substrate was added to the reaction system 5 minutes before luminescence detection.

[0048] Figure 27 HiBiT mutations E. coli The effect of HaloTag[3-19]-HiBiT on the fluorescence activity of the lysate. The HaloTag[3-19]-HiBiT construct containing the HiBiT mutation in... E. coli The lysate was expressed as a C-terminal fusion with FKBP. The lysate expressing the FKBP fusion was then compared with that expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or LgBiT-12xGly / Ser-HaloTag[22-297](M2F). E. coli Lysis mixture. 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 another 2 hours, followed by fluorescence detection.

[0049] Figure 28 The effect of the HaloTag[3-19]-HiBiT variant on the luminescence enhancement of LgBiT across the synthetic peptide concentration range. A titrant containing the HaloTag[3-19]-HiBiT synthetic peptide variant (bold) with a mutation from the native sequence (EIGTGFPFDPHYVEVLG) was added to purified LgBiT-6xHis protein. The reaction was incubated at 4°C for 30 min. Furimazine substrate was added to the reaction just before luminescence measurement. In this experiment, the peptide was conjugated to biotin at the N-terminus.

[0050] Figure 29The effect of the HaloTag[3-19]-HiBiT variant on the luminescence enhancement of HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT-6xHis across the synthetic peptide concentration range. Titration of the synthetic peptide containing the HaloTag[3-19]-HiBiT variant with a mutation (bold) from the native sequence (EIGTGFPFDPHYVEVLG) was added to purified HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT-6xHis protein. The reaction was incubated at 4°C for 30 min. Furimazine substrate was added to the reaction just before luminescence measurement. In this experiment, the peptide was conjugated to biotin at the N-terminus.

[0051] Figure 30 The effect of the HaloTag[3-19]-HiBiT variant on the luminescence enhancement of HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT-6xHis across peptide concentration ranges. Titration of the HaloTag[3-19]-HiBiT synthetic peptide containing a mutation (bold) from the native sequence (EIGTGFPFDPHYVEVLG) was added to purified HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT-6xHis protein. The reaction was incubated at 4°C for 30 min. Furimazine substrate was added to the reaction just before luminescence measurement. In this experiment, the peptide was conjugated to biotin at the N-terminus.

[0052] Figure 31 The effect of the HaloTag[3-19]-HiBiT variant on the luminescence enhancement of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis across the synthetic peptide concentration range. Titration of the synthetic peptide containing a sequence mutation from the native sequence (EIGTGFPFDPHYVEVLG) or additional linker residues (both in bold) of the HaloTag[3-19]-HiBiT variant was added to purified HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis protein. The reaction was incubated at 4 °C for 30 min. Furimazine substrate was added to the reaction just before luminescence measurement.

[0053] Figure 32The effect of the HaloTag[3-19]-HiBiT variant on the luminescence enhancement of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis across the synthetic peptide concentration range. Titration of the synthetic peptide containing the HaloTag[3-19]-HiBiT variant with a mutation (bold) from the native sequence (EIGTGFPFDPHYVEVLG) was added to purified HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis protein. The reaction was incubated at 4°C for 30 min. Furimazine substrate was added to the reaction just before luminescence measurement.

[0054] Figure 33 The effect of the HaloTag[3-19]-HiBiT variant on fluorescence enhancement of HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT-6xHis across the synthetic peptide concentration range. Titration of the synthetic peptide containing the HaloTag[3-19]-HiBiT variant with a mutation (bold) from the native sequence (EIGTGFPFDPHYVEVLG) was added to purified HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT-6xHis protein. The reaction was incubated at 4 °C for 30 min. JF646 HaloTag ligand was added to each reaction, and fluorescence was measured after incubation with the ligand at room temperature for 30 min. In this experiment, the peptide was conjugated to biotin at the N-terminus.

[0055] Figure 34 The effect of the HaloTag[3-19]-HiBiT variant on fluorescence enhancement of HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT across peptide concentrations when complementary to the synthetic peptide. Titration of the synthetic peptide containing a mutation (bold) of the HaloTag[3-19]-HiBiT variant from the native sequence (EIGTGFPFDPHYVEVLG) was added to purified HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT-6xHis protein. The reaction was incubated at 4°C for 30 min. JF646 HaloTag ligand was added to each reaction, and fluorescence was measured after incubation with the ligand at room temperature for 30 min. In this experiment, the peptide was conjugated to biotin at the N-terminus.

[0056] Figure 35The effect of the HaloTag[3-19]-HiBiT variant on fluorescence enhancement of HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT within the synthetic peptide concentration range. Titration of the synthetic peptide containing the HaloTag[3-19]-HiBiT variant containing a mutation (bold) from the native sequence (EIGTGFPFDPHYVEVLG) was added to purified HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT-6xHis protein. The reaction was incubated at 4 °C for 30 min. JF646 HaloTag ligand was added to each reaction, and fluorescence was measured after incubation with the ligand at room temperature for 30 min.

[0057] Figure 36 The effect of the HaloTag[3-19]-HiBiT variant on the fluorescence of HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT-6xHis within the synthetic peptide concentration range. Titration of the synthetic peptide containing the HaloTag[3-19]-HiBiT variant containing a mutation (bold) from the native sequence (EIGTGFPFDPHYVEVLG) was added to purified HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT-6xHis protein. The reaction was incubated at 4°C for 30 min. JF646 HaloTag ligand was added to each reaction, and fluorescence was measured after incubation with the ligand at room temperature for 30 min.

[0058] Figure 37 The effect of HaloTag[3-19] variants on the fluorescence intensity of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis within the synthetic peptide concentration range. The titration of HaloTag[3-19] synthetic peptides containing mutations (bold) or deletions (indicated by dashes) from the natural sequence (SEQ ID NO: 3061:EIGTGFPFDPHYVEVLG) was added to the solution expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. E. coli In the lysate. The reaction was incubated at room temperature for 2 hours. JF646 HaloTag ligand was added to the synthetic peptide / lysate mixture and incubated at room temperature for another 1.5 hours, followed by fluorescence detection.

[0059] Figure 38The effect of HaloTag[3-19] variants on the fluorescence intensity of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT within the synthetic peptide concentration range. The titration solution containing the HaloTag[3-19] synthetic peptide with a mutation (bold) from the natural sequence (SEQ ID NO: 3061:EIGTGFPFDPHYVEVLG) was added to the solution expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. E. coli In the lysate. The reaction was incubated at room temperature for 2 hours. JF646 HaloTag ligand was added to the synthetic peptide / lysate mixture and incubated at room temperature for another 1.5 hours, followed by fluorescence detection.

[0060] Figure 39 The effect of HaloTag[3-19] variants on fluorescence enhancement of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT within the synthetic peptide concentration range. The titration solution containing the HaloTag[3-19] synthetic peptide with a mutation (bold) from the natural sequence (SEQ ID NO: 3061:EIGTGFPFDPHYVEVLG) was added to the solution expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. E. coli In the lysate. The reaction was incubated at room temperature for 2 hours. JF646 HaloTag ligand was added to the synthetic peptide / lysate mixture and incubated at room temperature for another 1.5 hours, followed by fluorescence detection.

[0061] Figure 40 The effect of HaloTag[3-19] variants on the fluorescence intensity of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT within the synthetic peptide concentration range. The titration solution containing the HaloTag[3-19] synthetic peptide with a mutation (bold) from the natural sequence (SEQ ID NO: 3061:EIGTGFPFDPHYVEVLG) was added to the solution expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. E. coli In the lysate. The reaction was incubated at room temperature for 2 hours. JF646 HaloTag ligand was added to the synthetic peptide / lysate mixture and incubated at room temperature for another 1.5 hours, followed by fluorescence detection.

[0062] Figure 41The binding of HaloTag[22-297](M2F+F148M+V177E)-12xGly / Ser-LgBiT-6xHis to the biotin-HaloTag[3-19](I2R)-4xGly / Ser-VS-HiBiT synthetic peptide was measured using biolayer interferometry in PBST at 25°C with the biotin-HaloTag[22-297](M2F+F148M+V177E)-12xGly / Ser-LgBiT-6xHis at concentrations ranging from 0.62 to 150 nM (right side of dashed line).

[0063] Figure 42 Yeast surface display of HaloTag[3-19]-4xGly / Ser-VS-HiBiT. HaloTag[3-19]-4xGly / Ser-VS-HiBiT was fused with the yeast cell mating factor Aga2 for surface display. The resulting construct was transformed and... brewing yeast Cells were expressed in [a specific culture / organization]. Cells were incubated with HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis at room temperature for 45 min. Unbound HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis was washed, and cells were incubated with JF549 HaloTag ligand at room temperature for 45 min. Unbound ligand was washed away from the cells, and cells were then incubated with an anti-HA tag antibody from mice at room temperature for 30 min. After washing away excess primary antibody, cells were incubated with secondary antibody (goat anti-mouse IgG conjugated to Alexa Fluor 488) at room temperature for 30 min. Cells were analyzed using a Sony cell sorter after washing. The bar chart represents 10,192 recorded events from singlet cell positive phylogens. Cells in the AF488+ gate represent a population of cells that are positive for HaloTag[3-19]-4xGly / Ser-VS-HiBiT on the surface (left histogram, 51.23%). Cells in the JF549+ gate represent a population of cells that express HaloTag[3-19]-4xGly / Ser-VS-HiBiT and are complementary to HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis and bind to the JF549 HaloTag ligand (right histogram, 48.88%).

[0064] Figure 43Yeast surface display of EGFP-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT. EGFP-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT was fused with the yeast cell mating factor Aga2 for surface display. The resulting construct was transformed and... brewing yeast Expression was performed in the cells. Cells were incubated with HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis at room temperature for 45 min. Unbound HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis was washed, and cells were incubated with JF549 HaloTag ligand at room temperature for 45 min. After washing, cells were analyzed using a Sony cell sorter. The bar chart represents 10,569 recorded events. Cells in EGFP+ gating represent the EGFP-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT surface-positive cell population (left histogram, 48.07%). Cells in the JF549+ gate represent the cell population complementary to HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis bound to the JF549 HaloTag ligand (right histogram, 46.41%).

[0065] Figure 44 Yeast surface display of EGFP-GGSG-HaloTag[3-19]-GGSG-NVSGWRLFKKISN. The Aga2-EGFP-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT surface display construct was modified so that the VS-HiBiT sequence was replaced by the sequence NVSGWRLFKKISN, which is complementary to LgBiT and has a lower affinity than HiBiT. The resulting construct was transformed and... brewing yeastExpression was performed in the cells. Cells were incubated with HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis at room temperature for 45 min. Unbound HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis was washed, and cells were incubated with JF549 HaloTag ligand at room temperature for 45 min. After washing, cells were analyzed using a Sony cell sorter. The bar chart represents 11,216 recorded events. Cells in EGFP+ gated cells represent the positive cell population on the surface of EGFP-GGSG-HaloTag[3-19]-NVSGWRLFKKISN (left histogram, 44.57%). Cells in the JF549+ gate represent a population of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis complementary cells that bind to the JF549 HaloTag ligand (right histogram, 44.24%).

[0066] Figure 45 Yeast surface display of EGFP-GGSG-HaloTag[3-19]-GGSG-NVTGYRLFKKISN. The Aga2-EGFP-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT surface display construct was modified so that the VS-HiBiT sequence was replaced by the sequence NVTGYRLFKKISN, which is complementary to LgBiT and has a lower affinity than HiBiT. The resulting construct was transformed and... brewing yeast Expression was performed in the cells. Cells were incubated with HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis at room temperature for 45 min. Unbound HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis was washed, and cells were incubated with JF549 HaloTag ligand at room temperature for 45 min. After washing, cells were analyzed using a Sony cell sorter. The bar chart represents 11,203 recorded events. Cells in the EGFP+ gate represent the positive cell population on the surface of EGFP-GGSG-HaloTag[3-19]-GGSG-NVTGYRLFKKISN (left histogram, 46.47%). Cells in the JF549+ gate represent the cell population complementary to HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis bound to the JF549 HaloTag ligand (right histogram, 44.84%).

[0067] Figure 46Yeast surface display of EGFP-GGSG-HaloTag[3-19]-GGSG-VTGYRLFEKIS. The Aga2-EGFP-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT surface display construct was modified so that the VS-HiBiT sequence was replaced by the sequence VTGYRLFEKIS, which is complementary to LgBiT and has a lower affinity than HiBiT. The resulting construct was transformed and... brewing yeast Expression was performed in the cells. Cells were incubated with HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis at room temperature for 45 min. Unbound HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis was washed, and cells were incubated with JF549 HaloTag ligand at room temperature for 45 min. After washing, cells were analyzed using a Sony cell sorter. The bar chart represents 11,230 recorded events. Cells in EGFP+ gated cells represent the positive cell population on the surface of EGFP-GGSG-HaloTag[3-19]-GGSG-VTGYRLFEKIS (left histogram, 49.51%). Cells in the JF549+ gate represent a population of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-6xHis complementary cells that bind to the JF549 HaloTag ligand (right histogram, 6.96%).

[0068] Figure 47 When using the gene fusion of FKBP and HaloTag[3-19] peptide E. coli Complementation of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant in the lysate. HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or variants with the M49F+D53G mutation in... E. coli FKBP was expressed in lysates and combined with lysates of FKBP fused to different tag sequences. Reactions were incubated at room temperature for 2 hours and then labeled with a 50 nMJF646 HaloTag ligand before fluorescence detection. Data points represent independent cultures of each construct combined during the experiment, and error bars represent one standard deviation from the mean.

[0069] Figure 48 Using synthetic HaloTag[3-19] peptides in E. coliThe complementary and stabilizing effects of HaloTag[22-297](M2F+D53S+D56P)-12xGly / Ser-LgBiT in the lysis fraction. HaloTag[22-297](M2F+D53S+D56P)-12xGly / Ser-LgBiT in... E. coli The lysate was expressed and combined with an excess of synthetic HaloTag[3-19] peptide. The reaction was incubated at a specified time and temperature, then labeled with 50 nM JF646 HaloTag ligand, followed by fluorescence detection.

[0070] Figure 49 HaloTag[22-297](M2F)-12xGly / Ser-LgBiT after complementation with different synthetic peptides in... Escherichia coli bacteria Stability in pyrolysis products. HaloTag[22-297](M2F)-12xGly / Ser-LgBiT in E. coli The lysate was expressed and combined with 10 μmol or more of the indicated synthetic peptide. The reaction was incubated at the specified temperature for 10 min, then labeled with 50 nM JF646 HaloTag ligand, followed by fluorescence detection.

[0071] Figure 50 After complementation with different synthetic peptides, HaloTag[22-297](M2F+D53G+V177E)-12xGly / Ser-LgBiT in... E. coli Stability in the pyrolysis product. HaloTag[22-297](M2F+D53G+V177E)-12xGly / Ser-LgBiT in E. coli The lysate was expressed and combined with 10 μmol or more of the indicated synthetic peptide. The reaction was incubated at the specified temperature for 10 min, then labeled with 50 nM JF646 HaloTag ligand, followed by fluorescence detection.

[0072] Figure 51 After complementation with different synthetic peptides, HaloTag[22-297](M2F+M49F+S89A)-12xGly / Ser-LgBiT in... E. coli Stability in the pyrolysis product. HaloTag[22-297](M2F+M49F+S89A)-12xGly / Ser-LgBiT in E. coli The lysate was expressed and combined with 10 μmol or more of the indicated synthetic peptide. The reaction was incubated at the specified temperature for 10 min, then labeled with 50 nM JF646 HaloTag ligand, followed by fluorescence detection.

[0073] Figure 52 After complementation with different synthetic peptides, HaloTag[22-297](M2F+M49F+L57I)-12xGly / Ser-LgBiT in... E. coli Stability in the pyrolysis product. HaloTag[22-297](M2F+M49F+L57I)-12xGly / Ser-LgBiT in E. coli The lysate was expressed and combined with 10 μmol or more of the indicated synthetic peptide. The reaction was incubated at the specified temperature for 10 min, then labeled with 50 nM JF646 HaloTag ligand, followed by fluorescence detection.

[0074] Figure 53 After complementing synthetic peptides E. coli Tagging kinetics of HaloTag[22-297](M2F+D53G+V177E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis variants in lysates. The labeling kinetics of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis or its variants with the D53G+V177E mutation in... E. coli The lysate was expressed and combined with 200 μmol of HaloTag[3-19] synthetic peptide. The reaction mixture was incubated at room temperature for 3 h, and then added to 10 nM TMR HaloTag ligand. The fluorescence polarization of the ligand was continuously read to monitor the labeling. The lysate was added at the 3-minute time point in the figure to initially measure the baseline fluorescence polarization of the ligand, and then the increase in binding after the addition of the lysate was measured.

[0075] Figure 54 After complementing synthetic peptides E. coli Tagging kinetics of HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis variants in lysates. The labeling kinetics of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis or its variants with the K140E mutation in... E. coli The lysate was expressed and combined with 200 μmol of HaloTag[3-19] synthetic peptide. The reaction mixture was incubated at room temperature for 3 h, and then added to 10 nM TMR HaloTag ligand. The fluorescence polarization of the ligand was continuously read to monitor the labeling. The lysate was added at the 3-minute time point in the figure to initially measure the baseline fluorescence polarization of the ligand, and then the increase in binding after the addition of the lysate was measured.

[0076] Figure 55 After complementing synthetic peptides E. coli Tagging kinetics of HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis variants in lysis products. Tagging HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis or variants with K140E or D53G+K140E in... E. coli The lysate was expressed and combined with 200 μmol of HaloTag[3-19] synthetic peptide. The reaction mixture was incubated at room temperature for 3 h, and then added to 10 nM TMR HaloTag ligand. The fluorescence polarization of the ligand was continuously read to monitor the labeling. The lysate was added at the 3-minute time point in the figure to initially measure the baseline fluorescence polarization of the ligand, and then the increase in binding after the addition of the lysate was measured.

[0077] Figure 56 After complementing synthetic peptides E. coli The endpoint labeling activity of the HaloTag[22-297](M2F+D53G)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis variant in the lysate. The HaloTag[22-297](M2F+D53G)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis variant was... E. coli The lysate was expressed and combined with 200 μmol of 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 incubation with the ligand for 60 minutes.

[0078] Figure 57 Stability and labeling kinetics of purified HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis after complementation with synthetic peptides. 10 nM of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis was combined with 125 μmol of the HaloTag[3-19] synthetic peptide and incubated at 4°C or 32°C for 10 min. After pre-incubation with the peptide at the specified temperature, 2.5 nM TMR HaloTag ligand was injected into the reaction, and fluorescence polarization was monitored over time to observe the labeling kinetics.

[0079] Figure 58Stability and labeling kinetics of purified HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis after complementation with synthetic peptides. 10 nM of HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis was combined with 125 μmol of the HaloTag[3-19] synthetic peptide and incubated at 4 °C or 32 °C for 10 min. After pre-incubation with the peptide at the specified temperature, 2.5 nM TMR HaloTag® ligand was injected into the reaction, and fluorescence polarization was monitored over time to observe labeling kinetics.

[0080] Figure 59 Stability and labeling kinetics of purified HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis after complementation with synthetic peptides. 10 nM of HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis was combined with 125 μmol of the HaloTag[3-19] synthetic peptide and incubated at 4 °C or 32 °C for 10 min. After pre-incubation with the peptide at the specified temperature, 2.5 nM TMR HaloTag® ligand was injected into the reaction, and fluorescence polarization was monitored over time to observe labeling kinetics.

[0081] Figure 60 Stability and labeling kinetics of purified HaloTag[22-297](M2F+M49F+D53S+V177E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis after complementation with synthetic peptides. 10 nM of HaloTag[22-297](M2F+D53S+V177E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis was combined with 125 μmol of the HaloTag[3-19] synthetic peptide and incubated at 4 °C or 37 °C for 10 min. After pre-incubation with the peptide at the specified temperature, 2.5 nM TMR HaloTag ligand was injected into the reaction, and fluorescence polarization was monitored over time to observe the labeling kinetics.

[0082] Figure 61Stability and labeling kinetics of purified HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis after complementation with synthetic peptides. 10 nM of HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis was combined with 125 μmol of the HaloTag[3-19] synthetic peptide and incubated at 4 °C or 32 °C for 10 min. After pre-incubation with the peptide at the specified temperature, 2.5 nM TMR HaloTag ligand was injected into the reaction, and fluorescence polarization was monitored over time to observe the labeling kinetics.

[0083] Figure 62 The fluorescence intensity of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant co-expressing FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT in live mammalian cells was measured. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant and the FKBP-fused HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag ligand, and fluorescence was detected at specified time points. This figure shows the fluorescence intensity of the JF646 HaloTag ligand over time in a live-cell assay, comparing the fluorescence activity of cells simultaneously expressing HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants with cells expressing only HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. HT-7 refers to full-length HaloTag. Untransfected cells (NTC) controls were included as a reference. Each sample was measured in triplicate. Error bars represent the standard deviation (SD) of the data mean.

[0084] Figure 63The fluorescence intensity of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant co-expressing FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT in live mammalian cells was measured. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant and the FKBP-fused HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag ligand, and fluorescence was detected at specified time points. This figure shows the fluorescence intensity of the JF646 HaloTag ligand over time in a live-cell assay, comparing the fluorescence activity of cells simultaneously expressing HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants with cells expressing only HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. HT-7 refers to full-length HaloTag. Untransfected cells (NTC) controls were included as a reference. Each sample was measured in triplicate. Error bars represent the standard deviation (SD) of the data mean.

[0085] Figure 64 The fold-over response of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants co-expressing FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT in live mammalian cells was compared. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant and the FKBP-fused HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646HaloTag ligand, and fluorescence was detected at specified time points. The fold-over response for each assay condition was calculated as the ratio of the fluorescence signal of cells simultaneously expressing the HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants to the fluorescence signal of cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant. HT-7 refers to full-length HaloTag. Untransfected cells (NTC) controls were included as a reference. Error bars represent the standard deviation (SD) of the data mean.

[0086] Figure 65 Comparison of fluorescence intensity and fold response of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant. HeLa cells were transiently transfected with a single plasmid expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant and HaloTag[3-19]-5xGly / Ser-VS-HiBiT fused with FKBP. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag ligand. Fluorescence was detected after 5 hours of incubation with JF646 ligand. Fold-over-fold response was calculated as the ratio of the fluorescence signal of cells simultaneously expressing the FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants to the fluorescence signal of cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant. In this figure, M2F represents the template HaloTag[22-297](M2F)-12xGly / Ser-LgBiT construct. All double mutants were added to the template, but only the two added mutations are shown for each construct. Untransfected cells (NTC) controls are included as a reference. The figure shows a comparison of the fold-up response of each mutant to total fluorescence, highlighting the improved performance of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant in the presence of co-expressed FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT.

[0087] Figure 66Fluorescence intensity of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant in live-cell fluorescence assays. HeLa cells were transiently transfected with a single plasmid expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant and HaloTag[3-19]-5xGly / Ser-VS-HiBiT fused with FKBP. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag ligand. Fluorescence intensity was measured after 5 hours of incubation with JF646 HaloTag ligand. This figure shows the fluorescence intensity of the JF646 HaloTag ligand in a live-cell assay, comparing the fluorescence activity of cells simultaneously expressing the HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants with that of cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant. Each sample was measured in triplicate. Error bars represent the standard deviation (SD) of the data mean.

[0088] picture 67 Fluorescence response of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant in live-cell fluorescence assays. HeLa cells were transiently transfected with single plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused with FKBP. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. Fluorescence activity was detected after 5 hours of incubation with JF646 ligand. The fold-over response for each assay condition was calculated as the ratio of the fluorescence signal of cells simultaneously expressing the HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants to the fluorescence signal of cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant, and then normalized to the fold-over response of the template HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. Error bars represent the standard deviation (SD) of the data mean.

[0089] Figure 68The luminescent activity of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant when co-expressed with FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT in live cells. HeLa cells were transfected with a single plasmid expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant and the FKBP-fused HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment. Approximately 48 hours post-transfection, cells were treated with a luminescent live-cell substrate (Furimazine). The untransfected cell (NTC) control, except for the absence of the expression plasmid, was measured in the same manner. Each sample was measured in triplicate. Error bars represent the standard deviation (SD) of the data mean.

