Systems and methods for detecting biomolecular interactions

Abstract

Novel clickable and cleavable probes and methods of their use for detecting biomolecular interactions are described.

Description

SYSTEMS AND METHODS FOR DETECTING BIOMOLECULAR INTERACTIONSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority to U.S. Provisional Patent Application No. 63 / 662,864, filed June 21, 2024, the content of which is incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under R01 GM127633 awarded by the National Institutes of Health. The government has certain rights in the invention.TECHNICAL FIELD

[0003] Various embodiments and implementations described herein relate generally to novel clickable and cleavable probes for detecting biomolecular interactions including protein-protein, protein-RNA, RNA-RNA, protein-DNA, RNA-DNA, and DNA-DNA interactions.BACKGROUND

[0004] Proteins play a central role in cellular activities such as catalyzing reactions, transporting molecules, and facilitating immune responses, signal transduction between cells, and DNA replication to translation. These activities are regulated by the subcellular locations of the proteins and protein-protein interactions (PPIs). It is estimated that over 80% of the proteins cannot operate alone but in complexes, which further emphasizes the importance of PPIs.

[0005] In the past few decades, proteomic techniques for identifying and verifying PPIs have been developed, including approaches such as yeast two-hybrid (Y2H) assay, mass spectrometry, fused fluorescence tags (GFP, YFP, etc.), affinity tags, and FRET. These technologies provide the majority of what is known about PPIs today. However, these approaches require protein complexes to be physically separated or digested. Each technique has its limitations, making our understanding of cellular interactions far from complete. In some circumstances, only methods that employ direct protein binding reagents, such as antibodies, are sufficient. Immunohistochemistry is a well-established method for imaging the expression and distribution of proteins by microscopy. However, due to the optical limitation of microscopy, it is impossible to identify single protein molecules as well as protein complexes. Immunofluorescence also haslimitations, considering that the overlap of fluorescence signals of two protein targets when they interact can make achieving an accurate measurement difficult. Also, autofluorescence from formalin-fixed paraffin-embedded tissues can cause errors when identifying PPIs due to spatial overlapping of the signals. New approaches for characterizing PPIs in situ are desired.SUMMARY

[0006] In a first aspect, provided herein is a kit comprising: an alkyne-signal amplification probe comprising formula I or a salt thereof(i), wherein R1 - R5 are each independently selected from H, alkyl, alkenyl, alkynyl, halogen, carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronyl, aryl, and heteroaryl; wherein Linker comprises a cleavable linker; wherein Y is selected from hydrogen, a counter cation, and a moiety that can be cleaved by an enzyme, an ion, light, a base, or an acid; wherein m is an integer from 1 to 100; and a cleavable detection probe (CDP) comprising formula II or a salt thereof(ii), wherein X is O or S; wherein R1 - R4 are each independently selected from H, alkyl, alkenyl, alkynyl, halogen, carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronyl, aryl, and heteroaryl, wherein optionally R1 and R2 form a 3-10 member ring; wherein Y is selected from hydrogen, a counter cation and a moiety that can be cleaved by an enzyme, an ion, light, a base, or an acid; wherein TAG is a biologically detectable moiety comprising a fluorophore, a chromophore, or a metal isotope; wherein Linker 1 and Linker 2 each comprise a rigid chain or a flexible chain; and wherein n is an integer from 1 to 100.

[0007] In embodiments, the alkyne-signal amplification probe comprises: a terminal alkyne; and a terminal tyramide; and the cleavable linker may comprise at least one disulfide bond.

[0008] The alkyne-signal amplification probe may comprise formula la

[0009] The fluorophore of the CDP may be Cy3 or Cy5.

[0010] The kit may further comprise a fluorophore-tyramide comprising a fluorophore and a tyramide. The fluorophore of the CDP and the fluorophore of the fluorophore-tyramide may be different. The fluorophore of the fluorophore-tyramide may be Cy5.