[0090] Figure 69 The luminescent activity of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant when co-expressed with FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT in live cells was evaluated. HeLa cells were transfected with a single plasmid expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant fragment and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fused with FKBP. Approximately 48 hours post-transfection, cells were treated with a luminescent live-cell substrate (Furimazine). Each sample was measured in triplicate. Error bars represent the standard deviation (SD) of the data mean.

[0091] Figure 70 Expression of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant. HeLa cells were transiently transfected with a plasmid expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant. Approximately 48 hours post-transfection, cells were lysed by adding a final concentration of 200 nM HiBiT peptide and a lysing luminescent substrate (Furimazine), and the luminescence signal intensity was measured. To prevent signal saturation and improve signal linearity, the initial cell samples were diluted 80-fold with Opti-MEM medium. Untransfected cells (NTC) controls were included as a reference. Each sample was measured in triplicate. Error bars represent the standard deviation (SD) of the data mean.

[0092] Figure 71Comparison of fold-over response and expression of the LgHT-LgBiT HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant. To measure fold-over response, HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant fragment and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused with FKBP. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. Fluorescence was detected after 5 hours of incubation with JF646 HaloTag® ligand. Fold-over-fold response was calculated as the ratio of the fluorescence signal of cells simultaneously expressing the FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants to the fluorescence signal of cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant. In this figure, M2F represents the template HaloTag[22-297](M2F)-12xGly / Ser-LgBiT construct. All double mutants were added to the template, but only two added mutations are shown for each construct for brevity. Untransfected cells (NTC) controls are included for reference.

[0093] Figure 72 Expression of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant. HeLa cells were transiently transfected with a plasmid expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant. Approximately 48 hours post-transfection, cells were lysed by adding a final concentration of 200 nM HiBiT peptide and a lysing luminescent substrate (Furimazine), and the luminescence signal intensity was measured. To prevent signal saturation and improve signal linearity, the initial cell samples were diluted 80-fold with Opti-MEM medium. Untransfected cells (NTC) controls were included as a reference. Each sample was measured in triplicate. Error bars represent the standard deviation (SD) of the data mean.

[0094] Figure 73Expression of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant. HeLa cells were transiently transfected with a plasmid expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant fragment. Approximately 48 hours post-transfection, cells were lysed by adding a final concentration of 200 nM HiBiT peptide and a lysing luminescent substrate (Furimazine), and the luminescence signal intensity was measured. To prevent signal saturation and improve signal linearity, the initial cell samples were diluted 80-fold with Opti-MEM medium. Each sample was measured in four technical replicates. Error bars represent the standard deviation (SD) of the data mean.

[0095] Figure 74 Expression variability of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant in transfection replicate assays. HeLa cells were transiently transfected with a plasmid expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant fragment. Approximately 48 hours post-transfection, cells were lysed by adding a final concentration of 200 nM HiBiT peptide and a lysing luminescent substrate (Furimazine), and the luminescence signal intensity was measured. To prevent signal saturation and improve signal linearity, the initial cell samples were diluted 80-fold with Opti-MEM medium. Each sample was measured in triplicate. Error bars represent the standard deviation (SD) of the data mean.

[0096] Figure 75 Expression variability of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant in transfection replicate assays. HeLa cells were transiently transfected with a plasmid expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. Approximately 48 hours post-transfection, cells were lysed by adding a final concentration of 200 nM HiBiT peptide and a lysing luminescent substrate (Furimazine), and the luminescence signal intensity was measured. To prevent signal saturation and improve signal linearity, the initial cell samples were diluted 80-fold with Opti-MEM medium. Several technical replicate assays were performed for each transfection replicate assay. Error bars represent the standard deviation (SD) of the data mean. This figure shows the observed variability in the lysing luminescent plate assays. The expression levels of the template in multiple transfection replicate assays allowed for the normalization of experimental results obtained from different replicate assays to the template.

[0097] Figure 76. Density map of untransfected cells in cytometry assay, used to filter dead cells, cell debris, doublet cells, and cell aggregates in the 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 with DPBS medium for 30 minutes twice, 15 minutes each time. Subsequently, the cells were collected, and their fluorescence activity was measured using live cell cytometry. In the cytometry assay, the density map of untransfected cells was used to filter out dead cells, cell debris, doublet cells, and cell aggregates from singlet and live HeLa cell populations. A. Plotting the side-scattered area (SSC-A) of untransfected cells against the forward-scattered area (FSC-A) effectively filters out dead cells and cell debris. B. Density maps of forward scattering height (FSC-H) versus forward scattering area (FSC-A) for untransfected cells effectively filter out doublets and cell aggregates. In cytometry assays, the same gating strategy was applied to each individual sample to effectively remove dead cells, cell debris, and doublets, ensuring accurate analysis of singlet viable HeLa cell populations. In each cytometry experiment listed in this document, the total number of ungated population events was 10,000–12,000 cells. The numbers on the graphs indicate the percentage of cell populations within each depicted gate.

[0098] Figure 77 Gating strategy for non-transfected HeLa cells used for JF646-positive cell detection. Approximately 48 hours post-seeding, non-transfected HeLa cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with the ligand for 1 hour, cells were washed with DPBS medium for 30 minutes twice (15 minutes each time). Subsequently, cells were collected and their fluorescence activity was measured using viability cytometry. Non-transfected samples were used for gating, and the activity of transfected cells was measured and compared with non-transfected or non-fluorescently active cells. Histograms depict cell number on the Y-axis and their activity with the JF646 ligand on the X-axis. A consistent gating strategy based on baseline activity detected in non-transfected cells was applied to each transfected sample for accurate detection of its fluorescence activity. The initial number of events ranged from 10,000 to 12,000 cells. Specifically, for the analysis of JF646 activity, the number of events analyzed was typically between 2,000 and 4,000. Cells analyzed in JF646+ gating depend on the transfection efficiency of the mutant and the gating settings are based on JF646 activity.

[0099] Figure 78The fluorescence intensity of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant co-expressed with HaloTag[3-19]-5xGly / Ser-VS-HiBiT in JF646+ gated cells was measured by flow cytometry. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused with FKBP. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646HaloTag® ligand. After incubation with the ligand for 1 hour, cells were washed with DPBS medium for 30 minutes (twice, 15 minutes each). Cells were then collected and their fluorescence activity was measured using cytometry in a JF646+ gated array. Each sample was measured in triplicate. Error bars represent the standard deviation (SD) of the data mean.

[0100] Figure 79 The fluorescence fold response of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant co-expressing FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT was compared using flow cytometry. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant fragment and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused with FKBP. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646HaloTag® ligand. After incubation with the ligand for 1 hour, cells were washed with DPBS medium for 30 minutes twice (15 minutes each time). Subsequently, cells were collected, and their fluorescence activity was measured using cytometry in a JF646+-gated system. The fold-over response for each assay condition was calculated as the ratio of the mean fluorescence signal in the JF646+-gated region of cells simultaneously expressing the FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants to the mean fluorescence intensity in the same JF646+-gated region of cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant. Error bars represent the standard deviation (SD) of the data mean.

[0101] Figure 80Comparison of fluorescence intensity and fold response of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants as measured by flow cytometry. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused with FKBP. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with the ligand for 1 hour, cells were washed with DPBS medium for 30 minutes twice (15 minutes each time). Subsequently, cells were collected and their fluorescence activity was measured using cytometry in a JF646+-gated system. The fold-over response for each assay condition was calculated as the ratio of the mean fluorescence signal in the JF646+-gated region of cells simultaneously expressing the FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants to the mean fluorescence signal in the same JF646+-gated region of cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant. In this figure, M2F represents the template HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. All double mutants were added to the template, but only two added mutations are shown for each construct to fit the data. Untransfected cell (NTC) controls are included as a reference. The figure shows a comparison of the fold-up response of each mutant to the total fluorescence activity, highlighting the improved performance of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant when co-expressed with FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT.

[0102] Figure 81. Fluorescence intensity during co-expression of the FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants using flow cytometry. The histograms of the variants represent the higher fold response (A) and lower fold response (B) phenotypes, demonstrating how the mutations altered the performance of the template HaloTag[22-297](M2F)-12xGly / Ser-LgBiT as measured by flow cytometry. Cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant are shown in light gray, while cells co-expressing FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT are shown in dark gray. HeLa cells were transiently transfected with a single plasmid expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused with FKBP. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with the ligand for 1 hour, cells were washed with DPBS medium for 30 minutes twice (15 minutes each time). Subsequently, cells were collected and their fluorescence activity was measured using cytometry. In this figure, M2F represents the template HaloTag[22-297](M2F)-12xGly / Ser-LgBiT construct. All double mutants were added to the template, but only the two added mutations are shown for each construct to represent the data. The "Normalize to Pattern" on the Y-axis scales the event values ​​based on the most frequent values; in this graph, this value is the number of events in the JF646 negative gating. This enables standardized comparisons and highlights relative differences between different populations.

[0103] Figure 82The fluorescence intensity of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant co-expressed with FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT in JF646+ gated cells was measured by flow cytometry. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant fragment and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused with FKBP. Approximately 48 hours post-transfection, cells were labeled with 100 nMJF646 HaloTag® ligand. After incubation with the ligand for 1 hour, cells were washed with DPBS medium for 30 minutes (twice, 15 minutes each). Cells were then collected and their fluorescence activity was measured using flow cytometry in a JF646+ gated array. Each sample was measured in triplicate. Error bars represent the standard deviation (SD) of the data mean.

[0104] Figure 83 The fluorescence fold response of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT co-expressed with FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT in live mammalian cells was compared using flow cytometry. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant fragment and the FKBP-fused HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with the ligand for 1 hour, cells were washed with DPBS medium for 30 minutes twice (15 minutes each time). Cells were then collected and their fluorescence activity was measured using cytometry at JF646+ gates. The fold-over response for each assay condition was calculated as the ratio of the mean fluorescence signal in a JF646+ gate for cells simultaneously expressing the FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants to the mean fluorescence signal in the same JF646+ gate for cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant. Error bars represent the standard deviation (SD) of the data mean.

[0105] Figure 84The fluorescence intensity of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant co-expressed with FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT in JF646+ gated cells was measured by flow cytometry. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant fragment and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused with FKBP. Approximately 48 hours post-transfection, cells were labeled with 100 nMJF646 HaloTag® ligand. After incubation with the ligand for 1 hour, cells were washed with DPBS medium for 30 minutes (twice, 15 minutes each). Cells were then collected and their fluorescence activity was measured using cytometry in a JF646+ gated array. Each sample was measured twice using technical replicates. Error bars represent the standard deviation (SD) of the data mean.

[0106] Figure 85 The fluorescence fold response of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT co-expressed with FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT in live mammalian cells was compared using flow cytometry. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant fragment and the FKBP-fused HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with the ligand for 1 hour, cells were washed with DPBS medium for 30 minutes twice (15 minutes each time). Cells were then collected and their fluorescence activity was measured using cytometry at JF646+ gates. The fold-over response for each assay condition was calculated as the ratio of the mean fluorescence signal in a JF646+ gate for cells simultaneously expressing the HaloTag[3-19]-5xGly / Ser-VS-HiBiT and FKBP-HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants to the mean fluorescence signal in the same JF646+ gate for cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant. Error bars represent the standard deviation (SD) of the data mean.

[0107] Figure 86The fluorescence intensity of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant co-expressed with FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT in JF646+ gated cells was measured by flow cytometry during the extended wash period. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant fragment and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused with FKBP. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with the ligand for 1 hour, cells were washed with DPBS medium for 2 hours and 30 minutes (twice, 75 minutes each). Cells were then collected and their fluorescence activity was measured using flow cytometry in a JF646+ gated array. Each sample was measured twice using technical replicates. Error bars represent the standard deviation (SD) of the data mean.

[0108] Figure 87 The fluorescence fold response of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant co-expressed with FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT in live mammalian cells during extended wash periods was compared using flow cytometry. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant and the FKBP-fused HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with the ligand for 1 hour, cells were washed with DPBS medium for 2 hours and 30 minutes (twice, 75 minutes each). Cells were then collected and their fluorescence activity was measured using cytometry at JF646+ gates. The fold-over response for each assay condition was calculated as the ratio of the mean fluorescence signal in a JF646+ gate for cells simultaneously expressing the HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants to the mean fluorescence signal in the same JF646+ gate for cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant. Error bars represent the standard deviation (SD) of the data mean.

[0109] Figure 88The fluorescence activity of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant co-expressed with FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT in JF646+ gated cells was measured by flow cytometry. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant and the FKBP-fused HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646HaloTag® ligand. After incubation with the ligand for 1 hour, cells were washed with DPBS medium for 30 minutes (twice, 15 minutes each). Cells were then collected and their fluorescence activity was measured using cytometry at a JF646+ gate. Live-cell cytometry revealed relatively consistent fluorescence activity levels between the first and second transfection replicates of the complementary FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants, normalized to the template, HaloTag[22-297](M2F)-12xGly / Ser-LgBiT.

[0110] Figure 89 Fluorescence fold response of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant co-expressed with FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT in live mammalian cells was measured by flow cytometry. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant and the FKBP-fused HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with the ligand for 1 hour, cells were washed with DPBS medium for 1 hour (2 hours). Each time(15 minutes). Cells were then collected and their fluorescence activity was measured using cytometry at JF646+ gates. The fold-over response for each assay was calculated as the ratio of the mean fluorescence signal in the JF646+ gate of cells simultaneously expressing the FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants to the mean fluorescence signal in the same JF646+ gate of cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant. Flow cytometry revealed that the fluorescence fold-over response levels were relatively consistent between the first and second transfection repeat assays, normalized to the template, HaloTag[22-297](M2F)-12xGly / Ser-LgBiT construct.

[0111] Figure 90 The variability of fluorescence activity of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant co-expressed with FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT in JF646+-gated cells, as measured by flow cytometry in transfection repeat assays. HeLa cells were transiently transfected with separate plasmids expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and the FKBP-fused HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with the ligand for 1 hour, cells were washed with DPBS medium for 30 minutes (twice, 15 minutes each). Cells were then collected and their fluorescence activity was measured using flow cytometry in a JF646+-gated system. Template expression levels in multiple transfection replicates allowed for the normalization of experimental results from different replicates to the template.

[0112] Figure 91The fluorescence intensity and fold response of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT co-expressed with FKBP were compared using flow cytometry. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant and the FKBP-fused HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646HaloTag® ligand. After incubation with the ligand for 1 hour, cells were washed with DPBS medium for 30 minutes (twice, 15 minutes each). Cells were then collected and their fluorescence activity was measured using flow cytometry in JF646+-gated arrays. The fold-over response for each assay was calculated as the ratio of the mean fluorescence signal in the JF646+-gated array of cells simultaneously expressing the FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants to the mean fluorescence signal in the same JF646+-gated array of cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant. Four transfection replicate assays of the template HaloTag[22-297](M2F)-12xGly / Ser-LgBiT construct are depicted and 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 mutations added to each construct are shown. The figure highlights the comparison of the fold-over response of each mutant to the total fluorescence activity, emphasizing the improved performance of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant when co-expressed with FKBP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT.

[0113] Figure 92Gating of untransfected cells in flow cytometry for isolating single-positive and double-positive cells for JF646 and EGFP signals. Approximately 48 hours post-seeding, untransfected HeLa cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with the ligand for 1 hour, cells were washed with DPBS medium for 30 minutes twice (15 minutes each time). Cells were then collected, and their fluorescence activity was measured using viable cell cytometry. Gating regions were determined using density mapping of untransfected cells: JF646 negative (Q4) and JF646 positive (Q3), and negative EGFP expression (Q4) and positive EGFP expression (Q1). Therefore, 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.

[0114] Figure 93. Comparison of cells expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and co-expressing EGFP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT using flow cytometry. HeLa cells were transiently transfected with different plasmids expressing one of the following: (A) HaloTag[22-297](M2F)-12xGly / Ser-LgBiT alone, or (B) co-expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and EGFP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT on different plasmids. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with the ligand for 1 hour, the cells were washed with DPBS medium for 30 minutes (twice, 15 minutes each). The cells were then collected and their fluorescence activity was measured using flow cytometry. As depicted, when both the EGFP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag22-297-12xGly / Ser-LgBiT variants were expressed, a significant shift of cells from the Q3 quadrant to the Q2 quadrant was observed compared to HaloTag22-297-12xGly / Ser-LgBiT alone. This shift indicates the formation of a complex between the HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag22-297-12xGly / Ser-LgBiT variants, or the co-expression of the EGFP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag22-297-12xGly / Ser-LgBiT components in cells.

[0115] Figure 94 The fluorescence intensity of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant co-expressed with EGFP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT was measured by flow cytometry. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused with EGFP. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646HaloTag® ligand. After incubation with the ligand for 1 hour, cells were washed with DPBS medium for 30 minutes twice (15 minutes each time). Subsequently, cells were collected, and their fluorescence activity was measured by flow cytometry. Fluorescence intensity was determined based on the average intensity in the second (double-positive) quadrant, while the fluorescence intensity of individual samples of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant was measured based on the average fluorescence intensity in the third (JF646+) quadrant. Each sample was determined twice using technical replicates. Error bars represent the standard deviation (SD) of the data mean.

[0116] Figure 95The fluorescence fold response of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant co-expressed with EGFP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT in live mammalian cells was compared using flow cytometry with JF646 HaloTag ligand. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused with EGFP. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with the ligand for 1 hour, cells were washed with DPBS medium for 30 minutes (twice, 15 minutes each). Cells were then collected and their fluorescence activity was measured using flow cytometry. Fluorescence intensity in co-expressed samples was determined based on the average intensity in the second (double-positive) quadrant, while fluorescence activity of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT monovariate was measured based on the average fluorescence intensity in the third (JF646+) quadrant. The fold-over response 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.

[0117] Figure 96The fluorescence fold response of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant co-expressed with EGFP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT in live mammalian cells was compared using flow cytometry with JF635 HaloTag ligand. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant fragment and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused with EGFP. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF635 HaloTag® ligand. After incubation with the ligand for 1 hour, cells were washed with DPBS medium for 30 minutes (twice, 15 minutes each). Cells were then collected and their fluorescence activity was measured using flow cytometry. Fluorescence intensity in co-expressed samples was determined based on the average intensity in the second (double-positive) quadrant, while fluorescence activity of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT monovariate was measured based on the average fluorescence intensity in the third (JF646+) quadrant. The fold-over response 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 showed that the fold-over response was enhanced when using the more fluorescent ligand JF635 compared to the less fluorescent ligand JF646.

[0118] Figure 97Comparison of fluorescence intensity and fold response of selected HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants as measured by flow cytometry. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused with EGFP. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After incubation with the ligand for 1 hour, cells were washed with DPBS medium for 30 minutes twice (15 minutes each time). Subsequently, cells were collected and their fluorescence activity was measured by flow cytometry in JF646+-gated systems. The fold-over response for each assay condition was calculated as the ratio of the mean fluorescence signal in the JF646+-gated region of cells simultaneously expressing the EGFP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants to the mean fluorescence signal in the same JF646+-gated region of cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant. Two replicate assays of the template HaloTag[22-297](M2F)-12xGly / Ser-LgBiT are depicted and shown in dark gray. The figure highlights the improved fluorescence intensity of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant when co-expressed with EGFP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT.

[0119] Figure 98. Fluorescence of HeLa cells co-expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant with EGFP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT, using confocal imaging. HeLa cells were transiently transfected with separate plasmids expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fragment fused with EGFP. For comparison, cells were transfected separately with the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour of incubation, cells were imaged using confocal microscopy in the green channel (excitation wavelength 488 nm) and the far-red channel (excitation wavelength 640 nm). Laser and gain settings were optimized for each construct to minimize saturation pixels. The rows show the co-expression and single plasmid control signals of (A) HaloTag[22-297](M2F)-12xGly / Ser-LgBiT, (B) HaloTag[22-297](M2F+D53G+F148M)-12xGly / Ser-LgBiT, and (C) HaloTag[22-297](M2F+D53G+V177E)-12xGly / Ser-LgBiT.

[0120] Figure 99Quantitative fluorescence was measured in HeLa cells co-expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant with EGFP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT using confocal imaging. HeLa cells were transiently transfected with a single plasmid expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant and the HaloTag[3-19]-5xGly / Ser-VS-HiBiT fused with EGFP. Additionally, cells were transfected with a mutant of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant alone to measure background marker signal. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour of incubation, cells were imaged using a confocal microscope in the green channel (excitation wavelength 488 nm) and the far-red channel (excitation wavelength 640 nm). Laser and gain settings were optimized for each construct to minimize saturation pixels. To quantify the confocal images, multiple fields of view were acquired for each sample, and the intensity of individual cells in the co-expression signal and background signal was measured. The histogram represents the average of the data. The fold-over response of the samples shown above was calculated as the ratio of the mean specific signal of cells simultaneously expressing the EGFP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT and HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants to the mean signal intensity of cells expressing only the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant. This figure illustrates how template mutations improve the performance of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant when co-expressed with EGFP-HaloTag[3-19]-5xGly / Ser-VS-HiBiT in confocal imaging applications.

[0121] Figure 100CRISPR knock-in efficiency of endogenous PARP1 and CTNNB1 loci in the DLD-1 cell pool during the introduction of HaloTag[3-19]-VS-HiBiT variants. These variants included HaloTag[3-19]-VS-HiBiT without a adapter and HaloTag[3-19]-VS-HiBiT variants with a 4xGly / Ser adapter (GGSG-HaloTag[3-19]-GGSGVS-HiBiT). Data were collected using droplet digital polymerase chain reaction. Knock-in efficiency was calculated as the percentage of PCR amplicon copies per volume containing the tagged sequence compared to the untagged sequence at the target locus. Error bars represent the standard deviation (SD) of the data mean.

[0122] Figure 101 Expression of PARP1 and CTNNB1 in CRISPR pools was performed using either the HaloTag[3-19]-VS-HiBiT variant or HiBiT-tagged PARP1 and CTNNB1. Following cell lysis, the luminescence signal intensity of DLD-1 CRISPR cell pools (40,000 cells per pool) was measured by adding LgBiT protein and a lysis luminescent substrate (Furimazine). Six technical replicates were performed on each construct's CRISPR pool. The results confirmed the integration and functionality of the individual tags. Furthermore, it showed that adding the adapter to the HaloTag[3-19]-GGSG-VS-HiBiT variant improved detection. To ensure that luminescent activity was not affected by the knock-in efficiency of the inserted sequence in the CRISPR pools, pools showing relatively similar knock-in efficiencies were selected for this experiment. Error bars represent the standard deviation (SD) of the data mean.

[0123] Figure 102 Endogenous subexpression of PARP1 fused to the HaloTag[3-19]-VS-HiBiT variant or HiBiT in CRISPR clones. Following cell lysis, the luminescence signal intensity of DLD-1 CRISPR cell clones (40,000 cells / clone per replicate) was measured by adding LgBiT protein and a lysis luminescent substrate (Furimazine). Individual CRISPR clones of each construct were subjected to several technical replicates. The results confirmed the successful integration and functionality of the individual tags. Furthermore, it demonstrated that adding the adapter to the HaloTag[3-19]-GGSG-VS-HiBiT variant improved detection at the CRISPR clone level. Error bars represent the standard deviation (SD) of the data mean.

[0124] Figure 103. JF646-positive cell gating strategy for untransfected PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells. DLD-1 cell lines expressing PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT were labeled with 100 nM JF646 HaloTag® ligand approximately 48 hours post-seeding. After incubation with the ligand for 1 hour, the cells were washed with DPBS medium for 30 minutes (twice, 15 minutes each). Subsequently, the cells were detached, and their fluorescence intensity was measured using flow cytometry. In the cytometry assay, dead cells, cell debris, doublet cells, and cell aggregates were filtered from the singlet DLD-1 cell population using density mapping of the untransfected cells. (A) Plotting the side-scattered area (SSC-A) of untransfected cells against the forward-scattered area (FSC-A) effectively filters out dead cells and cell debris. (B) Density plotting the forward-scattered height (FSC-H) of untransfected cells against the forward-scattered area (FSC-A) effectively filters out doublets and cell aggregates. In cytometry assays, the same gating strategy was applied to each individual sample to effectively remove dead cells, cell debris, and doublets, ensuring accurate analysis of singlet viable DLD-1 cell populations. (C) The histogram represents the number of cells on the Y-axis and their intensity with the JF646 ligand on the X-axis. A consistent gating strategy determined using baseline activity detected with untransfected cells was applied to each transfected sample to accurately identify and quantify its fluorescence activity. Prior to this step, the gating strategy was consistent with previous cytometry experiments but excluded dead cells, cell debris, and cell aggregates. In each cytometry experiment listed in this document, the total number of events in the ungated population is 10,000–12,000 cells. The numbers on the graph indicate the percentage of cell populations within each depicted gate. Specifically, for the analysis of JF646 activity, the number of events analyzed is typically between 2,000 and 4,000. The number of cells analyzed in JF646+ gating depends on the transfection efficiency of the mutant and is based on the gating setting for JF646 activity.

[0125] Figure 104Comparison of fluorescence intensity of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT transiently expressed with 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 CMV or TK promoter control. Approximately 48 hours after transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour of incubation, cells were washed with DPBS medium for 30 minutes (twice, 15 minutes each time). Cells were then detached and their fluorescence activity was measured using flow cytometry. The gating strategy used was consistent with previous cytometry experiments, but dead cells, cell debris, and cell aggregates were excluded. Additionally, labeled, untransfected CRISPR cells were used to distinguish between JF646-positive and JF646-negative populations. The values ​​on the x-axis 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 represent the standard deviation (SD) of the data mean. This figure shows that, at all plasmid DNA concentrations, the fluorescence signal intensity in the JF646-positive gating was approximately 10-fold higher when the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant was expressed under the CMV promoter than when expressed under the TK promoter.

[0126] Figure 105Comparison of fold-over response and fluorescence intensity of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT transiently expressed with 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 CMV or TK promoter control. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour of incubation, cells were washed twice with DPBS medium for 30 minutes each time (15 minutes each time). Cells were then detached and their fluorescence activity was measured using flow cytometry. The fold-over response for each assay condition was calculated as the ratio of the mean fluorescence signal in the JF646+-gated region of 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 in the same JF646+-gated region of the parental DLD-1 cells transiently expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. Values ​​on the x-axis correspond to the amount of plasmid DNA expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT used for transient transfection of cells in each well of a 96-well plate. Error bars represent the standard deviation (SD) of the data mean. These results indicate that adjusting the concentration of plasmid DNA expressing HaloTag22-297-12xGly / Ser-LgBiT can improve the fold-up response, especially with promoters expressing lower amounts of HaloTag22-297-12xGly / Ser-LgBiT (such as the TK promoter).

[0127] Figure 106. Comparison of the effect of promoters tagged with HaloTag ligands on the percentage of cells in DLD-1 cells with endogenously tagged PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT and parental DLD-1 cells transiently expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. (A) and (B) Transient transfection of parental DLD-1 cells and DLD-1 cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT with different concentrations of plasmids expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT under the control of CMV (A) and TK (B) promoters. (C) DLD-1 cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT were transiently transfected with different concentrations of plasmids expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT under CMV or TK promoter control. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour of incubation, cells were washed with DPBS medium for 30 minutes twice (15 minutes each time). Subsequently, cells were detached, and their fluorescence activity was measured using flow cytometry. The values ​​on the x-axis 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 data mean. The figure shows that the percentage of DLD-1 cells overexpressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant under the CMV promoter in JF646+ gated cells is generally higher than the corresponding percentage of CRISPR cells overexpressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant under the TK promoter.

[0128] Figure 107The fold difference in the percentage of DLD-1 cells transiently expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT under different promoters was investigated. 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 CMV or TK promoter control. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour of incubation, cells were washed twice with DPBS medium for 15 minutes each time (30 minutes each time). Cells were then detached and their fluorescence activity was measured using flow cytometry. The fold-over response for each assay condition was calculated as the ratio of the parental frequency in the JF646+-gated region of PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells co-expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT to the parental frequency in the same JF646+-gated region of the transiently expressed HaloTag[22-297](M2F)-12xGly / Ser-LgBiT parental DLD-1 cells. Values ​​on the x-axis 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 data mean. This figure shows that although the total fluorescence intensity of the TK promoter was approximately one log lower than that of the CMV promoter, resulting in a lower percentage of cells in the JF646+ gating system (lower frequency of parental values), the TK promoter resulted in a higher fold change between tagged CRISPR cells and parental cells in the JF646+ gating system compared to the CMV promoter. When expressing with the TK promoter, the fold change response was maximized at a concentration of approximately 10–40 ng / well of HaloTag[22–297](M2F)-12xGly / Ser-LgBiT plasmid DNA.

[0129] Figure 108. Comparison of fluorescence intensity between HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT during flow cytometry. (A) Stacked histogram of PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells transiently expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT (light gray) or its variant HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT (dark gray). The "Normalize to Pattern" on the Y-axis scales event values ​​based on the most frequently occurring values; in this figure, this value represents the number of events in the JF646 negative gating. This allows for standardized comparisons and highlights relative differences between different populations. (B) Mean fluorescence intensity of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or its variant containing the K140E mutation in JF646 positive gating in PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT cells. Cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT were transiently transfected into 96-well plates at 20 ng / well with HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or its variant plasmid. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour of incubation, cells were washed with DPBS medium for 30 minutes twice (15 minutes each time). Subsequently, cells were detached and their fluorescence activity was measured using flow cytometry. Each construct was tested in duplicate. The gating strategy used to exclude dead cells, cell debris, and cell aggregates was consistent with previous cytometry experiments. The figure shows that the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants exhibited higher mean fluorescence intensity within the applied JF646 activity gating. Error bars represent the standard deviation (SD) of the data mean.

[0130] Figure 109. Comparison of fluorescence fold response in JF646-gated cells between HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT variants using flow cytometry. (A) Stacked histogram of PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells transiently expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT (left) or its variant HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT (right). For each variant histogram, the light gray histogram represents CRIPSR cells transiently expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant, and the dark gray histogram represents parental DLD-1 cells transiently expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant. The “Normalize to Pattern” on the Y-axis scales the event values ​​based on the most frequently occurring values, which in this figure is the number of events in the JF646 negative gating. This allows for standardized comparisons and highlights relative differences between different populations. (B) Fold-over-fold response of the HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT variant in the JF646 positive gating. Cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT were transiently transfected into the wells of 96-well plates at 20 ng / well using HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or a variant plasmid. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour of incubation, cells were washed twice with DPBS medium for 30 minutes each time (15 minutes each time). Subsequently, cells were detached and their fluorescence activity was measured using flow cytometry. The fold-over response for each assay condition was calculated as the ratio of the mean fluorescence signal in the JF646+-gated region of 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 in the same JF646+-gated region of the parental DLD-1 cells expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. The gating strategy used to exclude dead cells, cell debris, and cell aggregates was consistent with previous cytometry experiments.The error bars represent the standard deviation (SD) of the data mean.

[0131] Figure 110. Effect of K140E mutation in DLD-1 cells transiently expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT on JF646 positive cells. DLD-1 cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT were transiently transfected with plasmids expressing (A) 20 ng / well or (B) 40 ng / well HaloTag[22-297](M2F)-12xGly / Ser-LgBiT or HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT. Approximately 48 hours post-transfection, cells were labeled with 100 nMJF646 HaloTag® ligand. After 1 hour of incubation, cells were washed with DPBS medium for 30 minutes twice (15 minutes each time). Subsequently, cells were detached and their fluorescence activity was measured using flow cytometry. The fold-over response for each assay condition was calculated as the ratio of the parental frequency in JF646+-gated PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants to the parental frequency in DLD-1 parental cells expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants in the same JF646+-gated configuration. The gating strategy used to exclude dead cells, cell debris, and cell aggregates was consistent with previous cytometry experiments. Each construct was tested twice with technical replicates. The error bars represent the standard deviation (SD) of the data mean. It also shows that the parental frequency and the fold-over response of the parental frequency change with different amounts of plasmid DNA used to express HaloTag[22-297](M2F)-12xGly / Ser-LgBiT in transfected cells.

[0132] Figure 111Comparison of fluorescence intensity of transiently expressed HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mRNA in parental 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 post-transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour of incubation, cells were washed twice with DPBS medium for 15 minutes each time, for a total of 30 minutes. Subsequently, cells were detached, and their fluorescence activity was quantified using flow cytometry. The gating strategy used to exclude dead cells, cell debris, and cell aggregates remained consistent with previous cytometry experiments. For each sample, the JF646 positive gating setting was optimized to maximize the differentiation between tagged and untagged cells, varying with mRNA concentration to compare optimal performance at each mRNA concentration. Values ​​on the x-axis correspond to the mRNA concentration used for transfection in each well of the 96-well plate. Each construct was performed with two technical replicates. Error bars represent the standard deviation (SD) of the data mean. In the JF646 positive gating, the fluorescence intensity value for the no-mRNA control was zero.

[0133] Figure 112In the parental PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cell line, the fold-over response of transiently expressed HaloTag[22-297](M2F)-12xGly / Ser-LgBiT was compared. 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 post-transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour of incubation, cells were washed twice with DPBS medium for 15 minutes each time, for a total of 30 minutes. Subsequently, cells were detached, and their fluorescence activity was quantified using flow cytometry. The gating strategy used to exclude dead cells, cell debris, and cell aggregates remained consistent with previous cytometry experiments. However, the gating settings for distinguishing JF646-positive and negative populations varied depending on the mRNA concentration. Optimized settings were used to achieve maximum separation between tagged and parental cells. The fold-over response for each assay condition was calculated as the ratio of the mean fluorescence signal in the JF646+ gating of PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells co-expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT to the mean fluorescence intensity in the same JF646+ gating of DLD-1 parental cells expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. The values ​​on the x-axis correspond to the mRNA concentration used for transfection in each well of the 96-well plate. This figure shows that the fold-up response is higher at lower concentrations of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant mRNA. The error bars represent the standard deviation (SD) of the data mean. This observation suggests that at higher concentrations of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant mRNA, the abundance of the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variant exceeds that of the endogenously expressed PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT, leading to the accumulation of nonspecific signals and a decreased fold-up response.

[0134] Figure 113. Comparison of fluorescence intensity of transiently expressed HaloTag[22-297](M2F)-12xGly / Ser-LgBiT 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 (left) using transient transfection with different concentrations of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT mRNA. (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, 96-well plate). As a control, parental DLD-1 cells (dark gray) were transfected using the same mRNA. Approximately 24 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour of incubation, cells were washed twice with DPBS medium for 15 minutes each time, for a total of 30 minutes. Subsequently, cells were detached, and their fluorescence intensity was measured using flow cytometry. The gating strategy used to exclude dead cells, cell debris, and cell aggregates remained consistent with previous cytometry experiments. JF646-positive gating was observed for samples transfected using 5 ng / well mRNA. The "Normalize to Pattern" on the Y-axis scales the event values ​​based on the most frequent values; in this graph, this value is the number of events in the JF646 negative gating. This enables standardized comparisons and highlights relative differences between different populations.

[0135] Figure 114The fold difference in the percentage of cells labeled with HaloTag ligand when transiently expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT in DLD-1 cells using flow cytometry. DLD-1 cells expressing endogenous PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT were transfected with a specified amount of mRNA encoding HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. Approximately 24 hours post-transfection, cells were labeled with 100 nM HaloTag® ligand, JF646, and TMR. After 1 hour of incubation, cells were washed twice with DPBS medium for 15 minutes each time, for a total of 30 minutes. Cells were then detached, and their fluorescence activity was quantified using flow cytometry. Parental frequencies were measured under each assay condition using the exact same gating settings as those used for fluorescence intensity measurements. The fold-over response for each assay condition was calculated as the ratio of the parental frequency in a JF646+-gated control of PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT-DLD-1 cells co-expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT to the same JF646+-gated control of parental DLD-1 cells expressing HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. Values ​​on the x-axis correspond to the mRNA concentration used for transfection in each well of a 96-well plate. Error bars represent the standard deviation (SD) of the data mean.

[0136] Figure 115Comparison of fluorescence intensity of transiently expressed HaloTag[22-297](M2F)-12xGly / Ser-LgBiT 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 5 ng / well of plasmid DNA (dark gray) or mRNA (light gray) encoding HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. Approximately 24 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour of incubation, cells were washed twice with DPBS medium for 15 minutes each time, for a total of 30 minutes. Cells were then detached and their fluorescence activity was measured using flow cytometry. The gating strategy used to exclude dead cells, cell debris, and cell aggregates remained consistent with previous cytometry experiments. The figure shows the effect of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT expression on the fluorescence intensity of the entire cell population. When expressed via mRNA, expression variability was lower, resulting in a more pronounced peak in JF646-positive cells. In contrast, a broad peak was observed when comparing the same amount of plasmid DNA expression, indicating a wider expression range for HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. The “Normalize to Pattern” on the Y-axis scales event values ​​based on the most frequently occurring values, which in this figure represents the number of events in the JF646 negative gating. This allows for standardized comparisons and highlights relative differences between different populations.

[0137] Figure 116Confocal 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 (top panel) or parental DLD-1 cells (bottom panel) were transfected with plasmid DNA encoding HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. Approximately 48 hours post-transfection, cells were labeled with 100 nMJF646 HaloTag® ligand. After 1 hour of incubation, cells were imaged using confocal microscopy in the blue channel (excitation wavelength 405 nm), the far-red channel (excitation wavelength 640 nm), and DIC (differential interference contrast). Laser and gain settings were optimized to minimize saturation pixels in each channel. PARP1 protein is natively localized in the nucleus, and images of PARP1-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells showed far-red signal localization in the nucleus, while DAPI and JF646 were also clearly co-localized. These images provide evidence that tagging the endogenous target PARP1 with GGSG-HaloTag[3-19]-GGSG-VS-HiBiT does not affect the localization of the tagged protein in the nucleus.

[0138] Figure 117Quantification of confocal imaging in 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 post-transfection, cells were labeled with 100 nM JF646HaloTag® ligand. Confocal microscopy was performed using the blue channel (excitation wavelength 405 nm), the far-red channel (excitation wavelength 640 nm), and DIC (differential interference phase contrast). Laser and gain settings were carefully optimized to minimize saturated pixels, thus ensuring optimal image quality. These settings were consistently applied to collect specific and background signals. For quantification, single fields of view (FOV) of PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells were analyzed to measure the nuclear localization fluorescence intensity of PARP1, while individual FOVs of parental cells were assessed to quantify whole-cell background fluorescence intensity. The intensity of individual cells was analyzed using this strategy, generating data points in a graph. The bar graph represents the mean of the data. This graph illustrates the heterogeneity of plasmid DNA expression and highlights outliers due to higher expression that may affect the mean of the data. Imaging analysis was correlated with cytometry-based data analysis because the fold-down response of fluorescence in CRISPR cells did not differ significantly from the fluorescence intensity of parental cells. However, differentiating factors were the number of fluorescent cells limited within the gating range during cytometry or the threshold setting in imaging.

[0139] Figure 118Confocal 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 (top row) or parental DLD-1 cells (bottom row) were transfected with 5 ng / well mRNA encoding HaloTag[22-297](M2F)-12xGly / Ser-LgBiT. Approximately 24 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour of incubation, cells were imaged using confocal microscopy in the blue channel (excitation wavelength 405 nm), the far-red channel (excitation wavelength 640 nm), and DIC (differential interference phase contrast). Laser and gain settings were optimized to minimize saturation pixels in each channel. PARP1 protein is natively localized within the cell nucleus. Images of PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells show the localization of the far-red signal in the nucleus, as well as significant co-localization of DAPI and JF646. The background signal of non-complementary HaloTag[22-297](M2F)-12xGly / Ser-LgBiT labeling showed whole-cell localization, as excessive HaloTag[22-297](M2F)-12xGly / Ser-LgBiT expression also occurred in the cytoplasm. A comparison of confocal images depicting HaloTag[22-297](M2F)-12xGly / Ser-LgBiT expression using plasmid DNA versus mRNA showed that mRNA expression was significantly more homogeneous. 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.

[0140] Figure 119Quantification of confocal imaging in 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 post-transfection, cells were labeled with 100 nM JF646HaloTag® ligand and incubated for 1 hour. Confocal microscopy was performed using the blue channel (excitation wavelength 405 nm), the far-red channel (excitation wavelength 640 nm), and DIC (differential interference phase contrast). Laser and gain settings were carefully optimized to minimize saturated pixels, thus ensuring optimal image quality. These settings were consistently applied to collect specific signals and background signals. For quantification, single fields of view (FOV) of PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells were analyzed to measure the nuclear localization fluorescence intensity of PARP1, while individual FOVs of parental cells were assessed to quantify whole-cell background fluorescence intensity. The intensity of individual cells was analyzed using this strategy, generating data points in the graph. The bar chart represents the mean of the data. The graph shows high homogeneity of mRNA expression with only a few outliers. Therefore, the mean (1215.003) and median (1192.750) values ​​of the data are similar.

[0141] Figure 120. Dissociation kinetics of purified HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis or LgBiT after complementation with synthetic peptides. (A) The purified HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis (reporter gene) or (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 min. After pre-incubation with the peptide, the samples were diluted to 1 pM in a separate buffer or a buffer containing 100 nM of the tag peptide. At each time point, samples were transferred to a luminescent substate (furimazine) to monitor the dissociation of the complex. All measurements were performed in triplicate, and the error bar represents one standard deviation from the mean.

[0142] Figure 121. Bioluminescent resonance energy transfer (BRET) enabled the measurement of the occupancy of purified HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis ligands over a wide concentration range. Purified HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis at 200 nM was combined with 2 µM HaloTag[3-19](I2R)-4xGly / Ser-VS-HiBiT or VS-HiBiT synthetic peptides. After pre-incubation with the peptides, the complexes were serially diluted semi-logarithmically to buffers to concentrations between 0.1 pM and 10 nM, followed by separate buffer additions. These dilutions were added 1:1 to the furimazine substrate with or without a final concentration of 100 nM HaloTag® NanoBRET® 618 ligand. After incubation for approximately 1 min, luminescence was measured as follows: (A) luminescent donor signal measured using a 450 nm bandpass filter and (B) luminescent acceptor signal measured using a 600 nm longpass filter. The BRET ratio (C) was determined by dividing the acceptor signal by the donor signal for each sample. All samples were performed in triplicate, and the error bar represents one standard deviation from the mean.

[0143] Figure 122. Bioluminescent resonance energy transfer (BRET) enables the measurement of purified HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis stabilization via synthetic peptides. Purified HaloTag[22-297](M2F)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis (reporter gene) was combined with 2 µM HaloTag[3-19](I2R)-4xGly / Ser-VS-HiBiT (tag) or VS-HiBiT synthetic peptides. After pre-incubation with the peptides at 4°C, the solution was diluted 100-fold to buffer and incubated at room temperature. At multiple time points, samples were added 1:1 to buffers with or without 10 nM of the final HaloTag® NanoBRET® 618 ligand. For the reporter gene-only condition, samples were added 1:1 to a solution containing 10 nM of the final VS-HiBiT peptide, with or without 618 ligand for complementation with LgBiT, thereby creating a BRET donor. After the final time point, furimazine substrate was added, and the following measurements were taken: (A) the luminescent donor signal was measured using a 450 nm bandpass filter, and (B) the luminescent acceptor signal was measured using a 600 nm longpass filter. The BRET ratio was determined by dividing the acceptor signal by the donor signal for each sample.

[0144] Figure 123. Stability of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT activity in live cells in the presence of endogenously tagged PARP1 with HaloTag[3-19]-4xGly / Ser-HiBiT. Different mRNAs encoding (A) HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and EGFP or (B) HaloTag-LgBiT and EGFP were co-transfected into parental DLD-1 (light gray) or PARP1-HaloTag[3-19]-4xGly / Ser-VS-HiBiT DLD-1 (dark gray) cells. After 48 hours of expression, cells were labeled with 100 nM JF646 HaloTag® ligand for 60 minutes and then analyzed by flow cytometry. In the presence of PARP1-HaloTag[3-19]-4xGly / Ser-VS-HiBiT, stabilization of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT resulted in increased HaloTag® ligand markers in JF646 cells expressing these two fragments relative to the parental DLD-1 cells. Since the expression level and activity of HaloTag-LgBiT were unaffected by the presence or absence of DualTag, its HaloTag® activity showed an excellent proportional relationship with co-transfected EGFP, with very similar distributions in both cell lines. Therefore, EGFP signaling effectively indicates the expression level of the co-transfected reporter gene in each cell.

[0145] Figure 124The stability of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT activity in cells was detected using a luminescent assay in the presence of PARP1 endogenously tagged with HaloTag[3-19]-4xGly / Ser-HiBiT. The mRNA encoding HaloTag[22-297](M2F)-12xGly / Ser-LgBiT was transfected into parental DLD-1 cells or PARP1-HaloTag[3-19]-4xGly / Ser-VS-HiBiT DLD-1 cells. Twenty-four hours after expression, cells were treated with 50 µg / ml cycloheximide (0-hour time point), and samples were lysed and assayed repeatedly at subsequent time points. To measure changes in total reporter gene levels, lysis reagents containing excess HiBiT peptide and furimazine substrate were added at different time points. The proportion of luminescent signals in samples treated with + / - cyclohexamethyleneimide was calculated for each condition and then normalized to the initial proportion at 0 hours post-cyclohexamethyleneimide treatment. The half-life was calculated using a single-phase exponential decay model. Five replicate measurements were performed for each condition, and the error bar represents one standard deviation from the mean.

[0146] Figure 125 Gating settings to differentiate JF646-positive and negative cell populations. Histograms show the relationship between cell number (Y-axis) and their intensity (X-axis) of the JF646 ligand. A consistent gating strategy developed using baseline activity from untransfected cells was applied to all transfected samples to accurately identify and quantify fluorescent activity. This strategy is consistent with previous cytometry experiments but effectively excludes dead cells, cell debris, and aggregates.

[0147] Figure 126A-BComparison of fluorescence intensity at 24 and 48 hours post-transfection between parental and PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cell lines using transiently expressed HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT variants. 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 variants. At approximately 24 hours post-transfection (A) and 48 hours post-transfection (B), cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour of incubation, cells were washed twice with DPBS medium for 15 minutes each time, for a total of 30 minutes. Subsequently, cells were detached, and their fluorescence activity was quantified using flow cytometry. All experiments used a consistent gating strategy to exclude dead cells, debris, and aggregates, and the same gating settings were used to distinguish between JF646-positive and negative cohorts. The values ​​on the x-axis correspond to the concentration of mRNA used for transfection in each well of a 96-well plate. Each construct was performed in duplicate. Error bars represent the standard deviation (SD) of the data mean. The absence of a bar graph under any assay condition indicates no detectable signal in the JF646-positive gating 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. In the JF646-positive gating, the fluorescence intensity of the mRNA-free control was zero. These graphs illustrate that the fluorescence intensity at 48 hours post-transfection was approximately halved compared to 24 hours.

[0148] Figure 127A-B24 and 48 hours post-transfection, the fold-over response of transiently expressed HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+d53g+K140E)-12xGly / Ser-LgBiT variants was compared in parental and PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cell lines. 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 variants. At approximately 24 hours post-transfection (A) and 48 hours post-transfection (B), cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour of incubation, cells were washed twice with DPBS medium for 15 minutes each time, for a total of 30 minutes. Subsequently, cells were detached, and their fluorescence activity was quantified using flow cytometry. All experiments used a consistent gating strategy to exclude dead cells, debris, and aggregates, and employed the same gating settings to distinguish between JF646 positive and negative groups. The fold response for each assay condition was calculated as the ratio of the mean fluorescence signal in the JF646+ gate of PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells expressing both HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT variants to the mean fluorescence intensity in the same JF646+ gate of DLD-1 parental cells expressing both HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT variants. The values ​​on the x-axis correspond to the concentration of mRNA used for transfection in each well of the 96-well plate. The error bars represent the standard deviation (SD) of the data mean. The absence of a bar graph under any assay condition indicates no 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 indicate that there was no significant difference in fold-up response between assays run at 24 and 48 hours post-transfection. Furthermore, it also suggests a higher fold-up response at lower mRNA concentrations.