[0011] The kit may further comprise at least two antibodies, wherein each antibody is specific for a different protein, and wherein each antibody is conjugated to HRP or biotin. The kit may further comprise a click chemistry buffer. The kit may further comprise at least one of a tris(2- carboxyethyljphosphine (TCEP) solution and a l,3,5-triaza-7-phosphaadamantane (PTA) solution. The kit may further comprise a streptavidin-HRP solution.

[0012] In another aspect, provided herein is a method for characterizing the proximity of a first protein and a second protein in a fixed tissue, the method comprising: (a) contacting the tissue with an HRP -labeled first antibody that specifically binds to the first protein; (b) contacting the tissue with an alkyne-signal amplification probe comprising a cleavable linker, a fluorophore-tyramide comprising a fluorophore and a tyramide, and a tyramide signal amplification (TSA) buffer; (c) contacting the tissue with an HRP blocking solution; (d) contacting the tissue with an HRP-labeled second antibody that specifically binds to the second protein; (e) contacting the tissue with a cleavable detection probe (CDP) and the TSA buffer; (f) visualizing the fluorescence of the fixed tissue; (g) contacting the tissue with a click chemistry buffer; (h) contacting the tissue with a tris(2- carboxyethyljphosphine (TCEP) solution; (i) contacting the tissue with a l,3,5-triaza-7-phosphaadamantane (PT A) solution; and (j) visualizing the fluorescence of the fixed tissue; wherein the alkyne-signal amplification probe comprises formula I or a salt thereofwherein R1 - R5 are each independently selected from H, alkyl, alkenyl, alkynyl, halogen, carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronyl, aryl, and heteroaryl; wherein Linker comprises a cleavable linker; wherein Y is selected from hydrogen, a counter cation, and a moiety that can be cleaved by an enzyme, an ion, light, a base, or an acid; and wherein m is an integer from 1 to 100; and wherein the CDP comprises formula II or a salt thereofwherein X is O or S; wherein R1 - R4 are each independently selected from H, alkyl, alkenyl, alkynyl, halogen, carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronyl, aryl, and heteroaryl, wherein optionally R1 and R2 form a 3-10 member ring; wherein Y is selected from hydrogen, a counter cation and a moiety that can be cleaved by an enzyme, an ion, light, a base, or an acid; wherein TAG is a biologically detectable moiety comprising a fluorophore, a chromophore, or a metal isotope; wherein Linker 1 and Linker 2 each comprise a rigid chain or a flexible chain; and wherein n is an integer from 1 to 100.

[0013] The method may further comprise: (k) contacting the tissue with a fluorophore bleaching solution; and (1) repeating steps (a)-(k) at least one time; wherein each time at least one of the first protein and the second protein binds to a different first protein and a different second protein, respectively.

[0014] The fixed tissue may be a formalin-fixed paraffin-embedded tissue.

[0015] The alkyne-signal amplification probe may comprise: a terminal alkyne; a terminaltyramide; and the cleavable linker may comprise at least one disulfide bond.

[0016] The alkyne-signal amplification probe may comprise formula la

[0017] The fluorophore of the fluorophore-tyramide and the fluorophore of the CDP may be different. The fluorophore of the CDP may be Cy3 or Cy5. The fluorophore of the fluorophore- tyramide may be Cy5.

[0018] In another aspect, provided herein is a kit comprising: a first antibody that specifically binds to a first protein, wherein the first antibody is conjugated to an alkyne probe through a disulfide linker; a second antibody that specifically binds to a second protein, wherein the second antibody is conjugated to a tetrazine probe having an azide-based cleavable linker; a transcyclooctene (TCO)-labeled HRP; and a cleavable detection probe (CDP).

[0019] The tetrazine probe may comprise formula III(III),N3oA, wherein L is a linker, wherein Z is tetrazine, and wherein B is

[0020] The tetrazine probe may comprise formula (IV)(IV).

[0021] The alkyne probe may comprise formula Vwherein X is O or S; wherein R1 - R4 are each independently selected from H, alkyl, alkenyl, alkynyl, halogen, carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronyl, aryl, and heteroaryl, wherein optionally R1 and R2 form a 3-10 member ring; wherein Y is selected from hydrogen, a counter cation and a moiety that can be cleaved by an enzyme, an ion, light, a base, or an acid; wherein TAG is a biologically detectable moiety comprising a fluorophore, a chromophore, or a metal isotope; wherein Linker 1 and Linker 2 each comprise a rigid chain or a flexible chain; and wherein n is an integer from 1 to 100.

[0023] The fluorophore may be Cy3 or Cy5.

[0024] The kit may further comprise a click chemistry buffer. The kit may further comprise at least one of a tris(2-carboxyethyl)phosphine (TCEP) solution and a l,3,5-triaza-7- phosphaadamantane (PTA) solution.

[0025] In another aspect, provided herein is a method for characterizing the proximity of a first protein and a second protein in a fixed tissue, the method comprising: (a) contacting the tissue with (i) a first antibody that specifically binds to a first protein, wherein the first antibody is conjugated to an alkyne probe through a disulfide linker; and (ii) a second antibody that specifically binds to a second protein, wherein the second antibody is conjugated to a tetrazine probe having an azide- based cleavable linker; (b) contacting the tissue with a click chemistry buffer; (c) contacting the tissue with a tris(2-carboxyethyl)phosphine (TCEP) solution; (d) contacting the tissue with a 1,3,5- triaza-7-phosphaadamantane (PTA) solution; (e) contacting the tissue with a trans-cyclooctene (TCO) labeled HRP, a cleavable detection probe (CDP), and a tyramide signal amplification (TSA) buffer; (f) visualizing the fluorescence of the fixed tissue; (g) contacting the tissue with the TCEP solution; (h) contacting the tissue with the PTA solution; and (i) visualizing the fluorescence of the fixed tissue.

[0026] The method may further comprise: (j) contacting the tissue with a fluorophore bleaching solution; and (k) repeating steps (a)-(j) at least one time; wherein each time, at least one of the first antibody and the second antibody binds to a different first protein and a different second protein.

[0027] The fixed tissue may be a formalin-fixed paraffin-embedded tissue.

[0028] The tetrazine probe comprises formula III(III), wherein L is a linker, wherein Z is tetrazine, and wherein B is a reactive linker selected from

[0029] The tetrazine probe may comprise formula (IV)(IV).

[0030] The alkyne probe may comprise formula V(V).

[0031] The CDP may comprise formula II or a salt thereofwherein X is O or S; wherein R1 - R4 are each independently selected from H, alkyl, alkenyl, alkynyl, halogen, carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronyl, aryl, and heteroaryl, wherein optionally R1 and R2 form a 3-10 member ring; wherein Y is selected from hydrogen, a counter cation and a moiety that can be cleaved by an enzyme, an ion, light, a base, or an acid; wherein TAG is a biologically detectable moiety comprising a fluorophore, a chromophore, or a metal isotope; wherein Linker 1 and Linker 2 each comprise a rigid chain or a flexible chain; and wherein n is an integer from 1 to 100. The fluorophore may be Cy3 or Cy5.

[0032] The disclosure is not an extensive overview of all contemplated features, steps, or advantages of the disclosed embodiments and is intended neither to identify key or criticalelements of all aspects thereof nor to delineate the scope of any or all aspects of covered embodiments.

[0033] Aspects of the disclosure will become more fully understood upon a review of the drawings, detailed description, and attached appendices. Other aspects, features, and embodiments of the present disclosure will become apparent to those skilled in the art, upon reviewing the following (and attached) description of specific, example embodiments of the present disclosure in conjunction with the accompanying figures. While features of the present disclosure may be discussed relative to certain embodiments and figures, all embodiments of the present disclosure can include one or more of the advantageous features discussed herein. In other words, while one or more embodiments may be discussed as having certain advantageous features, one or more of such features may also be used in accordance with the various embodiments of the disclosure discussed herein. Similarly, while example embodiments may be discussed below as devices, systems, or methods embodiments it should be understood that such example embodiments can be implemented in various devices, systems, and methods.BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The patent or patent application file contains at least one drawing in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0035] Non-limiting embodiments of the present disclosure will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the disclosure shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure.