[0149] Figure 128A-BComparison of the percentage of cells labeled with HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT variants when transiently expressing mRNA in parental and PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cell lines at 24 and 48 hours post-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 variants. At approximately 24 hours post-transfection (A) and 48 hours post-transfection (B), cells were labeled with 100 nM JF646 HaloTag® ligand. After 1 hour of incubation, cells were washed twice with DPBS medium for 15 minutes each time, for a total of 30 minutes. Subsequently, cells were detached, and their fluorescence activity was quantified using flow cytometry. All experiments used a consistent gating strategy to exclude dead cells, debris, and aggregates, and the same gating settings were used to distinguish JF646 positive and negative populations. Parental frequencies were measured under each assay condition using the exact same gating settings as those used for fluorescence intensity measurements. The values ​​on the x-axis correspond to the concentration of mRNA used for transfection in each well of a 96-well plate. Each construct was performed in two technical replicates. Error bars represent the standard deviation (SD) of the data mean. The absence of a bar graph under any assay condition indicates that no detectable labeled cells were found in the JF646-positive gating 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. In the JF646-positive gating, the fluorescence intensity value for the mRNA-free control was zero. These figures show that, although fluorescence intensity decreased by approximately 50% at 48 hours post-transfection compared to 24 hours, the percentage of labeled PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells expressing the HaloTag® variant was significantly increased compared to labeled parental DLD-1 cells expressing the same variant at the JF646 positive phylum. Labeled parental cells were not detected at a mRNA concentration of 0.2 ng / well, and less than 1% were detected at 2 ng / well.In contrast, under these conditions, PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells were labeled on average by 15% and 40%, respectively. Therefore, for fluorescence sorting applications, it is recommended to perform assays 48 hours post-transfection. These results indicate that the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT variants performed similarly in cytometry assays. The latter variant showed a slight improvement in fluorescence and percentage of labeled cell fold response.

[0150] Figure 129A-BComparison of fluorescence intensities of HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants in DLD-1 cell lines as determined by time-delayed ligand incubation cytometry. Figure A depicts 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 variants expressed in the parental and PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cell lines. These variants were transiently expressed using mRNA in both the parental cell line and the PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cell line. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag® ligand and incubated for different times (0.5, 2, 8, and 24 hours), followed by isolation and quantitative fluorescence measurement by flow cytometry. A consistent gating strategy was used for all experiments to exclude dead cells, debris, and aggregates, and the same gating settings were used to distinguish between JF646-positive and negative cohorts. Figure B shows the fold-up fluorescence response of each variant at these time points. Fold-up responses were calculated by comparing the mean fluorescence intensity in the JF646+ gating of the PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cell line with the mean fluorescence intensity in the parental cell control at the same gating. Each construct was subjected to two technical replicates. Error bars represent the standard deviation (SD) of the data mean. In the JF646 positive gate, the fluorescence intensity value for the no-mRNA control was zero. These figures illustrate that the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT variants exhibited similar fluorescence intensities, slightly higher than the HaloTag[22-297](M2F+K140E)-12xGly / Ser-LgBiT variant. The fluorescence intensity peaked approximately two hours after ligand addition. Although the maximum fluorescence fold response was reached about two hours after ligand incubation, the fold response of the HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT variant reached its peak about 8 hours after ligand addition.

[0151] Figure 130A-BComparison of the percentage of cells labeled with the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants in the DLD-1 cell line as determined by time-delayed ligand incubation cytometry. Figure A shows the percentage of cells labeled with the parental and PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT variants expressed in the DLD-1 cell line, namely 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. These variants were transiently expressed using mRNA in both the parental line and the PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cell line. Approximately 48 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag® ligand and incubated for different times (0.5, 2, 8, and 24 hours), followed by separation and quantitative analysis by flow cytometry. A consistent gating strategy was used in all experiments to exclude dead cells, debris, and aggregates, and the same gating settings were used to distinguish between JF646-positive and negative cohorts. Figure B shows the fold-over response of the percentage of labeled cells (parental frequency) for each variant at these time points. The fold-over response for each assay condition was calculated as the ratio of the parental frequency in JF646+-gated PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants to the parental frequency in DLD-1 parental cells expressing the HaloTag[22-297](M2F)-12xGly / Ser-LgBiT variants in the same JF646+-gated configuration. Each construct was performed in duplicate. Error bars represent the standard deviation (SD) of the data mean. In the JF646-positive gated configuration, the fluorescence intensity was zero for the mRNA-free control. These plots suggest that longer ligand incubation times result in a better fold-over response in parental frequencies. At almost all time points, the HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT variant exhibited a slightly higher fold response compared to the other two HaloTag[22-297](M2F)-12xGly / Ser-LgBiT and HaloTag[22-297](M2F+ K140E)-12xGly / Ser-LgBiT variants.

[0152] Figure 131 Quantification of live PARP1-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells transiently transfected with mRNAs 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 variants using confocal imaging. 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 variants. Approximately 24 hours post-transfection, cells were labeled with 100 nM JF646 HaloTag ligand and incubated for 1 hour. Confocal microscopy was performed using the blue channel (excitation wavelength 405 nm), the far-red channel (excitation wavelength 640 nm), and DIC (differential interference phase contrast). Laser and gain settings were optimized to avoid pixel saturation and maintain uniform intensity across all variants to capture specific and background signals. For quantification of each variant, single fields of view (FOV) of PARP1-GGSG-HaloTag[3-19]-GGSG-VS-HiBiT DLD-1 cells were analyzed to measure the nuclear localization fluorescence intensity of PARP1, while individual FOVs of parental cells were assessed to quantify whole-cell background fluorescence intensity. The intensity of individual cells was analyzed using this strategy, generating data points in a graph. The bar chart represents the mean of the data. The graph shows high homogeneity of mRNA expression with only a few outliers. The results indicate similar fluorescence fold responses among the variants.

[0153] Figure 132The labeling kinetics of HaloTag7 and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis with HaloTag ligand JF549 were investigated. Fluorescence polarization was monitored over time to observe the labeling kinetics of HaloTag standard protein or HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis with HaloTag ligand JF549. Experiments were performed in Tris buffered saline + 0.01% CHAPS at a protein concentration of 10 nM and a ligand concentration of 2.5 nM.

[0154] Figure 133 The labeling kinetics of HaloTag7 and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis with HaloTag ligand JF503 were investigated. Fluorescence polarization was monitored over time to observe the labeling kinetics of HaloTag standard protein or HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis with HaloTag ligand JF503. The experiments were performed in Tris buffered saline + 0.01% CHAPS at a protein concentration of 10 nM and a ligand concentration of 2.5 nM.

[0155] Figure 134 The labeling kinetics of HaloTag7 and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis with HaloTag ligand JF554 were investigated. Fluorescence polarization was monitored over time to observe the labeling kinetics of HaloTag standard protein or HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis with the JFX554 HaloTag ligand. Experiments were performed in Tris buffered saline + 0.01% CHAPS at a protein concentration of 10 nM and a ligand concentration of 2.5 nM.

[0156] Figure 135The labeling kinetics of HaloTag7 and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis with the FAM HaloTag ligand Oregon Green. Fluorescence polarization was monitored over time to observe the labeling kinetics of HaloTag standard protein or HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis with the Oregon Green HaloTag ligand. Experiments were performed in Tris-buffered saline + 0.01% CHAPS at a protein concentration of 10 nM and a ligand concentration of 2.5 nM.

[0157] Figure 136 The labeling kinetics of HaloTag7 and HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis with FAM HaloTag ligands were investigated. Fluorescence polarization was monitored over time to observe the labeling kinetics of HaloTag standard protein or HaloTag[22-297](M2F+D53G+K140E)-12xGly / Ser-LgBiT-3xGly / Ser-6xHis with FAM HaloTag ligands. The experiments were performed in Tris buffered saline + 0.01% CHAPS at a protein concentration of 10 nM and a ligand concentration of 2.5 nM.

[0158] definition While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the embodiments described herein, this document describes some preferred methods, compositions, apparatuses, and materials. However, before describing the materials and methods of the invention, it should be understood that the invention is not limited to the specific molecules, compositions, methodologies, or schemes described herein, as these can vary based on routine experimentation and optimization. It should also be understood that the terminology used in the description is for the purpose of describing a particular version or embodiment only and is not intended to limit the scope of the embodiments described herein.

[0159] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of conflict, this specification (including the definitions) shall prevail. Therefore, the following definitions apply in the context of the embodiments described herein.

[0160] Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “described” as used herein and in the appended claims include plural indicators. Thus, for example, reference to “polypeptide” means one or more polypeptides and their equivalents known to those skilled in the art, etc.

[0161] As used herein, the term “and / or” includes any and all combinations of the listed items, including any one of the items listed individually. For example, “A, B and / or C” covers A, B, C, AB, AC, BC, and ABC, each of which should be considered as being described individually by the statement “A, B and / or C”.

[0162] As used herein, the term "comprising" and its variations indicate the presence of one or more listed features, elements, method steps, etc., without excluding the presence of additional features, elements, method steps, etc. Conversely, the term "consisting of" and its variations indicate the presence of one or more listed features, elements, method steps, etc., excluding any unlisted features, elements, method steps, etc., except for generally relevant impurities. The phrase "substantially consisting of" indicates the listed features, elements, method steps, etc., as well as any additional features, elements, method steps, etc., that do not substantially affect the fundamental properties of the composition, system, or method. Many embodiments described herein are described using the open-ended language of "comprising". Such embodiments encompass multiple closed-ended "consisting of" and / or "substantially consisting of" embodiments, which may alternatively use such language for claims or descriptions.

[0163] As used herein, the term "substantially" means that the described feature, parameter, and / or value does not need to be precisely achieved, but deviations or variations (including, for example, tolerances, measurement errors, measurement accuracy limitations, and other factors known to those skilled in the art) may occur in amounts that do not preclude the effect the feature is intended to provide. A substantially non-existent (e.g., substantially non-fluorescent) feature or characteristic may be within noise, against background, below the detection capability of the assay used, or only a fraction of a significant feature (e.g., fluorescence intensity of an active fluorophore) (e.g., <1%, <0.1%, <0.01%, <0.001%, <0.00001%, <0.000001%, <0.0000001%).

[0164] As used herein, when referring to an amino acid sequence or a position within an amino acid sequence, the term "corresponds to" refers to the relative position of an amino acid residue or fragment within the referred sequence, and not necessarily the specific identification of an amino acid at that position. For example, "peptide corresponding to positions 36 to 48 of SEQ ID NO: 1" may contain less than 100% sequence identity with positions 36 to 48 of SEQ ID NO: 1 (e.g., sequence identity >70%), but in the context of the described composition or system, the peptide is associated with these positions.

[0165] As used herein, the term "system" refers to multiple components (e.g., devices, compositions, etc.) for a specific purpose. For example, two individual biomolecules, whether present in the same composition or not, can constitute a system if they can be used together for a common purpose.

[0166] As used herein, the term "complementarity" refers to the property that two or more structural elements (e.g., peptides, polypeptides, nucleic acids, small molecules, etc.) can hybridize, dimerize, or otherwise form a complex. For example, "complementary peptides and polypeptides" can aggregate together to form a complex. Complementary elements may require assistance (facilitation) to form a complex (e.g., from interacting elements), such as placing the elements in the correct complementary conformation, placing the elements in the correct complementary proximity, co-locating complementary elements, reducing complementary interaction energies, overcoming insufficient affinity between them, etc.

[0167] As used herein, the term "complex" refers to a collection or aggregate of molecules (e.g., peptides, polypeptides, etc.) in direct and / or indirect contact with each other. On the one hand, "contact," or more specifically, "direct contact," means that two or more molecules are close enough that attractive non-covalent interactions (such as van der Waals forces, hydrogen bonding, ionic and hydrophobic interactions, etc.) dominate the molecular interactions. In this respect, molecular complexes (e.g., peptides, polypeptides, etc.) can form under assay conditions that make the complex thermodynamically more favorable (e.g., compared to the non-aggregated or non-complex state of the component molecules). As used herein, the term "complex," unless otherwise stated, refers to a combination of two or more molecules (e.g., peptides, polypeptides, etc.).

[0168] As used herein, the term "interacting element" refers to a portion that facilitates or promotes the binding of two or more structural elements (e.g., peptides, polypeptides, etc.) to form a complex. In some embodiments, a pair of interacting elements (also called an "interaction pair") is attached to a pair of structural elements (e.g., peptides, polypeptides, etc.), and the attractive interaction between the two interacting elements promotes the formation of a complex of the structural elements. Interacting elements can promote complex formation through any suitable mechanism (e.g., bringing structural elements close together, placing structural elements in a suitable conformation for stable interaction, lowering the activation energy for complex formation, their combination, etc.). Interacting elements can be proteins, polypeptides, peptides, small molecules, cofactors, nucleic acids, lipids, carbohydrates, antibodies, etc. Interacting pairs can consist of two identical interacting elements (i.e., homologous pairs) or two different interacting elements (i.e., heterologous pairs). In the case of heterologous pairs, the interacting elements can be parts of the same type (e.g., polypeptides) or can be two parts of different types (e.g., polypeptides and small molecules). In some implementations, when studying the formation of complexes of interaction pairs, the interaction pairs may be referred to as “target pairs” or “target elements”, while individual interaction elements may be referred to as “target elements” (e.g., “target peptide”, “target polypeptide”, etc.) or “target components” (e.g., “target peptide”, “target polypeptide”, etc.).

[0169] As used herein, the term “low affinity” describes an intermolecular interaction between two or more entities that is too weak to form a significant complex between these entities, except at concentrations substantially higher than physiological or assay conditions (e.g., 2, 5, 10, 100, 1000 or more), or facilitated by the formation of a second complex of linking elements (e.g., interacting elements).

[0170] As used herein, the term “high affinity” describes an intermolecular interaction between two or more (e.g., three) entities that is strong enough to produce a detectable complex under physiological or assay conditions without the need for a second complex formation facilitated by a connecting element (e.g., an interacting element).

[0171] As used herein, the term "pre-existing protein" refers to an amino acid sequence that was physically present prior to a certain event or date. A "peptide that is not a pre-existing protein fragment" is a short amino acid chain that is not a fragment or subsequence of a protein (e.g., synthetic or naturally occurring) that was physically present prior to the design and / or synthesis of the peptide.

[0172] As used herein, the term "fragment" refers to a peptide or polypeptide isolated or "fragmented" from a larger, whole entity (e.g., a protein, polypeptide, enzyme, etc.), or to a peptide or polypeptide prepared having the same sequence as that whole entity. Thus, a fragment is a subsequence of the whole entity (e.g., a protein, polypeptide, enzyme, etc.) from which it is made and / or designed. A peptide or polypeptide that is not a subsequence of a pre-existing whole protein is not a fragment (e.g., not a fragment of a pre-existing protein). A "peptide or polypeptide that is not a pre-existing protein fragment" is an amino acid chain that is not a subsequence of a protein (e.g., natural or synthetic) present in the entity prior to the design and / or synthesis of the peptide or polypeptide. As used herein, a hydrolase or dehalogenase fragment is a sequence smaller than the full-length sequence, but which cannot form a substrate binding site on its own and / or has substantially reduced or no substrate binding activity, but exhibits substantially increased substrate binding activity when closely adjacent to a second fragment of the hydrolase or dehalogenase. In one embodiment, the fragment of the hydrolase or dehalogenase is at least 5, 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 a mutant hydrolase, or a sequence having at least 70% sequence identity with the hydrolase, and does not necessarily include the N-terminal or C-terminal residues or N-terminal or C-terminal sequence of the corresponding full-length protein.

[0173] 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. The subsequence is a complete sequence match with a portion of the larger amino acid chain.

[0174] The term "amino acid" refers to natural amino acids, non-natural amino acids, and amino acid analogs, unless otherwise specified, and are D and L stereoisomers provided that their structures allow for such stereoisomerism.

[0175] The term "protein amino acid" 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 pyrrolidone can also be considered protein amino acids.

[0176] The term "non-protein amino acid" refers to an amino acid that is not naturally encoded or present in the genetic code of any organism and is not biosynthetically incorporated into proteins during translation. Non-protein amino acids can be "non-natural amino acids" (amino acids that do not exist in nature) or "naturally occurring non-protein amino acids" (e.g., valine, ornithine, homocysteine, etc.). Examples of non-protein amino acids include, but are not limited to: azacyclobutane carboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, naphthylalanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminohexanoic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, tert-butylglycine, 2,4-diaminoisobutyric acid, desmodium, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoproline, hydroxylysine, allohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmodium, alloisoleucine, N-methylalanine, N-alkylglycine, including N-methylglycine and N-methylisoleucine; N-alkylpentylglycine, including N-methylpentylglycine. N-methylvaline, naphthylalanine, n-valine, n-leucine (“Norleu”), octylglycine, ornithine, pentylglycine, piperidine, thioproline, high-lysine, and high-arginine. Non-proteins also include the D-amino acid form of any amino acid mentioned herein, as well as the non-α-amino acid forms (β-amino acids, γ-amino acids, δ-amino acids, etc.) of any amino acid mentioned herein, all of which are within the scope of this document and may be included in the peptides described herein.

[0177] The term "amino acid analog" refers to an amino acid (e.g., natural or non-natural, protein or non-protein) in which one or more of the C-terminal carboxyl group, N-terminal amino group, and side-chain bioactive groups have been chemically blocked (reversibly or irreversibly) or otherwise modified to form another bioactive group. For example, aspartic acid-(β-methyl ester) is an amino acid analog of aspartic acid; N-ethylglycine is an amino acid analog of glycine; or 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.

[0178] As used herein, unless otherwise specified, the terms “peptide” and “polypeptide” refer to polymer compounds consisting of two or more amino acids linked together by a peptide amide bond (--C(O)NH--). The term “peptide” generally refers to a short amino acid polymer (e.g., a chain having fewer than 30 amino acids), while the term “polypeptide” generally refers to a longer amino acid polymer (e.g., a chain consisting of more than 30 amino acids).

[0179] As used herein, the terms “artificial” or “synthetic” refer to compositions and systems designed or prepared by humans, rather than naturally occurring ones. For example, artificial or synthetic peptides, peptide-like substances, or nucleic acids refer to substances containing non-natural sequences (e.g., peptides that are not 100% identical to naturally occurring proteins or fragments thereof).

[0180] 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 contains amino acids that can be conservatively substituted for 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).

[0181] Amino acid residues can be classified into several categories based on common side-chain characteristics, such as: polar positive (or basic) (e.g., histidine (H), lysine (K), and arginine (R)); polar negative (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, "semi-conserved" amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid of the same class.

[0182] In some embodiments, unless otherwise specified, conserved or semi-conserved amino acid substitutions may also encompass non-naturally occurring amino acid residues having chemical properties similar to native residues. These non-natural residues are typically incorporated through chemical peptide synthesis rather than synthesis in biological systems. These include, but are not limited to, peptide mimics and other reversed or inverted forms of amino acid moieties. In some embodiments, the embodiments described herein may be limited to natural amino acids, non-natural amino acids, and / or amino acid analogs.

[0183] Non-conservative substitution can involve replacing a member of one class with a member of another class.

[0184] As used herein, the term "sequence identity" refers to the degree to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have a continuous composition of identical monomeric subunits. The term "sequence similarity" refers to the degree to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have similar polymer sequences. For example, similar amino acids refer to those amino acids that have the same biophysical properties and can be classified into different families, such as 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 best-aligned sequences within a comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specific window); (2) determining the number of positions containing the same (or similar) monomers (e.g., the same amino acid appears in both sequences, similar amino acids appear in both sequences) to produce the number of matching positions; (3) dividing the number of matching positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specific window); and (4) multiplying the result by 100 to produce the sequence identity percentage or sequence similarity percentage. For example, if peptide A and peptide B are both 20 amino acids long and have the same amino acid at all but one position, then peptide A and peptide B have 95% sequence identity. If the amino acids at different positions have the same biophysical properties (e.g., both are acidic), then peptide A and peptide B will have 100% sequence similarity. As another example, if peptide C is 20 amino acids long and peptide D is 15 amino acids long, and 14 of the 15 amino acids in peptide D are identical to a portion of the amino acids in peptide C, then peptide C and peptide D have 70% sequence identity. However, the optimal comparison window for peptide D and peptide C has 93.3% sequence identity. For the purpose of calculating the “percentage of sequence identity” (or “percentage of sequence similarity”) in this paper, any gaps in the aligned sequences are treated as mismatches at that position.

[0185] Any peptide / peptide described herein that has a specific percentage sequence identity or similarity (e.g., at least 70%) to the reference sequence ID number may also be expressed as having the maximum number of substitutions (or terminal deletions) relative to the reference sequence. For example, a sequence having at least Y% sequence identity (e.g., 90%) to SEQ ID NO: Z (e.g., 100 amino acids) may have up to X substitutions (e.g., 10) relative to SEQ ID NO: Z, and thus may also be expressed as "having X (e.g., 10) or fewer substitutions relative to SEQ ID NO: Z".

[0186] As used herein, the term "wild-type" refers to a gene or gene product (e.g., protein, polypeptide, peptide, etc.) that possesses the characteristics (e.g., sequence) of a gene or gene product isolated from a naturally occurring source and is most frequently observed in a population. In contrast, the terms "mutant" or "variant" refer to a gene or gene product that exhibits sequence modifications compared to the wild-type gene or gene product. It should be noted that "naturally occurring variants" refer to genes or gene products that exist in nature but whose sequence has been altered compared to the wild-type gene or gene product; they are not the most common sequences. "Artificial variants" or "synthetic variants" refer to genes or gene products whose sequence has been altered compared to the wild-type gene or gene product, and these genes or gene products do not exist in nature. Variant genes or gene products may be naturally occurring sequences that exist in nature but are not the most common variants of the gene or gene product, or they may be "synthetic," produced by human or experimental intervention.

[0187] As used herein, the term “physiological conditions” encompasses any conditions compatible with living cells, such as an environment primarily composed of water, whose temperature, pH, salinity, chemical composition, etc., are compatible with living cells.

[0188] As used herein, the term "sample" is used in its broadest sense. In one sense, it is intended to include samples or cultures obtained from any source, as well as biological and environmental samples. Biological samples can be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products such as plasma, serum, etc. A sample may also refer to cell lysates or purified forms of enzymes, peptides, and / or polypeptides described herein. Cell lysates may include cells or lysates that have been lysed with a lysing agent, such as rabbit reticulocytes or wheat germ lysates. Samples may also include cell-free expression systems. Environmental samples include environmental materials such as surface substances, soil, water, crystals, and industrial samples. However, such examples should not be construed as limiting the types of samples applicable to the present invention.

[0189] As used herein, the terms “fusion compound,” “fusion polypeptide,” and “fusion protein” refer to a chimeric protein containing a first target protein or polypeptide linked to a second different peptide, polypeptide, or protein (e.g., an interaction element).

[0190] As used herein, the terms “coupled” and “coupled” refer to the covalent connection of two molecular entities (e.g., post-synthesis and / or during synthetic production). An example of coupling is the chemical (e.g., “chemical” coupling) or enzymatic linking of peptide or small molecule tags to proteins or small molecules.

[0191] As used herein, the terms "peptide component" or "peptide component" are used synonymously with the terms "peptide component of [mutant dehalogenase] complex" or "peptide component of [mutant dehalogenase] complex". Generally, as used herein, the peptide component or peptide component is capable of forming a complex with a second component under appropriate conditions to form the desired complex.

[0192] As used herein, the term "dehalogenase" refers to an enzyme that catalyzes the removal of a halogen atom from a substrate. The term "haloalkyl dehalogenase" refers to an enzyme that catalyzes the removal of a halogen from a haloalkyl substrate to produce an alcohol and a halide. Dehalogenases and haloalkyl dehalogenases belong to the family of hydrolases, which may be referred to as hydrolases herein or elsewhere.

[0193] As used herein, the term "modified dehalogenase" refers to a dehalogenase variant (artificial variant) whose mutation 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 modified dehalogenases do not release the substrate, they are not reusable and are not classic enzymes. The HALOTAG system (Promega) is a commercially available modified dehalogenase and substrate system.

[0194] As used herein, the term "bioluminescence" refers to the generation and emission of light by a chemical reaction catalyzed or facilitated by an enzyme, protein, protein complex, or other biomolecule (e.g., a bioluminescent complex). 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; the substrate then emits light.

[0195] As used herein, the term "non-luminescent" refers to an entity (e.g., peptides, polypeptides, complexes, proteins, etc.) that does not emit detectable light in the visible spectrum (e.g., in the presence of a substrate). For example, an entity may be described as non-luminescent if it does not exhibit detectable luminescence in a given measurement. As used herein, the term "non-luminescent" is synonymous with the term "substantially non-luminescent." In some embodiments, an entity is considered "non-luminescent" if any light emission is sufficiently minimal to not introduce interfering background to a particular measurement.

[0196] As used herein, the terms "non-luminescent peptide" and "non-luminescent polypeptide" refer to peptides that, under standard conditions (e.g., physiological conditions, assay conditions, etc.) and using typical instruments (e.g., photometers, etc.), exhibit substantially no luminescence (e.g., in the presence of a substrate) or luminescence levels below noise (e.g., 100-fold, 200-fold, 500-fold, 1x10⁻⁶) compared to significant signals (e.g., bioluminescent complexes). 3 times, 1x10 4 times, 1x10 5 times, 1x10 6 times, 1x10 7 (Multiple times) peptides and polypeptides. In some embodiments, such non-luminescent peptides and polypeptides are assembled according to the criteria described herein to form a bioluminescent complex.

[0197] As used herein, the term "OgLuc" ("shrimp luciferase") refers to a luminescent polypeptide exhibiting significant sequence identity, structural conservation, and / or functional activity of luciferase, said luciferase being derived from the deep-sea shrimp *OgLuc*. Oplophorus gracilirostris ) generation and derivation. Specifically, OgLuc polypeptides refer to luminescent polypeptides with significant sequence identity, structural conservation and / or functional activity of the mature 19 kDa subunit of the scorpion luciferase protein complex (e.g., without a signal sequence), such as SEQ ID NO: 3034 (NanoLuc), which comprises 10 β chains (β1, β2, β3, β4, β5, β6, β7, β8, β9, β10) and utilizes substrates such as coelentrin or coelentrin derivatives or analogs to generate luminescence.

[0198] As used herein, the term "β9-like peptide" refers to a peptide (or peptide tag) that contains a functionally active β9 chain of an OgLuc polypeptide with significant sequence identity, structural conservation, and / or similarity. Specifically, a β9-like peptide is a peptide that is structurally complementary to an OgLuc polypeptide lacking a β9 chain, resulting in enhanced luminescence of the complex compared to an OgLuc polypeptide without a β9-like peptide. Other "βX-like peptides" may be similarly named (e.g., β1-like, β2-like, β3-like, β4-like, β5-like, β6-like, β7-like, β8-like, β9-like).

[0199] As used herein, the term "β10-like peptide" refers to a peptide (or peptide tag) that contains a functionally active β10 chain of an OgLuc polypeptide with significant sequence identity, structural conservation, and / or OgLuc peptide characteristics. Specifically, a β10-like peptide is a peptide that is structurally complementary to an OgLuc polypeptide lacking a β10 chain, resulting in enhanced luminescence of the complex compared to an OgLuc polypeptide without a β10-like peptide. Other "βX-like peptides" may be similarly named (e.g., β1-like, β2-like, β3-like, β4-like, β5-like, β6-like, β7-like, β8-like, β9-like).

[0200] As used in this article, the term "β" 1-8 "β-like polypeptides" refer to polypeptides that have sequence and structural similarity to the β (beta) chains 1-8 of the OgLuc polypeptide, but lack β chains 9 and 10. Other "β-like polypeptides"... Y-Z "Like polypeptides" can be named similarly (e.g., β-peptides). 1-4 Like polypeptide, β 2-8 Like polypeptide, β 5-10 (e.g., polypeptides).

[0201] As used in this article, the term "NANOLUC" refers to artificial luciferase or bioluminescent peptide commercially produced by Promega Corporation.

[0202] As used in this article, the term "LgBiT" refers to the term corresponding to β. 1-9 The polypeptide is a polypeptide that can be used, for example, for binary complementation to form a bioluminescent complex, and corresponds to SEQ ID NO: 3037.

[0203] As used in this article, the term "SmBiT" refers to the term corresponding to β. 10 The peptide is a type of peptide that can be used, for example, for binary complementation to form a bioluminescent complex, but has low affinity for LgBiT (e.g., requires to promote complex formation) and corresponds to SEQ ID NO: 3039.

[0204] As used in this article, the term "HiBiT" refers to the term corresponding to β. 10 The peptide is a type of peptide that can be used, for example, for binary complementation to form a bioluminescent complex, but has the high affinity of LgBiT (e.g., no need to promote complex formation). An exemplary HiBiT peptide corresponds to SEQ ID NO: 3038.

[0205] As used in this article, the term "LgTrip" refers to the term corresponding to β. 1-8 A polypeptide of the β9-like peptide. An exemplary LgTrip corresponds to SEQ ID NO: 3045 and can be used, for example, with β9-like peptides and β 10 Like peptides undergo three-part complementarity to form a bioluminescent complex, or with β9-10 The peptides undergo binary complementation to form a bioluminescent complex.

[0206] As used in this article, the term "SmTrip10" refers to the term corresponding to β. 10 The peptide is a peptide that can be used, for example, three-part complementarity to form a bioluminescent complex.

[0207] As used herein, the term “SmTrip9” refers to a peptide corresponding to a β9-like peptide, which can be used, for example, for triploidal complementarity to form a bioluminescent complex. As used herein, the term “sp” refers to a polypeptide that has been split into two fragments at a location within the original polypeptide. If the fragments of an sp polypeptide are structurally complementary and capable of forming an active complex, the activity of the original polypeptide can be reconstructed. The nomenclature used herein to refer to the split component of a polypeptide refers to the position number corresponding to the last residue of the N-terminal component of the split polypeptide in the complete polypeptide. For example, if the polypeptide is 100 residues long, the sp52 version of the polypeptide contains 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 a commercially available HALOTAG protein, wherein 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.

[0208] Alternatively, components of a split polypeptide may be expressed herein by referring to the name of its source polypeptide, the residues in the source polypeptide present in the component (in square brackets), and any substitutions relative to the source polypeptide (in parentheses). For example, a split component of a commercially available HALOTAG protein corresponding to positions 22-297 of the HALOTAG sequence can be written as HaloTag[22-297]. If the second position of the component contains an M to F substitution, the component may be referred to as HaloTag[22-297](M2F). Components may contain N-terminal methionine residues not present in the source sequence; such residues are counted when determining substitution positions but are not counted when numbering fragments in the source polypeptide.

[0209] As used herein, the term "gap" refers to a splitting variant of a polypeptide that lacks the original polypeptide fragment. For example, a "gap sp polypeptide" is a polypeptide that lacks the original sequence fragment that appears at the splitting site.

[0210] As used herein, the term "overlap" refers to a splitting variant of a polypeptide containing a repeating original polypeptide fragment. For example, an "overlapping sp polypeptide" refers to a polypeptide in which (repeated) original sequence fragments adjacent to the splitting site are present at the C-terminus of the first fragment and the N-terminus of the second fragment.

[0211] The term "binding moiety" refers to a domain that specifically binds to an antigen or epitope, independent of a separate epitope or antigen-binding domain. Binding moieties can be antibodies, antibody fragments, receptor domains that bind target ligands, proteins that bind to immunoglobulins (e.g., protein A, protein G, protein A / G, protein L, protein M), binding domains of proteins that bind to immunoglobulins (e.g., protein A, protein G, protein A / G, protein L, protein M), oligonucleotide probes, peptide nucleic acids, DARPin, aptamers, affimers, purified proteins (the analyte itself or proteins bound to the analyte), and analyte-binding domains of proteins, etc. Table A lists exemplary binding moieties that can be used alone or in various combinations in the methods, systems, and assays (e.g., immunoassays) described herein.

[0212] Table A. Exemplary Combination Section Detailed Implementation

[0213] This document provides compositions and systems comprising complementary tags and reporter genes for the labeling and detection of targets via luminescence and a second modality (e.g., fluorescence), as well as methods of using them. Specifically, the invention provides a tag comprising a fusion of a first component of a bioluminescent complex and a first component of a modified dehalogenase complex; a reporter gene comprising a second component of a bioluminescent complex and a second component of a modified dehalogenase complex; and systems and methods comprising the tags and reporter genes described herein for bimodal labeling and detection of targets.

[0214] In some embodiments, a system is provided herein 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 gene comprising a polypeptide component of a bioluminescent complex fused to a polypeptide component of the modified dehalogenase complex; wherein the bioluminescent complex and the modified dehalogenase complex are formed upon interaction (promoted or non-promoted) between the tandem peptide tag and the tandem polypeptide reporter; wherein the bioluminescent complex is capable of generating bioluminescence in the presence of a substrate (e.g., coelentrin, furimazine, etc.); and wherein the modified dehalogenase complex is capable of binding to a haloalkane ligand. In some embodiments, a high-affinity interaction between a pair of components to form a complex (e.g., a component of the bioluminescent complex or a component of the modified dehalogenase complex) is sufficient to promote the formation of a second complex (e.g., the modified dehalogenase complex of the bioluminescent complex).

[0215] HaloTag In some embodiments, this document provides 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 capable of interacting (e.g., promoted or non-promoted) to form an active modified dehalogenase complex, the active modified dehalogenase complex being capable of forming a covalent bond with a haloalkane ligand. In some embodiments, a first fusion comprising a complementary peptide fragment of a modified dehalogenase and a second fusion comprising a complementary polypeptide fragment of a modified dehalogenase, wherein, upon interaction (e.g., promoted or non-promoted), the complementary peptides and polypeptides form an active modified dehalogenase complex, the modified dehalogenase complex being capable of forming a covalent bond 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 as a fusion), wherein, upon interaction (e.g., promoted or non-promoted), the complementary peptide and polypeptide form an active modified dehalogenase complex capable of forming covalent bonds with a haloalkane ligand. In some embodiments, the complementary peptide and polypeptide are fragments of a cleavage mutant dehalogenase. In alternative embodiments, both fragments may be polypeptides.

[0216] The compositions, systems and methods provided herein that form part of this document are splitting mutant dehalogenases, such as dehalogenases derived from commercially available HALOTAG protein (Promega) and / or mutant dehalogenases disclosed in U.S. Publication No. 20060024808, the disclosure of which is incorporated herein by reference.

[0217] Although these mutant dehalogenases are technically not enzymes (no substrate turnover), their stable binding to the substrate depends on the correct protein structure. The consequences of rebinding the cleavage fragment of a mutant dehalogenase differ from those of a cleavage enzyme system because the labeling function of a mutant dehalogenase is retained on one of the fragments even after separation from its mate, whereas a cleavage enzyme is only active upon binding and leaves no trace of pre-activity after separation. In effect, the labeling reaction of a cleavage mutant dehalogenase provides molecular memory of protein-protein interactions. In the case of fluorescent ligands, the label is retained on one fragment but may be undetectable after dissociation of the complex (because the contact between the fluorescent activator and the protein may be disrupted / absent); therefore, the binding of a cleavage dehalogenase and a fluorescent ligand creates a unique condition of permanent labeling, but the retained label can be detected by dynamic (on / off) fluorescence.

[0218] Mutant dehalogenases provide effective labeling in living cells or their lysates. This labeling is conditional solely on the presence or expression of the protein and the presence of the labeled hydrolase substrate. In contrast, labeling of cleavage mutant dehalogenases depends on specific protein-protein interactions occurring within the cell and the presence of the labeled hydrolase substrate.

[0219] In some embodiments, a cleavage-modified dehalogenase is provided as a component of the compositions, systems, and methods described herein. In some embodiments, a first fragment of the mutant dehalogenase is fused to a first fragment of a luminescent protein (e.g., optionally a target protein or molecule), and a second fragment of the mutant dehalogenase is fused to a second fragment of the luminescent protein (e.g., optionally a target protein or molecule). In some embodiments, at least one mutant dehalogenase fragment has a substituent that, if present in the full-length modified dehalogenase having both fragment sequences, would form a bond with a haloalkane ligand. In some embodiments, the first and second fragments of the mutant dehalogenase are capable of interacting (e.g., promoted or non-promoted) to form an active modified dehalogenase complex.

[0220] HALOTAG is a 297-residue self-labeled polypeptide (33 kDa) derived from a bacterial hydrolase (dehalogenase) that is modified to covalently bind to its ligand (haloalkyl moiety). The HALOTAG ligand can be attached to a solid surface (e.g., a bead) or a functional group (e.g., a fluorophore). The HALOTAG polypeptide can be fused with various target proteins, covalently linking the target protein to a solid surface or functional group.

[0221] HALOTAG peptide is a modified dehalogenase with a genetically modified active site that specifically binds to chloroalkyl linkers with enhanced and increased ligand binding rates (Pries et al.). The Journal of Biological Chemistry270(18):10405–11; incorporated by reference). Under physiological conditions, the reaction between the protein tag and the chloroalkane linker is rapid and essentially irreversible (Waugh DS (June 2005)). Trends in Biotechnology. 23(6):316–20; incorporated by reference). In native hydrolases, nucleophilic attack on the chloroalkane linker leads to the substitution of the halogen with an amino acid residue, forming a covalent alkylase intermediate. This intermediate is then hydrolyzed by the amino acid residue in the wild-type hydrolase (Chen et al. (February 2005) CurrentOpinion in Biotechnology. 16(1):35–40; incorporated by reference). This leads to post-reaction enzyme regeneration. However, for modified haloalkane dehalogenases like HALOTAG, the intermediate cannot be hydrolyzed due to enzyme mutation, thus preventing the second reaction. This allows the intermediate to persist as a stable covalent adduct without any associated reverse reaction (Marks et al. (August 2006) Nature Methods. 3(8): 591–6; incorporated by reference in its entirety).

[0222] 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; incorporated by reference). Because the HALOTAG peptide is a relatively small protein and the reaction is foreign to mammalian cells, it is not affected by endogenous mammalian metabolic responses (Naested et al., The Plant Journal. 18(5):571–6; incorporated by reference). Once the fusion protein is expressed, a wide range of potential experimental applications are available, including enzyme analysis, cell imaging, protein arrays, determination of subcellular localization, and many other possibilities (Janssen DB (April 2004) Current Opinion in Chemical Biology. 8(2):150–9; incorporated by reference).

[0223] 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 2009 / 0253131; U.S. Patent Application 2010 / 0273186; 20130337539; U.S. Patent Application 2012 / 0258470; U.S. Patent Application 2012 / 0252048; U.S. Patent Application 2011 / 0201024; and U.S. Patent No. 2014 / 0322794. Each of these documents is incorporated herein by reference in its entirety.

[0224] In some embodiments, the modified dehalogenase fragments, complementary peptides, complementary polypeptides, etc., described herein are based on the HALOTAG complementation 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 comprising two or more peptide or polypeptide components described herein correspond to the HALOTAG protein and are capable of binding to haloalkyl ligands in a similar manner.

[0225] In some embodiments, such as those described in U.S. Provisional Application Nos. 63 / 338,323 and 18 / 312,117 (all of which are incorporated herein by reference in their entirety), extensive experiments have been conducted to demonstrate the feasibility of generating HALOTAG fragments (and variants thereof) capable of interacting to form modified dehalogenase complexes that can bind to haloalkyl ligands, and to optimize variants of the HALOTAG fragments to obtain desired properties. As described herein, embodiments are not limited to the HALOTAG sequence. In some embodiments, this document provides cleaving modified dehalogenases with sequences different from HALOTAG (SEQ ID NO: 1).

[0226] In some embodiments, compositions and systems are provided comprising components of a cleavage modified dehalogenase, such as cleavage HALOTAG (“spHT”) or variants thereof. In some embodiments, the systems and compositions herein comprise spHT peptides and polypeptides (e.g., as part of the fusions described herein).

[0227] In some embodiments, compositions (e.g., fusions) and systems (e.g., multiple fusions with suitable ligands and substrates) are provided, comprising polypeptides, peptides, fragments, and combinations thereof derived from a modified dehalogenase sequence of SEQ ID NO: 1 (HALOTAG): MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETF QAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISG.

[0228] In some embodiments, the spHT component described herein lacks mutations (e.g., 272 and / or 106) that produce covalent bonds with haloalkane substrates. Such sp dehalogenases are true enzymes capable of substrate turnover, but otherwise contain the sequences and features of the embodiments described herein.

[0229] In some embodiments, the spHT peptides and polypeptides described herein (e.g., as part of the fusion described herein, as a standalone reporter gene, or tag, etc.) contain at least 70% sequence identity with a portion of SEQ ID NO: 1 (e.g., >70% sequence identity, >75% sequence identity, >80% sequence identity, >85% sequence identity, >90% sequence identity, >95% sequence identity, >96% sequence identity, >97% sequence identity, >98% sequence identity, >99% sequence identity). In some embodiments, the spHT peptides and polypeptides described herein (e.g., as part of the fusion described herein, as a standalone reporter gene, or tag, etc.) contain 100% sequence identity with all or part of SEQ ID NO: 1. In some embodiments, the spHT peptides and polypeptides described herein (e.g., as part of the fusion compound described herein, as a standalone reporter gene, or tag, etc.) contain at least 70% sequence similarity to all or part of SEQ ID NO: 1 (e.g., >70% sequence similarity, >75% sequence similarity, >80% sequence similarity, >85% sequence similarity, >90% sequence similarity, >95% sequence similarity, >96% sequence similarity, >97% sequence similarity, >98% sequence similarity, >99% sequence similarity). In some embodiments, the spHT peptides and polypeptides described herein (e.g., as part of the fusion compound described herein, as a standalone reporter gene, or tag, etc.) contain 100% sequence similarity to all or part of SEQ ID NO: 1.

[0230] In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound, as an independent reporter gene, or tag, etc.) is contained at position A corresponding to position 2 of SEQ ID NO: 1. In other embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound, as an independent reporter gene, or tag, etc.) is contained at position S corresponding to position 2 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound, as an independent reporter gene, or tag, etc.) is contained at position V corresponding to position 47 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound, as an independent reporter gene, or tag, etc.) is contained at position T corresponding to position 58 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound, as an independent reporter gene, or tag, etc.) is contained at position G corresponding to position 78 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound, as an independent reporter gene, or tag, etc.) is contained at position F corresponding to position 88 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound, as an independent reporter gene, or tag, etc.) is contained at position M corresponding to position 89 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound, as an independent reporter gene, or tag, etc.) is contained at position F corresponding to position 128 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound, as an independent reporter gene, or tag, etc.) is contained at position T corresponding to position 155 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound, as an independent reporter gene, or tag, etc.) is contained at position K corresponding to position 160 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion herein, as an independent reporter gene, or a tag, etc.) is contained at position V corresponding to position 167 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion herein, as an independent reporter gene, or a tag, etc.) is contained at position T corresponding to position 172 of SEQ ID NO: 1.In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound, as an independent reporter gene, or tag, etc.) is contained at position M corresponding to position 175 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound, as an independent reporter gene, or tag, etc.) is contained at position G corresponding to position 176 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound, as an independent reporter gene, or tag, etc.) is contained at position N corresponding to position 195 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound, as an independent reporter gene, or tag, etc.) is contained at position E corresponding to position 224 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound, as an independent reporter gene, or tag, etc.) is contained at position D corresponding to position 227 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion herein, as an independent reporter gene, or tag, etc.) is contained at position 257 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion herein, as an independent reporter gene, or tag, etc.) is contained at position 264 corresponding to SEQ ID NO: 1. In some embodiments, the peptide or polypeptide described herein is contained at position 272 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion herein, as an independent reporter gene, or tag, etc.) is contained at position 273 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion herein, as an independent reporter gene, or tag, etc.) is contained at position 291 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion herein, as an independent reporter gene, or a tag, etc.) is contained at position T corresponding to position 292 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion herein, as an independent reporter gene, or a tag, etc.) is contained at position E corresponding to position 294 of SEQ ID NO: 1.In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound described herein, as an independent reporter gene, or a tag, etc.) is contained at position I corresponding to position 295 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound described herein, as an independent reporter gene, or a tag, etc.) is contained at position S corresponding to position 296 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound described herein, as an independent reporter gene, or a tag, etc.) is contained at position G corresponding to position 297 of SEQ ID NO: 1.

[0231] In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound described herein, as an independent reporter gene, or tag, etc.) does not have an S at position 2 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound described herein, as an independent reporter gene, or tag, etc.) does not have an L at position 47 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound described herein, as an independent reporter gene, or tag, etc.) does not have an S at position 58 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound described herein, as an independent reporter gene, or tag, etc.) does not have a D at position 78 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound described herein, as an independent reporter gene, or tag, etc.) does not have a Y at position 88 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound described herein, as an independent reporter gene, or tag, etc.) does not have the L corresponding to position 89 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound described herein, as an independent reporter gene, or tag, etc.) does not have the C corresponding to position 128 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound described herein, as an independent reporter gene, or tag, etc.) does not have the A corresponding to position 155 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound described herein, as an independent reporter gene, or tag, etc.) does not have the E corresponding to position 160 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound described herein, as an independent reporter gene, or tag, etc.) does not have the A corresponding to position 167 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion herein, as an independent reporter gene, or a tag, etc.) does not have an A corresponding to position 172 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion herein, as an independent reporter gene, or a tag, etc.) does not have a K corresponding to position 175 of SEQ ID NO: 1.In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound described herein, as an independent reporter gene, or tag, etc.) does not have a C corresponding to position 176 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound described herein, as an independent reporter gene, or tag, etc.) does not have a K corresponding to position 195 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound described herein, as an independent reporter gene, or tag, etc.) does not have an A corresponding to position 224 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound described herein, as an independent reporter gene, or tag, etc.) does not have an N corresponding to position 227 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound described herein, as an independent reporter gene, or tag, etc.) does not have an E corresponding to position 257 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound described herein, as an independent reporter gene, or a tag, etc.) does not have a T corresponding to position 264 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound described herein, as an independent reporter gene, or a tag, etc.) does not have an H corresponding to position 272 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound described herein, as an independent reporter gene, or a tag, etc.) does not have a Y corresponding to position 273 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound described herein, as an independent reporter gene, or a tag, etc.) does not have a P corresponding to position 291 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion compound described herein, as an independent reporter gene, or a tag, etc.) does not have an A corresponding to position 292 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion herein, as an independent reporter gene, or a tag, etc.) does not have the amino acid corresponding to position 294 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion herein, as an independent reporter gene, or a tag, etc.) does not have the amino acid corresponding to position 295 of SEQ ID NO: 1.In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion herein, as an independent reporter gene, or a tag, etc.) does not have the amino acid corresponding to position 296 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of the fusion herein, as an independent reporter gene, or a tag, etc.) does not have the amino acid corresponding to position 297 of SEQ ID NO: 1.

[0232] In some embodiments, the sp dehalogenase (e.g., spHT) comprises two peptide and / or polypeptide components, said two peptide and / or polypeptide components collectively containing at least 70% sequence similarity or identity with all or part of SEQ ID NO: 1 (e.g., >70% sequence similarity or identity, >75% sequence similarity or identity, >80% sequence similarity or identity, >85% sequence similarity or identity, >90% sequence similarity or identity, >95% sequence similarity or identity, >96% sequence similarity or identity, >97% sequence similarity or identity, >98% sequence similarity or identity, >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 identity with 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 identity with the second part). In some embodiments, the sp dehalogenase (e.g., spHT) comprises two fragments that together contain 100% sequence similarity or identity with all or part of SEQ ID NO: 1. For example, the first fragment of the sp polypeptide has 100% sequence similarity or identity with the first part of SEQ ID NO: 1, and the second fragment of the sp polypeptide has 100% sequence similarity or identity with the second part of SEQ ID NO: 1.