[0036] FIG. 1 shows a schematic presentation of the basic working principle of click chemistry protein-protein interaction (PPI) assay, according to an aspect as disclosed herein.

[0037] FIG. 2 shows the chemical structure of a cleavable fluorescent tyramide probe (CFT) and alkyne probe for PPI characterization, according to an embodiment disclosed herein.

[0038] FIG. 3 shows a synthetic route for an alkyne probe described herein.

[0039] FIG. 4 shows a schematic workflow presentation for multiplexed in-situ PPIcharacterization with a CFT probe (also referred to as a CDP) and click chemistry, according to an aspect as disclosed herein.

[0040] FIGS. 5A-5C show a demonstration of PPI characterization using click chemistry with the alkyne probe (Cy5 for visualization) staining histone H4 and Cy3-Ns-Tyr staining histone H3. Fluorescence images of Cy3 channel (A) before and (B) after click chemistry / cleavage and (C) the intensity profde.

[0041] FIGS. 6A-6C show a demonstration of click chemistry with Cy3-Ns-Tyr restaining histone H3 and H4 after the cleavage condition was applied to remove the alkyne probe deposited on histone H4. Fluorescence images of Cy3 channel (A) before and (B) after click chemistry / cleavage and (C) the intensity profile.

[0042] FIGS. 7A-7C show a demonstration of PPI characterization using click chemistry with the alkyne probe (Cy5 for visualization) staining H3S10P and Cy3-N3-Tyr staining Na+ / K+-ATPAse. Fluorescence images of Cy3 channel (A) before and (B) after click chemistry / cleavage and (C) the intensity profile.

[0043] FIGS. 8A-8C show a demonstration of PPI characterization using click chemistry with the alkyne probe (Cy5 for visualization) staining CD79a and Cy3-N3-Tyr staining HLADRA1. Fluorescence images of Cy3 channel (A) before and (B) after click chemistry / cleavage and (C) the intensity profile.

[0044] FIGS. 9A-9D show zoomed in fluorescence images for protein sets: (A) histone H3 and H4. (B) H3S10P and Na+ / K+-ATPase. (C) CD79a and HLADRA1. (D) Boxplot of cleavage efficiency for the three protein sets.

[0045] FIGS. 10A-10K showPD-1 (Cy5 / alkyne) andPD-Ll (Cy3) expression in (A) control tonsil tissue, (B) control breast tissue, (C) cancer breast tissue, (D) control colon tissue, (E) cancer colon tissue, (F) control liver tissue, (G) cancer liver tissue, (H) control lung tissue, (I) cancer lung tissue, (J) control prostate tissue, (K) cancer prostate tissue.

[0046] FIGS. 11A-1 IF show fluorescence images and intensity profiles of PD-1 (Cy5 / alkyne) and PD-L1 (Cy3) on (A) control tonsil, (B) breast cancer, (C) colon cancer, (D) liver cancer, (E) lung cancer, and (F) prostate cancer tissues. Scale bar, 50 pm.

[0047] FIGS. 12A-12F show fluorescence images and intensity profiles of CD3 (Cy5 / alkyne) and CD4 (Cy3) on (A) control tonsil, (B) breast cancer, (C) colon cancer, (D) liver cancer, (E) lung cancer, and (F) prostate cancer tissues. Scale bar, 50 pm.

[0048] FIGS. 13A-13K show the CD45 (Cy5 / alkyne) and HLADRA1 (Cy3) expression in (A) control tonsil tissue, (B) control breast tissue, (C) cancer breast tissue, (D) control colon tissue, (E) cancer colon tissue, (F) control liver tissue, (G) cancer liver tissue, (H) control lung tissue, (I) cancer lung tissue, (J) control prostate tissue, (K) cancer prostate tissue.