[0233] In some embodiments, the sp dehalogenase (e.g., present as part of a fusion herein, as a separate reporter gene, or a tag, etc.) contains an sp site. The sp site is an internal location in the parental sequence that defines the C-terminus of the first component or fragment of the sp dehalogenase and the N-terminus of the second component or fragment. For example, if a theoretically 100-amino acid polypeptide is split between residues 57 and 58 of the parental polypeptide using an sp site (referred to herein as the sp site of 57), the first component polypeptide will correspond to positions 1-57, and the second component polypeptide will correspond to positions 58-100. In some embodiments herein, the sp site within SEQ ID NO: 1 may be present at any position from position 5 to position 290 of SEQ ID NO: 1. In some embodiments, SEQ ID NO: 2-577 are exemplary components of an spHT polypeptide having 100% sequence identity with SEQ ID NO: 1. In some embodiments, the active spHT complex is formed between two fragments that collectively contain amino acids corresponding to each position in SEQ ID NO: 1. For example, a peptide having the sequence SEQ ID NO: 26 and a peptide having the sequence SEQ ID NO: 27 together comprise amino acids corresponding to each position in SEQ ID NO: 1. Any peptide and polypeptide pair (or two peptides) corresponding to two of SEQ ID NO: 2-577 and together comprising amino acids corresponding to each position in SEQ ID NO: 1 (with or without positional deletions or repetitions) may be used in the embodiments described herein. In some embodiments, the spHT dehalogenase (e.g., the fusion used herein or as a standalone reporter gene or tag) comprises any pair of the following fragments: SEQ ID NO: 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 W 121, 121, 122 W 123, 124 W 125, 126 W 127, 128 W 129, 130 W 131, 132 W 133, 134 W 135, 136 W 137, 138 W 139, 140 W 141, 142 W 143, 144 W 145, 146 W 147, 148 W 149, 150 W 151, 152 W 153, 154 W 155, 156 W 157, 158 W 159, 160 W 161, 172 W 173, 174 W 175, 176 W 177, 178 W 179, 180 W 181, 182 W 183, 184 W 185, 186 W 187, 188 W 189, 190 W 1 91, 192 and 193, 194 and 195, 196 and 197, 198 and 199, 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, 2 22 W 223, 224 W 225, 226 W 227, 228 W 229, 300 W 301, 302 W 303, 304 W 305, 306 W 307, 308 W 309, 310 W 311, 312 W 313, 314 W 315, 316 W 317, 318 W 319, 320 W 321, 322 W 32 3, 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, 35 4-355, 356-357, 358-359, 360-361, 362-363, 364-365, 366-367, 368-369, 370-371, 372-373, 374-375, 376-377, 378-379, 380-381, 382-383, 384-385 , 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 Wa 417, 418 Wa 419, 420 Wa 421, 422 Wa 423, 424 Wa 425, 426 Wa 427, 428 Wa 429, 430 Wa 431, 432 Wa 433, 434 Wa 435, 436 Wa 437, 438 Wa 439, 440 Wa 441, 442 Wa 443, 444 Wa 445, 446 Wa 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, 4 64 and 465, 466 and 467, 468 and 469, 470 and 471, 472 and 473, 474 and 475, 476 and 477, 478 and 479, 48 0 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 5 29, 530 and 531, 532 and 533, 534 and 535, 536 and 537, 538 and 539, 540 and 541, 542 and 543, 544 and 54 5, 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. ,

[0234] In some embodiments, spHT comprises a pair of peptides and polypeptides (or two polypeptides) corresponding to two of SEQ ID NO: 2-577, collectively containing amino acids corresponding to each position in SEQ ID NO: 1, but with a deletion of up to 40 amino acid lengths (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or a range thereof) at the C-terminus or N-terminus of one or both fragments. For example, the pair corresponding to SEQ ID NO: 7 and 28 corresponds to the position in SEQ ID NO: 1, but with 11 residues missing. In some embodiments, any pair of SEQ ID NO: 2-577, collectively corresponding to the sequence of SEQ ID NO: 1, but with a deletion of up to 40 amino acids, is within the scope of spHT herein. In some embodiments, the deletion site is adjacent to the splitting site. In some embodiments, the deletion corresponds to the N-terminus or C-terminus of SEQ ID NO: 1.

[0235] In some embodiments, spHT comprises a pair of peptides and polypeptides (or two polypeptides) corresponding to two of SEQ ID NO: 2-577, collectively containing amino acids corresponding to each position in SEQ ID NO: 1, but repeating up to 40 amino acid lengths (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or a range thereof) at the C-terminus or N-terminus of one or both segments. For example, the pair corresponding to SEQ ID NO: 6 and 29 corresponds to the position in SEQ ID NO: 1, but repeats 11 residues. In some embodiments, any pair of SEQ ID NO: 2-577, collectively corresponding to the sequence of SEQ ID NO: 1, but having a repeat of up to 40 amino acids, all within the scope of spHT herein. In some embodiments, the repeat site is adjacent to the splitting site. In some embodiments, the repeat corresponds to the N-terminus or C-terminus of SEQ ID NO: 1.

[0236] Using any sp site fragment, such as the fragment corresponding to the position between position 5 and position 290 of SEQ ID NO: 1, is readily conceived and is within the scope of this document.

[0237] In some implementations, spHT has a corresponding SEQ ID NO: The sp bits at the following positions of 1: 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, 14 8, 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, 3 19, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, or 290.

[0238] In some embodiments, spHT has sp sites corresponding to the positions between the following positions of SEQ ID NO: 1: 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.

[0239] In some embodiments, the spHT peptides and polypeptides described herein (e.g., fusions described herein or as standalone reporter genes or tags) contain one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75 or more) substitutions or deletions relative to SEQ ID NO: 2-557. In some embodiments, sp peptides and polypeptides (e.g., within the fusions described herein or as standalone reporter genes or tags) are provided having 70%-100% sequence identity with one of SEQ ID NO: 2-557 (e.g., >70% sequence identity, >75% sequence identity, >80% sequence identity, >85% sequence identity, >90% sequence identity, >95% sequence identity, >96% sequence identity, >97% sequence identity, >98% sequence identity, >99% sequence identity). In some embodiments, the provided sp peptide and polypeptide have 70%-100% sequence similarity to one of SEQ ID NO: 2-557 (e.g., >70% sequence similarity, >75% sequence similarity, >80% sequence similarity, >85% sequence similarity, >90% sequence similarity, >95% sequence similarity, >96% sequence similarity, >97% sequence similarity, >98% sequence similarity, >99% sequence similarity).

[0240] In some embodiments, pairs of sp peptides and / or polypeptides are provided (e.g., in the fusions described herein or as standalone reporter genes or tags) capable of forming an active sp dehalogenase complex (active spHT complex). In some embodiments, such pairs contain at least 70% sequence identity or similarity to two of the sequences in SEQ ID NO: 2-557 and together contain residues corresponding to 100% of the positions in SEQ ID NO: 1, thereby allowing up to 40 deletions or repetitions at the C-terminus or N-terminus of the peptide / polypeptide.

[0241] In some implementations, the first fragment of the spHT complementary pair corresponds to SEQ ID NO: Position 1 to 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, 8 4, 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, 14 8, 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, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, or 290.

[0242] In some implementations, the second fragment of the spHT complementary pair corresponds to SEQ ID NO: Position 1: 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, 15 0, 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, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 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 290 to position 294.

[0243] In some implementations, the repeating portion of the spHT complementary pair has a length of 1-40 amino acids (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 thereof).

[0244] In some implementations, the length of the missing portion of the spHT complement pair is 1-40 amino acids (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 thereof).

[0245] The exemplary spHT fragment sequences of SEQ ID NO: 2-577 contain 100% sequence identity with a portion of SEQ ID NO: 1; these sequences do not contain a portion having 100% sequence identity with SEQ ID NO: 1. However, as described herein, spHT peptides and polypeptides may have less than 100% sequence identity with SEQ ID NO: 1 (e.g., >70%, >75%, >80%, >85%, >90%, >95%, >96%, >97%, >98%, >99%, but less than 100% sequence identity). Therefore, this document provides peptides and polypeptides having less than 100% sequence identity with one or SEQ ID NO: 2-577 (e.g., >70%, >75%, >80%, >85%, >90%, >95%, >96%, >97%, >98%, >99%, but less than 100% sequence identity) and may be used in complementary pairs and complexes herein.

[0246] In some embodiments, the spHT complement pair herein comprises a peptide corresponding to SEQ ID NO: 578 and a polypeptide corresponding to SEQ ID NO: 1188. SEQ NO: 578 and 1188 are fragments of SEQ ID NO: 1 and have 100% sequence identity with the portion of SEQ ID NO: 1. In some embodiments, the spHT complement pair comprises a peptide having 100% sequence identity with SEQ ID NO: 578; this peptide is referred to herein as “SmHT”. In some embodiments, the spHT complement pair comprises a polypeptide having 100% sequence identity with SEQ ID NO: 1188; this polypeptide is referred to herein as “LgHT”. Extensive experiments were conducted during the development of the embodiments herein to analyze variants of SmHT and LgHT. SEQ ID NO: 579-1187 correspond to peptide variants in which SEQ ID NO: 588 is substituted at at least one position and at most all positions. Each peptide of SEQ ID NO: 578-1187 was synthesized and its various properties were tested, including its ability to form an active complex with complementary LgHT variant peptides. SEQ ID NO: 1189-3033 correspond to peptide variants having one or more substitutions relative to SEQ ID NO: 1188. Each peptide of SEQ ID NO: 1188-3033 was synthesized and its various properties were tested, including its ability to form an active complex with complementary SmHT variant peptides.

[0247] In some embodiments, this document provides variant SmHT peptides having one or more substitutions relative to a reference SmHT peptide sequence (e.g., peptides of SEQ ID NO: 3061, 3064-3066, and 3079-3091).

[0248] In some embodiments, this document provides (e.g., in fusions herein or as a standalone reporter gene or tag, etc.) a SmHT peptide or a SmHT variant peptide that has at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence similarity (e.g., conserved or semi-conserved similarity) to one of SEQ ID NO: 578-1187, 3061, 3064-3066, and 3079-3091. In some embodiments, the peptide (e.g., in fusions herein or as a standalone reporter gene or tag, etc.) corresponds to SmHT (SEQ ID NO: 578) but has one or more substitutions relative to one or more of SEQ ID NO: 588-1187 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or a range therebetween). In some implementations, the SmHT variant (e.g., in the fusions described herein or as a standalone reporter gene or tag, etc.) has 1 to 8 (e.g., a range of 1, 2, 3, 4, 5, 6, 7, 8 or between) nonconservative substitutions relative to one of SEQ ID NO: 578-1187.

[0249] In some embodiments, the peptide component of the modified dehalogenase (e.g., in the fusions described herein or as a standalone reporter gene or tag, etc.) corresponds to HT[3-19] (SEQ ID NO: 3061), or has one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or a range thereof) of one or more of SEQ ID NO: 3061, 3064-3066, and 3079-3091. In some embodiments, the HT[3-19] variant (e.g., in the fusions described herein or as a standalone reporter gene or tag, etc.) has 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or a range thereof) nonconservative substitutions relative to one of 3061, 3064-3066, and 3079-3091.

[0250] In some embodiments, this document provides (e.g., in the fusions described herein or as standalone reporter genes or tags, etc.) SmHT peptides or SmHT variant peptides comprising the following: 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) X14 (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 (SEQ ID NO: 3035); Each X is any amino acid (e.g., a protein amino acid).

[0251] In some embodiments, this document provides an LgHT polypeptide or LgHT variant polypeptide (e.g., in the fusions described herein or as a standalone reporter gene or tag, etc.) having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence similarity (e.g., conserved or semi-conserved similarity) to one of SEQ ID NO: 1188-3033. In some embodiments, the polypeptide (e.g., in the fusions described herein or as a standalone reporter gene or tag, etc.) corresponds to LgHT (SEQ ID NO: 1188) but has one or more substitutions relative to one or more of SEQ ID NO: 1188 and SEQ ID NO: 1189-3033 (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 therebetween). In some embodiments, the LgHT variant (e.g., in the fusions described herein or as a standalone reporter gene or tag, etc.) has at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therein) sequence identity with one of SEQ ID NO: 1188-3033. In some embodiments, the LgHT polypeptide or LgHT variant polypeptide comprises a substitution relative to the reference LgHT sequence (SEQ ID NO: 1188), said substitution corresponding to a substitution present in the LgHT portion of one or the construct of SEQ ID NO: 3110-4064.

[0252] In some embodiments, this document provides spHT complementary pairs (e.g., each component of the pair is present in a separate fusion) comprising: (a) a SmHT peptide or a SmHT variant peptide having: (1) at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100% or a range thereof) sequence similarity (e.g., conserved or semi-conserved similarity) to one of SEQ ID NO: 578-1187, 3061, 3064-3066 and 3079-3091; (2) amino acid substitutions relative to one or more of SEQ ID NO: 578 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or a range thereof); and / or (3) relative to SEQ ID NO: 578. (a) One to eight (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or a range thereof) nonconservative substitutions of one of SEQ ID NO: 578-1187, 3061, 3064-3066 and 3079-3091; (b) an LgHT polypeptide or an LgHT variant polypeptide having: (1) at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100% or a range thereof) sequence similarity (e.g., conserved or semi-conserved similarity) to one of SEQ ID NO: 578-1187, 3061, 3064-3066 and 3079-3091; (c) relative to SEQ ID NO: One or more of 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 a range thereof) are substituted; and / or (3) have at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100% or a range thereof) sequence identity with one of SEQ ID NO: 1188-3033.

[0253] The formation of an spHT complex from two complementary fragments can be reversible or irreversible. In some embodiments, the spHT complex is capable of denaturing, refolding, and restoring its activity. In some embodiments, such spHT can be used in methods that expose a sample containing spHT to denaturing conditions (e.g., manufacturing conditions, storage conditions, etc.) prior to substrate binding.

[0254] NanoLuc In some embodiments, this document provides compositions (e.g., fusion peptides and peptides) and systems (e.g., multiple complementary fusion peptides and peptides, substrates, ligands, etc.) comprising peptide / peptide fragments capable of interacting (e.g., promoted or non-promoted) to form an active luminescent protein capable of generating luminescence using a suitable substrate.

[0255] In some embodiments, this document provides compositions (e.g., fusion peptides and peptides) and systems (e.g., multiple complementary fusion peptides and peptides, substrates, ligands, etc.) comprising complementary peptide / peptide fragments capable of interacting (e.g., promoted or non-promoted) to form an active bioluminescent complex capable of emitting light upon interaction with a suitable luminescent substrate. In some embodiments, a first fusion comprising a complementary peptide fragment of a luminescent protein and a second fusion comprising a complementary peptide fragment of a luminescent protein are provided, wherein the complementary peptide and peptide form an active bioluminescent complex upon interaction (e.g., promoted or non-promoted) capable of emitting light upon interaction with a suitable luminescent substrate. In some embodiments, the complementary peptide and peptide are fragments of a cleaved luminescent protein (e.g., luciferase). In alternative embodiments, both fragments can be peptides.

[0256] As a component of the compositions, systems, and methods described herein, this article provides two- or multi-part bioluminescent complexes, such as complexes derived from commercially available NANOLUC protein (Promega) and / or NANOBIT (Promega) or NANOTRIP structural complementary systems.

[0257] natural Small-horned shrimpBoth OgLuc and the commercially available NANOLUC luciferase (Promega Corporation) contain polypeptides comprising 10 β (beta) chains (β1, β2, β3, β4, β5, β6, β7, β8, β9, β10). U.S. Patent No. 9,797,889 (incorporated in its entirety by reference) describes the development and use of complementary systems comprising β1-9-like polypeptides and β10-like peptides (some of the polypeptide and peptide sequences in U.S. Patent No. 9,797,889 based on OgLuc / NANOLUC differ from their counterparts in NANOLUC and wild-type natural OgLuc). Similarly, U.S. Patent Application Serial No. 16 / 439,565 (incorporated in its entirety by reference) describes the development and use of complementary systems comprising two or more OgLuc / NANOLUC peptides and / or polypeptides (some of the OgLuc / NANOLUC-based polypeptides and peptide sequences in U.S. Patent No. 16 / 439,565 differ from the corresponding sequences in NANOLUC and wild-type natural OgLuc).

[0258] In some embodiments, this document provides a peptide component of a binary bioluminescent complex (e.g., in the fusion herein or as a standalone reporter gene or tag, etc.) having a sequence identity greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%, 100%) with SEQ ID NO: 3036, wherein when the peptide component of the binary bioluminescent complex is contacted with a polypeptide composed of SEQ ID NO: 3037 (e.g., in the fusion herein or as a standalone reporter gene or tag, etc.), a detectable bioluminescent signal (e.g., greater luminescence than the component of the complex in the presence of the substrate) is generated in the presence of the bioluminescent complex. In some embodiments, the peptide has a sequence identity less than 100% with SEQ ID NO: 3036. In some embodiments, when the peptide component of the binary bioluminescent complex contacts the polypeptide component of the binary bioluminescent complex, a detectable bioluminescent signal is generated, wherein the polypeptide component has a sequence identity greater than 40% with SEQ ID NO: 3037 (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%, 100%). In some embodiments, when the peptide binds to a polypeptide comprising or composed of SEQ ID NO: 3037, a detectable bioluminescent signal is generated or substantially increased. In a preferred embodiment, when combined with a polypeptide composed of SEQ ID NO: 3037, the peptide exhibits a change (e.g., enhancement) in one or more properties compared to the peptide of SEQ ID NO: 3038 or 3039, wherein said properties are selected from: affinity of the polypeptide composed of SEQ ID NO: 3037; expression, intracellular solubility, intracellular stability, and bioluminescent activity.

[0259] For example, U.S. Patent No. 9,797,889 (incorporated in its entirety by reference) describes exemplary sequences of peptide components of binary bioluminescent complexes that can be used in embodiments herein. While the peptide components of binary bioluminescent complexes herein are not limited to these sequences, in some embodiments, the peptide components of binary bioluminescent complexes herein may be selected from the amino acid sequences of SEQ ID NOs: 3-438 and 2162-2365 of U.S. Patent No. 9,797,889 (incorporated in its entirety by reference).

[0260] In some embodiments, this document provides a peptide component of a binary bioluminescent complex (e.g., in the fusion herein or as a standalone reporter gene or tag, etc.) having a sequence identity greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%, 100%) with SEQ ID NO: 3038, wherein when the peptide component of the binary bioluminescent complex is contacted with a polypeptide composed of SEQ ID NO: 3037 (e.g., in the fusion herein or as a standalone reporter gene or tag, etc.), a detectable bioluminescent signal (e.g., greater luminescence than the component of the complex in the presence of the substrate) is generated in the presence of the bioluminescent complex. In some embodiments, the peptide has a sequence identity less than 100% with SEQ ID NO: 3036. In some embodiments, a detectable bioluminescent signal is generated when the peptide component of the binary bioluminescent complex contacts the polypeptide component of the binary bioluminescent complex, wherein the polypeptide component has a sequence identity greater than 40% with SEQ ID NO: 3037 (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%, 100%). In some embodiments, a detectable bioluminescent signal is generated or substantially increased when the peptide binds to a polypeptide comprising or composed of SEQ ID NO: 3037. In some embodiments, the peptide exhibits a high affinity for the polypeptide of SEQ ID NO: 3037 and is capable of forming a bioluminescent complex without promotion. In a preferred embodiment, when combined with a peptide consisting of SEQ ID NO: 3037, the peptide exhibits a change (e.g., enhancement) in one or more properties compared to the peptide of SEQ ID NO: 3036, wherein said properties are selected from: affinity of the peptide consisting of SEQ ID NO: 3037; expression, intracellular solubility, intracellular stability, and bioluminescent activity.

[0261] In some embodiments, this document provides a peptide component of a binary bioluminescent complex (e.g., in the fusion herein or as a standalone reporter gene or tag, etc.) having a sequence identity greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%, 100%) with SEQ ID NO: 3039, wherein when the peptide component of the binary bioluminescent complex is contacted with a polypeptide composed of SEQ ID NO: 3037 (e.g., in the fusion herein or as a standalone reporter gene or tag, etc.), a detectable bioluminescent signal (e.g., greater luminescence than the component of the complex in the presence of the substrate) is generated in the presence of the bioluminescent complex. In some embodiments, the peptide has a sequence identity less than 100% with SEQ ID NO: 3036. In some embodiments, a detectable bioluminescent signal is generated when the peptide component of the binary bioluminescent complex contacts the polypeptide component of the binary bioluminescent complex, wherein the polypeptide component has a sequence identity greater than 40% with SEQ ID NO: 3037 (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%, 100%). In some embodiments, a detectable bioluminescent signal is generated or substantially increased when the peptide binds to a polypeptide comprising or composed 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 promotion. In a preferred embodiment, when combined with the peptide consisting of SEQ ID NO: 3037, the peptide exhibits a change (e.g., enhancement) in one or more properties compared to the peptide of SEQ ID NO: 3036, wherein said properties are selected from: affinity (or low affinity) of the peptide consisting of SEQ ID NO: 3037; expression, intracellular solubility, intracellular stability, and bioluminescent activity.In some embodiments, this document provides a polypeptide component of a binary bioluminescent complex (e.g., in the fusion herein or as a standalone reporter gene or tag, etc.) having a sequence identity greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%, 100%) with SEQ ID NO: 3037, wherein when the polypeptide is contacted with a peptide composed of SEQ ID NO: 3036 (e.g., in the fusion herein or as a standalone reporter gene or tag, etc.), a detectable bioluminescent signal (e.g., greater luminescence than the component of the complex in the presence of the substrate) is generated in the presence of the bioluminescent complex. In some embodiments, the polypeptide component of the binary bioluminescent complex (e.g., in the fusion herein or as a standalone reporter gene or tag, etc.) has a sequence identity less than 100% with SEQ ID NO: 3037. In some embodiments, a detectable bioluminescent signal is generated when the polypeptide is contacted with a peptide having a sequence identity greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%, 100%) with SEQ ID NO: 3036. In some embodiments, when combined with peptides consisting of SEQ ID NO: 3036, 3038, or 3039, the polypeptide exhibits a change (e.g., enhancement) in one or more properties compared to the peptide of SEQ ID NO: 3037, wherein said properties are selected from: affinity for the peptides consisting of SEQ ID NO: 3036 or 3038; expression, intracellular solubility, intracellular stability, and bioluminescent activity.

[0262] For example, U.S. Patent No. 9,797,889 (incorporated in its entirety by reference) describes exemplary sequences of peptide components of binary bioluminescent complexes that can be used in embodiments herein. While the peptide components of binary bioluminescent complexes herein are not limited to these sequences, in some embodiments, the peptide components of binary bioluminescent complexes herein may be selected from the amino acid sequences of SEQ ID NO: 441-2156 of U.S. Patent No. 9,797,889 (incorporated in its entirety by reference).

[0263] In some embodiments, the present invention provides a bioluminescent complex (formed between the fusions described herein) comprising: (a) a peptide comprising a peptide amino acid sequence having greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%, 100%) sequence identity with SEQ ID NO: 3036, 3038, or 3039; and (b) a polypeptide comprising a peptide amino acid sequence having greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%, 100%) sequence identity with SEQ ID NO: 3037. (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%, 100%) polypeptide amino acid sequences with sequence identity, wherein the bioluminescent complex exhibits detectable luminescence in the presence of the substrate of the bioluminescent complex (e.g., greater luminescence than the components of the complex in the presence of the substrate).

[0264] In some embodiments, this document provides a polypeptide component of a binary bioluminescent complex (e.g., in the fusions described herein or as a standalone reporter gene or tag, etc.) having a sequence identity greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%, 100%) with SEQ ID NO: 3037, wherein when the polypeptide is contacted with a peptide composed of SEQ ID NO: 3036, 3038, or 3039 (e.g., in the fusions described herein or as a standalone reporter gene or tag, etc.), a detectable bioluminescent signal (e.g., greater luminescence than the component of the complex in the presence of the substrate) is generated in the presence of the bioluminescent complex.

[0265] In some embodiments, this document provides (alone and / or in the fusions described herein) components of a bioluminescent complex comprising a first component having 40% or higher (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher (e.g., 100%), or a range therebetween) sequence identity with a first fragment of SEQ ID NO: 3040 or SEQ ID NO: 3041; and one or more complementary fragments collectively comprising a sequence identity with a first fragment of SEQ ID NO: 3040 or SEQ ID NO: 3041. The complementary portion of 3041 has a sequence identity of 40% or higher (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher (e.g., 100%), or in between); wherein the bioluminescent signal generated by the bioluminescent complex assembled from the components in the presence of coelentrin or coelentrin derivative substrates is substantially increased when compared to the bioluminescent signal generated by coelentrin substrates and by a single component alone. In some embodiments, the first component contains 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more (e.g., 100%), or a range thereof) of sequence identity with SEQ ID NO: 3042, and one or more complementary components collectively contain 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more (e.g., 100%), or a range thereof) of sequence identity with SEQ ID NO: 3046. In some embodiments, the first component contains 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more (e.g., 100%), or a range thereof) of sequence identity with SEQ ID NO: 3043, and one or more complementary components collectively contain 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more (e.g., 100%), or a range thereof) of sequence identity with SEQ ID NO: 3047.In some embodiments, the first component contains 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more (e.g., 100%), or a range thereof) of sequence identity with SEQ ID NO: 3044, and one or more complementary components collectively contain 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more (e.g., 100%), or a range thereof) of sequence identity with SEQ ID NO: 3048. In some embodiments, the first component contains 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more (e.g., 100%), or a range thereof) of sequence identity with SEQ ID NO: 3045, and one or more complementary components collectively contain 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more (e.g., 100%), or a range thereof) of sequence identity with SEQ ID NO: 3049. In some embodiments, the first component contains 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more (e.g., 100%), or a range thereof) of sequence identity with SEQ ID NO: 3042, and one or more complementary components collectively contain 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more (e.g., 100%), or a range thereof) of sequence identity with SEQ ID NO: 3050. In some embodiments, the first component contains 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more (e.g., 100%), or a range thereof) of sequence identity with SEQ ID NO: 3043, and one or more complementary components collectively contain 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more (e.g., 100%), or a range thereof) of sequence identity with SEQ ID NO: 3051.In some embodiments, the first component contains 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more (e.g., 100%), or a range thereof) of sequence identity with SEQ ID NO: 3044, and one or more complementary components collectively contain 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more (e.g., 100%), or a range thereof) of sequence identity with SEQ ID NO: 3052. In some embodiments, the first component contains 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more (e.g., 100%), or a range thereof) of sequence identity with SEQ ID NO: 3045, and one or more complementary components collectively contain 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more (e.g., 100%), or a range thereof) of sequence identity with SEQ ID NO: 3053. In some embodiments, when the first component binds to one or more complementary components, the bioluminescent signal substantially increases.

[0266] Exemplary sequences of peptide and polypeptide components of binary or multipart bioluminescent complexes that can be used in embodiments herein are described, for example, in U.S. Application Serial No. 16 / 439,565 (incorporated entirely by reference). Although the peptide and polypeptide components of binary or multipart bioluminescent complexes herein are not limited to these sequences, in some embodiments, the peptide or polypeptide components of binary or multipart bioluminescent complexes herein may be selected from the amino acid sequence of SEQ ID NO: 1-804 of U.S. Application Serial No. 16 / 439,565 (incorporated entirely by reference).

[0267] In some embodiments, this document provides a polypeptide (e.g., in the fusions described herein) comprising 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more (e.g., 100%), or a range thereof) of one of SEQ ID NO: 790, 791, 792 or 793.

[0268] In some embodiments, this document provides peptides (e.g., in the fusions described herein) comprising SEQ ID NO: 3054-3060. In some embodiments, this document provides peptides (e.g., in the fusions described herein) comprising 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more (e.g., 100%), or a range thereof) of one of SEQ ID NO: 3054-3060.

[0269] In some embodiments, this document provides β6-7-like peptides (e.g., in the fusions described herein) comprising SEQ ID NO: 3054 and 3055. In some embodiments, this document provides β6-7-like peptides having a sequence identity of 40% or higher (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher (e.g., 100%), or a range thereof) with SEQ ID NO: 3054 and 3055.

[0270] In some embodiments, this document provides a β7-8-like peptide (e.g., in the fusions described herein) comprising SEQ ID NO: 3055 and 3056. In some embodiments, this document provides a β7-8-like peptide (e.g., in the fusions described herein) having a sequence identity of 40% or higher (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher (e.g., 100%), or a range thereof, with SEQ ID NO: 3055 and 3056.

[0271] In some embodiments, this document provides a β8-9-like peptide (e.g., in the fusions described herein) comprising SEQ ID NO: 3056 / 3059 or 3056 / 3060. In some embodiments, this document provides a β8-9-like peptide (e.g., in the fusions described herein) having a sequence identity of 40% or higher (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher (e.g., 100%), or a range therebetween) with SEQ ID NO: 3056 / 3059 or 3056 / 3060.

[0272] In some embodiments, this document provides a β9-10-like peptide (e.g., in the fusions described herein) comprising SEQ ID NO: 3059 / 3057, 3059 / 3058, 3060 / 3057, or 3060 / 3058. In some embodiments, this document provides a β8-9-like peptide (e.g., in the fusions described herein) having a sequence identity of 40% or higher (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher (e.g., 100%), or a range therebetween) with SEQ ID NO: 3059 / 3057, 3059 / 3058, 3060 / 3057, or 3060 / 3058.

[0273] In some embodiments, this document provides β6-8-like peptides or polypeptides (e.g., in the fusions described herein) comprising SEQ ID NO: 3054-3056. In some embodiments, this document provides β6-8-like peptides or polypeptides (e.g., in the fusions described herein) having a sequence identity of 40% or higher (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher (e.g., 100%), or a range therebetween) with SEQ ID NO: 3054-3056.

[0274] In some embodiments, this document provides a β7-9-like peptide or polypeptide (e.g., in the fusions described herein) comprising SEQ ID NO: 3055 / 3056 / 3059 or 3055 / 3056 / 3060. In some embodiments, this document provides a β7-9-like peptide or polypeptide (e.g., in the fusions described herein) having a sequence identity of 40% or higher (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher (e.g., 100%), or a range thereof, with SEQ ID NO: 3055 / 3056 / 3059 or 3055 / 3056 / 3060.

[0275] In some embodiments, this document provides β8-10-like peptides or polypeptides (e.g., in the fusions described herein) comprising SEQ ID NO: 3056 / 3059 / 3057, 3056 / 3059 / 3058, 3056 / 3060 / 3057, or 3056 / 3060 / 3058. In some embodiments, this document provides a β7-9-like peptide or polypeptide (e.g., in the fusions described herein) having a sequence identity of 40% or higher (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher (e.g., 100%), or a range thereof) with NO:SEQ ID NO: 3056 / 3059 / 3057, 3056 / 3059 / 3058, 3056 / 3060 / 3057 or 3056 / 3060 / 3058.

[0276] Tandem peptide tags In some embodiments, this document provides a tandem peptide tag comprising (i) a peptide component of a bioluminescent complex, said peptide component being fused with (ii) a peptide component of a modified dehalogenase complex. In some embodiments, the peptide component of the bioluminescent complex is capable of interacting (e.g., promoted or non-promoted) with the polypeptide component of the bioluminescent complex to form a bioluminescent complex. In some embodiments, the peptide component of the modified dehalogenase complex is capable of interacting (e.g., promoted or non-promoted) with the polypeptide component of the modified dehalogenase complex to form a modified dehalogenase complex. In some embodiments, the bioluminescent complex and the modified dehalogenase complex are formed after interaction (promoted or non-promoted) between the tandem peptide tag and a tandem polypeptide reporter gene (e.g., comprising a polypeptide component of the bioluminescent complex fused with the polypeptide component of the modified dehalogenase complex). In some embodiments, the bioluminescent complex is capable of generating bioluminescence in the presence of a substrate (e.g., coelentrin, furimazine, etc.), and the modified dehalogenase complex is capable of binding to a haloalkane ligand.

[0277] In some embodiments, the tandem peptide tag herein includes a first component that is a component of a bioluminescent complex, as described herein and throughout U.S. Patent No. 9,797,889 and U.S. Application Serial No. 16 / 439,565, both of which are incorporated herein by reference in their entirety. Any component of the bioluminescent complex described herein or incorporated herein by reference may be used in the tandem peptide tag described herein. In a particular embodiment, the tandem peptide tag herein comprises a peptide component of a bioluminescent complex.

[0278] 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 Application Nos. 63 / 338,323 and 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 herein by reference may be used in the tandem peptide tag described herein. In a particular embodiment, the tandem peptide tag herein comprises a peptide component of a modified dehalogenase complex.

[0279] In some embodiments, a tandem peptide tag comprising a component of a bioluminescent complex and a component of a modified dehalogenase complex is fused to or otherwise linked to a target peptide or protein. In some embodiments, the tandem peptide tag is expressed as a fusion with the target peptide or protein. In some embodiments, the target peptide or protein is a cellular target that will be detected, quantified, or otherwise characterized using the systems and methods described herein.

[0280] In some embodiments, the tandem peptide tag herein comprises at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity with 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 comprises one or more substitutions of the reference SmHT peptide relative to any SmHT variant peptide herein and / or the SmHT moiety of one or more of 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 of a reference HiBiT peptide relative to any HiBiT variant peptide herein and / or one or more of the HiBiT portions of SEQ ID NO: 3062-3063, 3067-3078, 3092, 3094-3109 and 4177-4181.

[0281] Tandem polypeptide reporter gene In some embodiments, this document provides a tandem polypeptide reporter gene comprising a polypeptide component of a bioluminescent complex fused with a polypeptide component of a modified dehalogenase complex. In some embodiments, the polypeptide component of the bioluminescent complex is capable of interacting (e.g., promoted or non-promoted) with the peptide component of the bioluminescent complex to form the bioluminescent complex. In some embodiments, the polypeptide component of the modified dehalogenase complex is capable of interacting (e.g., promoted or non-promoted) with the peptide component of the modified dehalogenase complex to form the modified dehalogenase complex. In some embodiments, the bioluminescent complex and the modified dehalogenase complex are formed after interaction (promoted or non-promoted) between the tandem polypeptide reporter gene and the tandem peptide tag (e.g., comprising a peptide component of the bioluminescent complex fused with the peptide component of the modified dehalogenase complex). In some embodiments, the bioluminescent complex can produce bioluminescence in the presence of a substrate (e.g., coelentrin, furimazine, etc.), and the modified dehalogenase complex is capable of binding to a haloalkane ligand.

[0282] In some embodiments, the tandem peptide reporter gene described herein includes a first component that is a component of a bioluminescent complex, as described herein and throughout U.S. Patent No. 9,797,889 and U.S. Application Serial No. 16 / 439,565, both of which are incorporated herein by reference in their entirety. Any component of the bioluminescent complex described herein or incorporated herein by reference may be used in the tandem peptide reporter gene described herein. In a particular embodiment, the tandem peptide reporter gene described herein comprises a peptide component of a bioluminescent complex.

[0283] In some embodiments, the tandem peptide reporter gene described herein includes a second component, which is a component of a modified dehalogenase complex, as described herein and throughout U.S. Provisional Application Nos. 63 / 338,323 and 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 herein by reference may be used in the tandem peptide reporter gene described herein. In a specific embodiment, the tandem peptide reporter gene described herein comprises a peptide component of a modified dehalogenase complex.

[0284] In some embodiments, the tandem polypeptide reporter gene described herein comprises at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity with one or more of SEQ ID NO: 3110-4064. In some embodiments, the polypeptide component of the modified dehalogenase moiety (e.g., LgHT) of the tandem polypeptide reporter gene described herein comprises one or more substitutions of a reference LgHT peptide relative to any LgHT variant peptide described herein and / or the LgHT moiety of one or more of SEQ ID NO: 3110-4064. In some embodiments, the polypeptide component of the bioluminescent complex moiety (e.g., HiBiT) of the tandem polypeptide reporter gene described herein comprises one or more substitutions of a reference HiBiT peptide relative to any HiBiT variant peptide described herein and / or the HiBiT moiety of one or more of SEQ ID NO: 3110-4064.

[0285] Other tandem tags and reporter genes The embodiments described herein primarily describe tags comprising a fusion peptide component of a bioluminescent and modified dehalogenase complex, and a reporter gene comprising a fusion peptide component of the complex. However, embodiments within the scope of this document also include tags (e.g., for linkage / fusion with a target) comprising: a peptide component of a bioluminescent and modified dehalogenase complex, a peptide component of a bioluminescent complex and a peptide component of a modified dehalogenase complex, or a peptide component of a bioluminescent complex and a peptide component of a modified dehalogenase complex; and a reporter gene comprising a peptide component of a bioluminescent and modified dehalogenase complex, a peptide component of a bioluminescent complex and a peptide component of a modified dehalogenase complex, or a peptide component of a bioluminescent complex and a peptide component of a modified dehalogenase complex. Any suitable arrangement of the components described herein is within the scope.

[0286] connector This document describes various fusions. In some embodiments, the components of the fusion are directly linked (e.g., C-terminus to N-terminus). In other embodiments, the fusions described herein include peptide or polypeptide linkers between the components. Such linkers can have any suitable sequence and are up to 100 amino acids in length (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 therebetween). In some embodiments, the tandem peptide tag described herein includes a linker sequence between a peptide component of a modified dehalogenase complex and a peptide component of a bioluminescent complex. In some embodiments, the tandem peptide tag is linked to a target element via a suitable linker sequence. In some embodiments, the tandem detector described herein includes a linker sequence between a peptide component of a modified dehalogenase complex and a peptide component of a bioluminescent complex. Exemplary linker sequences in tandem tags and tandem detectors are illustrated herein, but other linker sequences are also within the scope of this document. Any peptide / peptide sequence that can link the components of the constructs described herein is within the scope of this document, including but not limited to the sequences specifically illustrated herein, both in terms of length and amino acid composition.

[0287] Halogenated alkyl ligands As described herein, the spHT system (e.g., within the fusions described herein) utilizes a haloalkane ligand. In some embodiments, the ligand has the formula (I): R-linker-AX, where R is a solid surface, one or more functional groups, or is absent, where the linker is a polyatomic straight or branched chain comprising C, N, S, or O, or a group containing one or more rings, such as saturated or unsaturated rings, such as one or more aromatic rings, heteroaromatic rings, or any combination thereof, where AX is a ligand for the dehalogenase, hydrolase, HALOTAG, or spHT system described herein (e.g., where A is (CH2)). 4-20 X is a halide (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, all of which are incorporated herein by reference in their entirety. In some embodiments, X in formula (I) is a methanesulfonamide or trifluoromethanesulfonamide, rather than a halide; such embodiments yield exchangeable 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, all of which are incorporated herein by reference in their entirety.

[0288] In some embodiments, R is one or more functional groups (such as fluorophores, biotin, luminescent groups, or fluorescent or luminescent molecules). Exemplary functional groups used in this invention include, but are not limited to: amino acids, proteins (e.g., enzymes, antibodies, or other immunogenic proteins), radionuclides, nucleic acid molecules, drugs, lipids, biotin, avidin, streptavidin, magnetic beads, solid carriers, electronically opaque molecules, chromophores, MRI contrast agents, dyes (e.g., xanthones), calcium-sensitive dyes (e.g., 1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxy-9-xanthonyl)-phenoxy]-2-( 2'-Amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid (Fluo-3)), sodium-sensitive dyes (e.g., 1,3-phthalic acid, 4,4'-[1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diylbis(5-methoxy-6,2-benzofurandiyl)]bis(PBFI)), NO-sensitive dyes (e.g., 4-amino-5-methylamino-2',7'-difluorescein) or other fluorophores. In one embodiment, the functional group is an immunogenic molecule, i.e., a molecule that binds to a specific antibody against that molecule.

[0289] In some embodiments, the ligands of the present invention can penetrate into the cell membrane (i.e., can enter the cell from the outside of the cell (e.g., eukaryotic cells, prokaryotic cells) without chemical, enzymatic or mechanical disruption of the cell membrane).

[0290] In some embodiments, the ligands herein comprise a cleavable connector, such as the connector described in U.S. Patent No. 10,618,907, which is incorporated herein by reference in its entirety.

[0291] In some implementations, the ligand comprises a fluorescent functional group (R). Suitable fluorescent functional groups include, but are not limited to, stilbazolium derivatives (Marquesa et al., Mechanism-Based Strategy for Optimizing HaloTag Protein Labeling. ChemRxiv. Cambridge: Cambridge OpenEngage; 2021; incorporated by reference), xanthones (e.g., fluorescein, rhodamine, Oregon Green, eosin, Texas Red, etc.), cyanine derivatives (e.g., cyanine, indolecarbazine, oxacarbazine, thiacarbazine, benzocyanine, etc.), naphthalene derivatives (e.g., dansyl and prodan derivatives), oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, etc.), pyrene derivatives (e.g., cascade blue), oxazine derivatives (e.g., Nile Red, Nile Blue, cresol violet, oxazine 170, etc.), acridine derivatives (e.g., proflavin, acridine orange, acridine yellow, etc.), arylmethyl derivatives (e.g., auramine, crystal violet, malachite green, etc.), tetrapyrrole derivatives (e.g., porphyrin, 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 dye (SETA BioMedicals), QUASAR and CAL FLUOR dye (Biosearch Technologies), SURELIGHT dye (APC, RPE, PerCP, phycobilisomes) (Columbia Biosciences), APC, APCXL, RPE, BPE (Phyco-Biotech), autofluorescent proteins (e.g., YFP, RFP, mCherry, mKate), quantum dot nanocrystals, etc.

[0292] In some embodiments, the ligand comprises a fluorescent functional group (R). A fluorescent functional group is a functional group that produces an enhanced fluorescence signal when the ligand binds to a target (e.g., a haloalkane binds to a modified dehalogenase). Background signal problems can be mitigated by generating a significantly increased fluorescence (e.g., 10-fold, 31-fold, 50-fold, 100-fold, 310-fold, 500-fold, 1000-fold, or more) upon target engagement. Exemplary fluorescent dyes that can be used in the embodiments described herein include the JANELIA FLUOR family of fluorophores, such as: Janelia Fluor® (JF) 549 Janelia Fluor® (JF) 646 Janelia Fluor® (JFX) 554 Janelia Fluor® (JFX) 650 Janelia Fluor® (JF) 503 Janelia Fluor® (JF) 585 Janelia Fluor® (JF) 635 Janelia Fluor® (JF) 552 Janelia Fluor® (JF) 608 Janelia Fluor® (JF) 669 Janelia Fluor® (JFX) 673 Janelia Fluor® (JF) 525 PA Janelia Fluor® (JF) 646, and PA Janelia Fluor® (JF) 549, JF549i, and JF635i (See, for example, U.S. Patent Nos. 9,933,417; 10,018,624; 10,161,932; and 10,495,632; each of these documents is incorporated herein by reference in its entirety.) In some embodiments, exemplary conjugates of JANELIA FLUOR 549 and JANELIA FLUOR 646 with a haloalkane ligand (e.g., HALOTAG) modified with a dehalogenase 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.; Wang et al., Nat Chem. 3120 Feb; 12(2):165-172; all of which are incorporated herein by reference in their entirety.

[0293] In some embodiments, the fluorophore or fluorescent dye described herein is rhodamine and / or p-aminophenol (rhodol) dye.

[0294] In some implementations, the fluorophore (R) has the following structure: Where Y is C, O, or Si, and if Y is C or Si, it is replaced by two CH3 groups; where R 1 R 2 R 3 R 4 and R 5 Each is independently H or F (e.g., R) 1 -R 5 Both are H; R 1 -R 5 Both are F; R 1 and R 2 For F and R 3 For H; R 1 -R 3 For F and R 4 -R 5 For H; R 1 and R 2 For F and R 3 -R 5 For H, etc.). In some embodiments, the aziridine is further substituented at the 3-position by one or two non-hydrogen substituents (e.g., CO). 2 (H, CH3, F, etc.) substitution. In some embodiments, the exemplary compounds described herein have the following structures: (where R) 1-5 As defined above, Y and aziridine are optionally substituted as described above, and alternative linkers and AX groups are also included.

[0295] In some implementations, the fluorophore (R) has the following structure: Where Y is C, O, or Si, and if Y is C or Si, it is replaced by two CH3 groups; where R 1 R 2 R 3 R 4 and R 5 Each is independently H or F (e.g., R) 1 -R 5 Both are H; R 1 -R 5 Both are F; R 1 and R 2 For F and R 3 For H; R 1 -R 3 For F and R 4 -R 5 For H; R 1 and R 2 For F and R 3 -R 5 (For H, etc.); and each of them It contains either azahexacyclobutane or per-deuterated pyrrolidine. In some embodiments, the azahexacyclobutane, when present, is further substituented at the 3-position by one or two non-hydrogen groups (e.g., CO). 2 (H, CH3, F, etc.) substitution. In some embodiments, the exemplary compounds described herein have the following structures: ;where R 1-5 Y and As defined above, Y and aziridine may optionally be substituted as described above, and alternative linkers and AX groups are also included.

[0296] In some embodiments, the linker is a polyatomic straight or branched chain comprising C, N, S, or O, or a group containing one or more rings, such as saturated or unsaturated rings, such as one or more aromatic rings, heteroaromatic rings, or any combination thereof. In some embodiments, the linker comprises 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 a range therebetween). In some embodiments, the length of the linker used to tether the haloalkane to R can be optimized for proximity and geometry (e.g., for binding of the modified dehalogenase complex to the haloalkane, for the function of the functional part (e.g., fluorescence), etc.). The scope of the embodiments described herein is not limited to the types of linkers available. The fluorophore and AX can be directly linked (e.g., the linker consists of a single covalent bond) or linked via a suitable linker. Implementations are not limited to any specific linker group. A variety of linker groups are considered, and suitable linker groups may include, but are not limited to, alkyl, methylene carbon chains, ethers, polyethers, alkylamide linkers, peptide linkers, modified peptide linkers, polyethylene glycol (PEG) linkers, streptavidin-biotin or avidin-biotin linkers, polyamino acids (e.g., polylysine), functionalized PEG, polysaccharides, glycosaminoglycans, dendritic polymers (WO93 / 06868 and Tomalia et al. Angew. Chem. Int. Ed. Engl. 29:138-175 (1990), which are incorporated herein by reference in their entirety), PEG-chelate polymers (W94 / 08629, WO94 / 09056 and WO96 / 26754, which are incorporated herein by reference in their entirety), oligonucleotide linkers, phospholipid derivatives, alkenyl chains, alkynyl chains, disulfide bonds, or combinations thereof. In some embodiments, the linker is cleavable (e.g., enzymatic cleavage (e.g., TEV protease site), chemical cleavage, photoinduced cleavage, etc.). In some embodiments, the cleavable linker comprises an allyl heteroatom group or an acetylacetyl heteroatom group, such as the linker described, for example, in U.S. Application No. 16 / 813,295; the entire document is incorporated herein by reference.

[0297] The haloalkyl ligands described herein include haloalkyl groups, linkers, and functional groups. Exemplary compounds comprising rhodamine dyes linked to haloalkyl groups include: , , , , , , , , , , , , , and Other ligands containing different halogens (e.g., Br), A groups of different lengths, and R groups (e.g., biotin, other fluorophores, etc.) are within the scope of this document. Other non-limiting examples of haloalkyl ligands that can be used in the embodiments described herein include: , , , , , , , , , , , , , , , , , , , , (Among them, substitute R, linker groups and AX groups are also included).

[0298] Bioluminescent substrates In some embodiments, the system described herein (comprising a bioluminescent complex and / or a component thereof) utilizes an imidazopyrazine luminescent substrate to generate bioluminescence. In some embodiments, the substrate is coelenterate: .

[0299] In some embodiments, the substrate is a coelentin derivative, such as furimazine, furimazine analogs (e.g., fluorofurimazine), coelentin-n, coelentin-f, coelentin-h, coelentin-hcp, coelentin-cp, coelentin-c, coelentin-e, coelentin-fcp, dideoxycoelentin (“coelentin-hh”), coelentin-i, coelentin-icp, coelentin-v, and 2-methylcoelentin, as well as substrates disclosed in WO 2003 / 040100; U.S. Application Serial No. 12 / 056,073 (paragraph

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

[0300] In some implementations, the substrate is furimazine: .