[0049] FIGS. 14A-14F show fluorescence images and intensity profiles of CD45 (Cy5 / alkyne) and HLADRA1 (Cy3) on (A) control tonsil, (B) breast cancer, (C) colon cancer, (D) liver cancer, (E) lung cancer, and (F) prostate cancer tissues. Scale bar, 50 pm.

[0050] FIGS. 15A-15F show fluorescence images and intensity profiles of histone H3 (Cy5 / alkyne) and histone H4 (Cy3) on (A) control tonsil, (B) breast cancer, (C) colon cancer, (D) liver cancer, (E) lung cancer, and (F) prostate cancer tissues. Scale bar, 50 pm.

[0051] FIGS. 16A-16F show fluorescence images and intensity profiles of Ku80 (Cy5 / alkyne) and Vimentin (Cy3) on (A) control tonsil, (B) breast cancer, (C) colon cancer, (D) liver cancer, (E) lung cancer, and (F) prostate cancer tissues. Scale bar, 50 pm.

[0052] FIGS. 17A-17B. (A) shows highly multiplexed single-cell in situ PPI profiling by clickable and cleavable probes. (B) shows a click chemistry reaction quenches the cleavable tetrazine when PPI is detected by the antibody pair.

[0053] FIGS. 18A-18F show (A) protein H3 SI OP (purple) and Na / K ATPase (yellow) stained with cleavable alkyne and tetrazine conjugated antibodies, or (B) unconjugated primary antibodies. (C) Protein H3 (purple) and H4 (yellow) are stained with cleavable alkyne and tetrazine conjugated antibodies, or (D) unconjugated primary antibodies. (E) PPI between H3 and H4 along with (F) H3S10P and Na / K ATPase are analyzed sequentially on the same human formalin-fixed paraffin- embedded (FFPE) tissue using our method. Scale bars, 5 pm.

[0054] FIG. 19 shows the structures of (A) azido-based linkers and (B) allyl-based linkers.

[0055] FIG. 20 shows an example of the highly multiplexed imaging of a hub protein interactome, according to an aspect of the disclosure herein.

[0056] FIG. 21 shows an example of a synthetic scheme of cleavable oligonucleotides conjugated antibodies, according to an aspect of the disclosure herein.

[0057] FIG. 22 shows a schematic presentation of the working principle of prior art method PLA.DETAILED DESCRIPTION

[0058] Disclosed herein are compounds, compositions and methods for the characterization ofprotein -protein interactions (PPIs) in situ using click chemistry. The methods can also be used to detect interactions between any two biomolecules, including, but not limited to, proteins, DNA, RNA, metabolites, etc. The method for the detection of protein-protein interactions involves using a cleavable detection probe (CDP) and click chemistry in order to test if two proteins are in proximity of each other. One of the proteins of interest contains an azido group, which participates in the click chemistry, but is also the cleavable site of the CDP, while the other protein contains the alkyne group. When “clicked” together, the CDP becomes non-cleavable as the azide is now incorporated into the triazole click product. The two probes now produce a fluorescent signal which cannot be removed under conditions that would otherwise cleave off the fluorophore probe. This method allows for comprehensive PPI profiling both in solution and in tissues, as this method allows for both single cell in situ, comprehensive, FFPE tissue, and hub protein interactome analysis.

[0059] In a first aspect, provided herein is a kit comprising: an alkyne-signal amplification probe comprising a cleavable linker; and a cleavable detection probe (CDP). The alkyne-signal amplification probe may comprise: a terminal alkyne; a terminal tyramide; and a cleavable linker between the terminal alkyne and the terminal tyramide, wherein the cleavable linker comprises at least one disulfide bond. The alkyne-signal amplification probe may be modified with disulfide bonds on both the tyramine end and the alkyne end to render it cleavable.