[0301] In some implementations, the substrate is fluorofurimazine: .

[0302] Other reporter peptides In some embodiments, the bioluminescent and / or modified dehalogenase complex is a three- or multi-part complex (e.g., containing three or more peptide / peptide components). In such embodiments, the first component of the complex may be present in a tandem peptide tag, the second component of the complex may be present in a tandem peptide reporter gene, and the third (or fourth or more) component of the complex may be present as an additional reporter gene. In some embodiments, the additional reporter gene, the tandem peptide reporter gene, and the tandem peptide tag (promoted or non-promoted) interact to result in the formation of the modified dehalogenase and bioluminescent complex.

[0303] Additional reporter genes can exist as fusions with one or more additional elements or amino acid sequences (e.g., interacting elements, target proteins, etc.). In some embodiments, the additional amino acid sequence is selected from the group consisting of target proteins, interacting elements, co-localization elements, and binding moieties. In some embodiments, the additional amino acid sequence is a binding moieties selected from the group consisting of: antibodies (polyclonal, monoclonal, and / or recombinant antibodies), antibody fragments, protein A, the Ig-binding domain of protein A, protein G, the Ig-binding domain of protein G, protein A / G, the Ig-binding domain of protein A / G, protein L, the Ig-binding domain of protein L, protein M, the Ig-binding domain of protein M, peptide nucleic acids, DARPin, affimer, purified proteins (either the analyte itself or a protein to which the analyte is bound), and the analyte-binding domain of proteins. In some embodiments, the additional amino acid sequence is a first interacting polypeptide configured to form a complex with a second interacting polypeptide upon contact between the first and second interacting polypeptides. In some embodiments, the additional amino acid sequence is a first colocalizing polypeptide configured to colocalize with a second colocalizing polypeptide in a cellular compartment, cell, tissue, or organism. In some embodiments, the additional amino acid sequence is a target protein and a candidate drug target. In some embodiments, the additional amino acid sequence is a fluorescent protein (e.g., GFP and its variants), a luciferase (e.g., firefly or sea kidney), SPYTAG, SPYCATCHER, SNAP tag, CLIP tag, etc.

[0304] System Description In some embodiments, systems comprising bioluminescent and modified dehalogenase complexes and their components are provided. In some embodiments, protein complementation systems (and their components) are provided herein, which are capable of luminescent and fluorescence detection based on a combination of split NanoLuc® and split HaloTag® technologies. Certain embodiments are described in combination with split NANOLUC-based systems and components (e.g., NanoBiT, NanoTrip, smBiT, HiBiT, LgBiT, etc., and variants thereof) and split HALOTAG-based systems and components (e.g., spHT, SmHT, LgHT, etc., and variants thereof). Experiments have been performed to tandemly combine two small peptide tags from each system (e.g., SmHT-HiBiT), demonstrating that the tandem peptide tags can promote complementarity with both technologies, either individually or simultaneously, by adding their homologous large sequences LgHT or LgBiT. Furthermore, experiments have demonstrated the fusion of reporter peptides into a single tandem reporter peptide (e.g., LgHT-LgBiT). The advantage of the LgHT-LgBiT reporter peptide is that the high affinity of the HiBiT:LgBiT interaction promotes SmHT:LgHT complementarity in the complex, thereby providing the functionality of the high affinity / spontaneous division HaloTag® system.

[0305] In some embodiments, the tag and reporter gene described herein are not limited to combinations of peptide and polypeptide components. For example, a peptide component from a NanoLuc®-based complementation system can be fused with a polypeptide component from a HaloTag®-based complementation system to achieve complementarity between the NanoLuc®-based polypeptide component and the HaloTag®-based peptide component fusion. Furthermore, various components can be combined in different configurations or orientations, such as SmHT-LgBiT and HiBiT-LgHT. In some embodiments, the fusions described herein include linkers to facilitate optimized geometries for complementarity, ligand / substrate binding, etc. In some embodiments, the sequences of the HaloTag®-based component and the NanoLuc®-based component are not limited to commercially or publicly available sequences from HaloTag, NanoLuc, or their binary multipart complementation systems.

[0306] In some embodiments described herein, a peptide component of a bioluminescent complex is fused with a peptide component of a modified dehalogenase complex to form a tandem peptide tag. In some systems, methods, assays, etc., this fusion configuration offers certain advantages, where two peptide components are fused to form a tandem tag, and two peptide components are fused to form a tandem reporter gene. The small tag fusion remains a peptide-sized sequence (e.g., 28 amino acids (17 amino acids SmHT + 11 amino acids HiBiT)). This is still within the size range (84 nucleotides), facilitating synthesis as a single-stranded donor DNA oligonucleotide and efficient delivery during CRISPR genome engineering to introduce the tandem tag onto endogenous protein targets. In some embodiments, the small tag minimizes interference with the target protein fusion partner, which is also a potential benefit. Exemplary configurations of this tandem peptide tag include different versions, including SmBiT, HiBiT, and other sequence variants that alter their affinity. There are hundreds of sequence variants of SmHT, all of which maintain a low affinity for LgHT and are functional in complementary systems. In some embodiments, any SmHT variant having the properties desired for in vivo and in vitro use (such as altered charge and hydrophobicity or chemically modifiable cysteine / arginine residues) can be used alone or in tandem with SmBiT / HiBiT (or variants thereof) in the context of an SmHT-HiBiT fusion tag to maintain similar physical properties along the length of the peptide tag. In some embodiments, it is advantageous to introduce a linker between the SmHT and SmBiT / LgBiT in the fusion tag or to change their orientation in order to minimize any potential spatial limitations on complementarity, orientation, or flexibility. Alternative orientations include using each tag at different ends (N- or C-) of the target protein, for example, in proteins with closely adjacent ends, whereby the LgHT-LgBiT detector peptide can still form complementary complexes with both tags simultaneously.

[0307] In some implementations, users may only want to use one technology while another is not used / does not interfere. For example, in some implementations, the fluorescent component of this technology allows for fluorescent cell sorting after CRISPR editing, resulting in more efficient / faster separation of tagged cells, after which the user may be primarily interested in downstream measurements of intracellular biological events using the tagged HiBiT portion. Another example is the concept that high-affinity LgBiT:HiBiT interactions spontaneously promote SmHT:LgHT interactions in complementary complexes, thereby effectively creating a novel high-affinity version of split HaloTag. In this case, if the user only desires a spontaneously completed split HaloTag® system for targeting tagged protein targets with chloroalkanes, then no luminescent technology is used except for assembling the split HaloTag® fragments together. In another implementation, the HiBiT component of the tandem peptide tag will be detected with LgBiT alone (i.e., not fused with LgHT). In the event of an overabundance of LgBiT-LgHT detectors in cells, it may amplify the signal. This can happen if a molecule in the detector binds to the dipeptide tag and forms a complex, and the LgHT sequence is fluorescently labeled but subsequently dissociates from the dipeptide tag. This allows the previously unlabeled LgBiT-LgHT to bind to the dipeptide tag and become labeled.

[0308] In some embodiments of this technology utilizing LgHT-LgBiT peptides or variants thereof, LgHT-LgBiT fusions are primarily conceived as configurations that fuse two proteins at their ends, as this fusion imparts multiple functionalities for the detection and manipulation of tagged target proteins. In some embodiments, the properties and performance of LgHT-LgBiT fusions can be further optimized by modifying the linker between them and their relative orientation (LgHT-LgBiT vs. LgBiT-LgHT). Other potential configurations include circular arrangement of one or both proteins in the fusion, or insertion of one sequence / arranged sequence into another. The latter is similar to our work creating “chimeras” between HaloTag and NanoLuc / LgBiT / LgTrip, where the luminescent protein is inserted into HaloTag, providing the advantages of close proximity and specific geometry between their respective substrate binding sites, resulting in exceptionally efficient energy transfer for BRET. The “split chimera” version of LgHT-LgBiT can be configured similarly, potentially providing the correct orientation so that it still forms a complementary complex with the SmHT-HiBiT peptide tag.

[0309] Some advantages of this invention in configuring LgHT-LgBiT (or variants thereof) into fusion peptides include its introduction as a single detection reagent, compatibility with all NanoLuc® and HaloTag® substrates / ligands, sequential or simultaneous luminescence / fluorescence measurements, and a variety of different measurements (quantification, interaction, localization, etc.) of tagged targets. As a single detection reagent, the LgHT-LgBiT fusion peptide (or variants thereof) can be delivered as a purified protein to assays or cells, encoded by plasmids or mRNA, or uniformly expressed from chromosomes in stable or CRISPR cell lines. As a purified protein, it can be fused or conjugated to a solid support / bead / surface or other protein (such as an antibody). The latter is advantageous for biochemical or immunoassays, where LgHT-LgBiT (or variants thereof) will be conjugated with an antibody and configured as a detection reagent that relies on the recognition of an antibody target in vitro or in diagnostic form.

[0310] The LgHT-LgBiT peptide (or its variants) is compatible with all NanoLuc® substrates and HaloTag® ligands, offering exceptional versatility in detection modality (luminescent or fluorescent), emission wavelength (UV to IR), and functionality (non-fluorescent HaloTag® ligands). One example application is in animal models, such as mice expressing the LgHT-LgBiT detection peptide (or its variants), where the introduction of protein targets tagged with SmHT-HiBiT (e.g., via viral infection, where small tags encoded on their genomes are advantageous) enables sensitive detection with low background luminescence, allowing for the quantification of target protein abundance, while the far-red, brain-penetrating fluorescent HaloTag® ligand enables sophisticated neuronal imaging at another wavelength (with the potential for BRET). This example highlights the trade-offs researchers are currently making in animal imaging, where one detection technique offers some, but not all, benefits, and the implementation described in this paper addresses these issues, enabling both to be achieved in a single model system without changing the tag or reengineering the cells / animal / host. For animal imaging, existing research has demonstrated the wide applicability of targeted PET, SPECT, sensors, and MRI. Each of these can be combined with tandem peptide-tagged bioluminescence imaging.

[0311] In some embodiments, the system described herein can be used with non-fluorescent HaloTag® ligands. For example, biotinylated chloroalkyl ligands enable the capture of dual-tagged target proteins. Another application is targeting molecules to induce protein proximity, with particularly relevant examples being the targeting of chimeric molecules (PROTAC, LyTAC, PhosTAC, AuTAC, etc.) and molecular gels. Because these molecules can be configured as chloroalkyl (i.e., HaloPROTAC), they provide specific use cases where the invention is novel. For example, the abundance of SmHT-HiBiT-tagged target proteins and the luminescent activity of an LgHT-LgBiT detector can be monitored in real time, and then the binding of HaloPROTAC to the complex via the SmHT:LgHT interaction initiates the recruitment of an E3 ligase, leading to proteasome ubiquitination and degradation, and consequently a decrease in the real-time luminescent signal. This highlights the simultaneous use of splitting NanoLuc® and splitting HaloTag® functions, which are currently not achievable using either function alone. HaloTag®-HiBiT tags can be used for this purpose; however, there are instances where using full-length HaloTags as fusion tags increases target protein expression and stability, which is particularly detrimental under conditions of target stability / degradation. Furthermore, it necessitates adding significantly larger HaloTag®-HiBiT tags to the target protein. Therefore, SmHT-HiBiT, unique to this invention, can manipulate the protein target with minimal interference while complementing the LgHT-LgBiT detector.

[0312] In some implementations, the techniques described herein, particularly the fluorescent output component, provide a more efficient workflow for engineered HiBiT-tagged endogenous gene cell lines through cell sorting. The ability to high-throughput sort positive cells (targeted tagged genes) can significantly reduce the time and effort required for manual screening of positive pools or clones. This can have a particularly significant impact when working with targets that are difficult to modify using CRISPR editing, or when working with cells that are difficult to propagate (such as primary cells). Fluorescent ligands can be used to screen cells or cell pools via fluorescently activated cell sorting, and then, once clones or clonal pools are identified, the same or different ligands can be used for actual imaging experiments on the cells. In some implementations, the system described herein can be used in conjunction with a variety of surface display techniques.

[0313] In some embodiments, biomolecules (such as proteins / DNA / RNA) are immobilized on the surface of cells, beads, or other surfaces via fusion with a DualTag or Dual Detector, leveraging the system's high affinity and extremely low dissociation rate. In some embodiments, cells or microbeads are shown as platforms for high-throughput capture or measurement methods (such as magnetic bead sorting or flow cytometry) to measure the binding or modification of a target fusion analyte. Examples of target analytes include a variety of biomolecules (such as protein variants, adaptor variants, small molecules, nucleic acids, or binding proteins). Such embodiments also allow for the simultaneous use of additional reporter genes, dyes, tags, or markers on the cell or bead surface to enable multiplex detection of signals on the surface, such as in the case of protein:protein interactions between two tagged / labeled substances. A distinguishing feature of the system described herein is not due to the high affinity interaction and slow dissociation of the tandem peptide-tagged detector, but also to the ability to facilitate fluorescence detection by labeling with fluorescent HaloTag® ligands, where the fluorescence signal is particularly useful for measurements using high-speed cell or bead cytometry or sorting applications.

[0314] In some implementations, the ability of the system described herein to facilitate cell surface display can be used for bioassays, for example, for monitoring cell surface receptors. Cell surface receptors, such as GPCRs, are valuable drug targets and are widely used to manipulate cell behavior and physiology. These receptors can be genetically modified to express tandem peptide tags linked to the present invention. The tandem peptide tags of the present invention are small in size and have minimal perturbation expected, enabling the display of the tandem tags external to the cell surface or internal to the cytoplasm. When combined with the tandem detector of the present i...

Claims

1. A dual reporter gene system comprising: (a) A tandem peptide tag comprising (i) a peptide component of a bioluminescent complex fused with (ii) a peptide component of a modified dehalogenase complex; as well as (b) A tandem polypeptide reporter gene comprising (i) a polypeptide component of a bioluminescent complex and (ii) a polypeptide component of a modified dehalogenase complex; The peptide and polypeptide components of the bioluminescent complex are capable of interacting to form the bioluminescent complex, and the bioluminescent complex is capable of generating bioluminescence in the presence of its substrate. and The peptide and polypeptide components of the modified dehalogenase complex are capable of interacting to form the modified dehalogenase complex, and the modified dehalogenase complex is capable of forming covalent bonds with a haloalkyl ligand.

2. The system of claim 1, wherein the tandem peptide reporter gene exhibits high affinity for the tandem peptide tag.

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

4. The system of claim 3, wherein the high affinity is K. d Less than 1 µM.

5. The system of claim 4, wherein the high affinity is K. d Less than 1 nM.

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

7. The system of claim 6, wherein the peptide component of the bioluminescent complex is 100% identical to SEQ ID NO:3038.

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

9. The system of claim 8, wherein the polypeptide component of the bioluminescent complex is 100% identical to SEQ ID NO:3037.

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

11. The system of claim 10, wherein the peptide component of the modified dehalogenase complex is 100% identical to SEQ ID NO: 3034.

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

13. The system of claim 12, wherein the polypeptide component of the modified dehalogenase complex is 100% identical to SEQ ID NO: 1188.

14. The system of claim 1, wherein the tandem peptide tag has at least 70% identity with one of SEQ ID NO: 3062-3063, 3067-3078, 3092, 3094-3109 or 4177-4181.

15. The system of claim 14, wherein the tandem peptide tag is 100% identical to one of SEQ ID NO: 3062-3063, 3067-3078, 3092, 3094-3109 or 4177-4181.

16. The system of claim 1, wherein the tandem polypeptide reporter gene has at least 70% identity with one of SEQ ID NO: 3110-4064.

17. The system of claim 16, wherein the tandem polypeptide reporter gene has 100% identity with one of SEQ ID NO: 3110-4064.

18. The system of claim 1, further comprising the substrate of the bioluminescent complex.

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

20. The system of claim 19, wherein the haloalkyl ligand comprises a haloalkyl moiety attached to a fluorophore.

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

22. The system of claim 1, wherein the tandem peptide tag is attached to the target element.

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

24. A tandem peptide tag comprising a peptide component of a bioluminescent complex fused with a peptide component of a modified dehalogenase complex; The peptide component of the bioluminescent complex is capable of interacting with the polypeptide component of the bioluminescent complex to form the bioluminescent complex, and the bioluminescent complex is capable of generating bioluminescence in the presence of the substrate of the bioluminescent complex. and The peptide component of the modified dehalogenase complex is capable of interacting with the polypeptide component of the modified dehalogenase complex to form the modified dehalogenase complex, and the modified dehalogenase complex is capable of forming a covalent bond with a haloalkyl ligand.

25. The tandem peptide tag of claim 24, wherein the tandem peptide tag has at least 70% identity with one of SEQ ID NO: 3062-3063, 3067-3078, 3092, 3094-3109 or 4177-4181.

26. The tandem peptide tag of claim 24, wherein the tandem peptide tag is 100% identical to one of SEQ ID NO: 3062-3063, 3067-3078, 3092, 3094-3109 or 4177-4181.

27. The tandem peptide tag of 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 of claim 27, wherein the peptide component of the bioluminescent complex is 100% identical to that of SEQ ID NO: 3038.

29. The tandem peptide tag of 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 of 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 as described in any one of claims 24-30.

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

33. A polynucleotide encoding a fusion polypeptide as described in claim 30 or 31.

34. An expression vector comprising the polynucleotide as described in claim 33.

35. A system comprising a tandem peptide tag as described in any one of claims 24-30 or a fusion polypeptide as described in claim 30 or 31, and the polypeptide component of the bioluminescent complex.

36. The system of claim 35, further comprising a substrate of the bioluminescent complex.

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

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

39. The system of 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 gene polypeptides.

40. The system of 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 a tandem polypeptide reporter gene.

41. A system comprising the tandem peptide tag as described in claim 24, and the polypeptide component of the modified dehalogenase complex.

42. The system of claim 41, further comprising a substrate of the bioluminescent complex.

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

44. A tandem polypeptide reporter gene comprising a polypeptide component of the bioluminescent complex and a polypeptide component of the modified dehalogenase complex; The polypeptide component of the bioluminescent complex is capable of interacting with the peptide component of the bioluminescent complex to form the bioluminescent complex, and the bioluminescent complex is capable of generating bioluminescence in the presence of the substrate of the bioluminescent complex. and The polypeptide component of the modified dehalogenase complex is capable of interacting with the peptide component of the modified dehalogenase complex to form the modified dehalogenase complex, and the modified dehalogenase complex is capable of forming a covalent bond with a haloalkyl ligand.

45. The tandem polypeptide reporter gene of claim 44, wherein the tandem polypeptide reporter gene has at least 70% identity with one of SEQ ID NO: 3110-4064.

46. ​​The tandem polypeptide reporter gene of claim 46, wherein the tandem polypeptide reporter gene has at least 100% identity with one of SEQ ID NO: 3110-4064.

47. The tandem polypeptide reporter gene of 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 gene of claim 47, wherein the polypeptide component of the modified dehalogenase complex is 100% identical to SEQ ID NO: 1188.

49. The tandem polypeptide reporter gene of 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 gene of claim 49, wherein the polypeptide component of the bioluminescent complex is 100% identical to that of SEQ ID NO: 3037.

51. A system comprising the tandem peptide reporter gene as described in claim 44, and the peptide component of the bioluminescent complex.

52. The system of claim 51, further comprising a substrate of the bioluminescent complex.

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

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

55. The system of 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 gene peptides.

56. The system of 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 a tandem polypeptide reporter gene.

57. A system comprising the tandem polypeptide reporter gene as described in claim 44, and the peptide component of the modified dehalogenase complex.

58. The system of claim 57, further comprising a substrate of the bioluminescent complex.

59. A polynucleotide encoding a tandem polypeptide reporter gene as described in claim 44.

60. An expression vector comprising the polynucleotide as described in claim 59.

61. A method for detecting a target component in a system, the method comprising: (a) Linking the target component to a tandem peptide tag, the tandem peptide tag comprising a peptide component of a bioluminescent complex fused with a peptide component of a modified dehalogenase complex, wherein the peptide component of the bioluminescent complex is capable of interacting with a polypeptide component of the bioluminescent complex to form the bioluminescent complex, wherein the bioluminescent complex is capable of generating bioluminescence in the presence of a substrate, wherein the peptide component of the modified dehalogenase complex is capable of interacting with a polypeptide component of the modified dehalogenase complex to form the modified dehalogenase complex, and wherein the modified dehalogenase complex is capable of forming a covalent bond with a haloalkyl ligand. (b) Under conditions that enable the formation of a modified dehalogenase complex, the target component attached to the tandem peptide tag is brought into contact with the polypeptide component of the modified dehalogenase complex. (c) Contacting the modified dehalogenase complex with a haloalkyl ligand comprising a haloalkane linked to a fluorophore; and (d) Detect fluorescence intensity.

62. A method for detecting a target component in a system, the method comprising: (a) Linking the target component to a tandem peptide tag, the tandem peptide tag comprising a peptide component of a bioluminescent complex fused with a peptide component of a modified dehalogenase complex, wherein the peptide component of the bioluminescent complex is capable of interacting with a polypeptide component of the bioluminescent complex to form the bioluminescent complex, wherein the bioluminescent complex is capable of generating bioluminescence in the presence of a substrate, wherein the peptide component of the modified dehalogenase complex is capable of interacting with a polypeptide component of the modified dehalogenase complex to form the modified dehalogenase complex, and wherein the modified dehalogenase complex is capable of forming a covalent bond with a haloalkyl ligand. (b) Under conditions that enable the formation of a bioluminescent complex, the target component attached to the tandem peptide tag is brought into contact with the polypeptide component of the bioluminescent complex. (c) Contacting the bioluminescent complex with its substrate; and (d) Detect bioluminescence.

63. A method for detecting a target component in a system, the method comprising: (a) Linking the target component to a tandem peptide tag, the tandem peptide tag comprising a peptide component of a bioluminescent complex fused with a peptide component of a modified dehalogenase complex, wherein the peptide component of the bioluminescent complex is capable of interacting with a polypeptide component of the bioluminescent complex to form the bioluminescent complex, wherein the bioluminescent complex is capable of generating bioluminescence in the presence of a substrate, wherein the peptide component of the modified dehalogenase complex is capable of interacting with a polypeptide component of the modified dehalogenase complex to form the modified dehalogenase complex, and wherein the modified dehalogenase complex is capable of forming a covalent bond with a haloalkyl ligand. (b) Under conditions that enable the formation of the bioluminescent complex and the modified dehalogenase complex, the target component attached to the tandem peptide tag is contacted with a tandem polypeptide reporter gene, the tandem polypeptide reporter gene comprising (i) a polypeptide component of the bioluminescent complex and (ii) a polypeptide component of the modified dehalogenase complex. (c) Contacting the bioluminescent complex with a substrate of the bioluminescent complex and / or a haloalkyl ligand comprising a haloalkane linked to a fluorophore; and (d) Detect bioluminescence and / or fluorescence.

64. A method for detecting a target component in a system, the method comprising: (a) Linking the target component to a tandem peptide tag, the tandem peptide tag comprising a peptide component of a bioluminescent complex fused with a peptide component of a modified dehalogenase complex, wherein the peptide component of the bioluminescent complex is capable of interacting with a polypeptide component of the bioluminescent complex to form the bioluminescent complex, wherein the bioluminescent complex is capable of generating bioluminescence in the presence of a substrate, wherein the peptide component of the modified dehalogenase complex is capable of interacting with a polypeptide component of the modified dehalogenase complex to form the modified dehalogenase complex, and wherein the modified dehalogenase complex is capable of forming a covalent bond with a haloalkyl ligand. (b) Under conditions that enable the formation of the bioluminescent complex and the modified dehalogenase complex, the target component linked to the tandem peptide tag is contacted with an independent polypeptide, the tandem polypeptide reporter gene comprising (i) a polypeptide component of the bioluminescent complex and (ii) a polypeptide component of the modified dehalogenase complex. (c) Contacting the bioluminescent complex with a substrate of the bioluminescent complex and / or a haloalkyl ligand comprising a haloalkane linked to a fluorophore; and (d) Detect bioluminescence and / or fluorescence.

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