[0060] In embodiments, the alkyne-signal amplification probe comprises Formula I or a salt thereof

[0061] R1 - R5 each independently are H, alkyl, alkenyl, alkynyl, halogen, carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronyl, aryl, or heteroaryl, and optionally R1 and R2 form a 3-10 member ring. In embodiments, Y is hydrogen. In other embodiments, Y is a counter cation when the O is negatively charged. In other embodiments, Y is a moiety that can be cleaved by an enzyme, an ion, light, a base, or an acid. The Y group can be removed to provide a tyramide-like structure thatis able to participate in a tyramide signal amplification (TSA) reaction. In other words, when Y is present in the probe, it may be a caged compound, in which a trigger (e.g., presence of an enzyme, light, acid, base) causes the Y group at the end of the probe to be removed, the tyramide-like structure becomes available to be activated by HRP / peroxide, and the probe can be deposited around the biomolecule by the TSA reaction. In embodiments alkyne-signal amplification probe includes a terminal alkyne where R5 is H and a terminal tyramide where Y is H.

[0062] The Linker may be a cleavable linker. In embodiments, the cleavable linker is a disulfide bond. The Linker may further include rigid chains and / or flexible chains. In embodiments, the Linker includes substituted or unsubstituted Ci-Cio alkyl, Ci-Cio alkenyl, Ci-Cio alkynyl, aryl, heteroaryl groups. In embodiments, the Linker includes a polyethylene glycol chain having the. . . formula, wherein a is an integer between 1 and 20. In embodiments, the Linker comprises amino, amido, carbonate, ester, ether, thioether, and / or carbamate groups.

[0063] In embodiments, the Linker comprises a moiety having a bond that cleaves in the presence of an acid, a base, ions, a reducing reagent, an oxidative reagent, light, or a specific enzyme. In formula I, m is an integer from 1 to 100 or any integer or range in between (e.g., 1 to 5).

[0064] In embodiments, the alkyne-signal amplification probe comprises formula la

[0065] In embodiments, the CDP comprises formula 11 or a salt thereof:(II). X is O or S. R1 -R4 are each independently selected from H, alkyl, alkenyl, alkynyl, halogen, carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronyl, aryl, and heteroaryl, and optionally R1 and R2 form a 3-10 member ring. In embodiments, Y is hydrogen. In embodiments, Y is a counter cation when the O is negatively charged. In other embodiments, Y is a moiety that can be cleaved by an enzyme, an ion, light, base or acid. TAG is a biologically detectable moiety comprising a fluorophore, a chromophore, or a metal isotope, n is an integer from 1 to 100 (e.g. 1 to 5). The Y selected for the CDP can be different from the Y selected for the alkyne-signal amplification probe. Linker 1 and Linker 2 may each comprise rigid chains and / or flexible chains. In embodiments, Linker 1 and Linker 2 include substituted or unsubstituted Ci-Cio alkyl, Ci-Cio alkenyl, Ci-Cio alkynyl, aryl, or heteroaryl groups. In embodiments, Linker 1 and Linker 2 include a polyethylene glycol linker having the formula, wherein a is an integer between 1 and 20. In embodiments,Linker 1 and Linker 2 can include amine, amide, carbonate, ester, ether, thioether, or carbamate groups. TAG is a biologically detectable moiety. TAG can include a fluorophore, chromophore, metal isotope, or a combination thereof.

[0067] In embodiments, the CDP comprises formula Ila or a salt thereof:wherein A is a fluorophore.

[0068] Appropriate fluorophores and chromophores for use in the probes and methods of this disclosure include, without limitation, Cy2, Cy 3, Cy3.5, Cy 5, Cy5.5, Cy7, TAMRA (labeled with tetramethylrhodamine or “TMR”), ALEXA FLUOR™ 594, and ATTO 647N and ATTO 700 fluorophores (ATTO-TEC, Germany). Other fluorophores appropriate for use according to the methods provided herein include, without limitation, quantum dots, ALEXA FLUOR™ 350, ALEXA FLUOR™ 532, ALEXA FLUO® 546, ALEXA FLUOR™ 568, ALEXA FLUOR™ 647, BODIPY 493 / 503, BODIPY FL, BODIPY R6G, BODIPY 530 / 550, BODIPY TMR, BODIPY558 / 568, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY630 / 650, BODIPY 650 / 665, Cascade Blue, Cascade Yellow, Dansyl, lissamine rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, rhodamine 6G, rhodamine green, rhodamine red, tetramethyl rhodamine, DYLIGHT™ DYES (e.g., DYLIGHT™ 405, DYLIGHT™ 488, DYLIGHT™ 549, DYLIGHT™ 594, DYLIGHT™ 633, DYLIGHT™ 649, DYLIGHT™ 680, DYLIGHT™ 750, DYLIGHT™ 800 and the like), and Texas Red.

[0069] Appropriate metal isotopes for use in the probes and methods of this disclosure include, without limitation, isotopes of any of Y, Cu, Pd, In, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, and B. Metal isotopes can be incorporated into the TAG using a chelating agent.

[0070] The kit may further comprise a fluorophore-tyramide comprising a fluorophore and a tyramide. When the CDP comprises a fluorophore, the fluorophore of the CDP and the fluorophore of the fluorophore-tyramide may be different. In exemplary embodiments, the fluorophore of the CDP is Cy3 or Cy5. In exemplary embodiments, the fluorophore of the fluorophore-tyramide is Cy5.

[0071] The kit may further comprise at least two antibodies, wherein each antibody is specific for a different protein. In embodiments, the antibodies are conjugated to horseradish peroxidase (HRP). In other embodiments, the antibodies are conjugated to biotin. The antibodies may be selected based on their likelihood to bind each other or be in proximity to one another. In exemplary embodiments, an antibody may be specific for PD-1, PD-L1, CD3, CD4, CD45, HLADRA1, histone H3, histone H4, and Ku80, Vimentin, CD79a, Na+ / K+-ATPAse, or phosphorylated histone H3.

[0072] Conjugation of molecules may be done by formation of a covalent bond, such as an amide, an ester, a carbonate, an ether, or a thioether.

[0073] The kit may further comprise a click chemistry buffer. A click chemistry buffer is a buffer comprising components necessary to carry out a click chemistry reaction. The click chemistry buffer may comprise a copper reagent, such as CuBr, Cui, Q1NO3, or Q1SO4. The click chemistry buffer may also comprise a stabilizing ligand, such as tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), tris[(l-benzyl-lH-l,2,3-triazol-4-yl)methyl]amine (BTA), or bis(triazolyl)acetic acid (BTTAA). In exemplary embodiments, the click chemistry buffer is a copper(II) sulfate solution comprising CuSO4, tris-hydroxypropyltriazolylmethylamine (THPTA) ligand, and sodium ascorbate. The buffer may comprise about 0.6 mM CuSCh, about 3 mM THPTA in phosphate buffered saline (PBS), and about 4 mM sodium ascorbate (freshly made and added to CuSCLimmediately before use). However, any buffer that facilitates a click chemistry reaction may be used.

[0074] The kit may further comprise a reducing agent that cleaves disulfide bonds, including at least one of a tris(2-carboxyethyl)phosphine (TCEP) solution and a l,3,5-triaza-7- phosphaadamantane (PTA) solution. The TCEP solution may be about 0.1 M TCEP in PBT, and the pH may be tuned with about IM sodium hydroxide to about pH 9. The PTA solution may be about 0.1M PTA in PBT. Other reducing agents include dithiothreitol, 2-mercaptoethanol, dithiobutylamine, ammonium thioglycolate, glutathione, cysteine, sodium borohydride, or electrochemical methods.

[0075] The kit may further comprise a streptavidin-HRP solution comprising HRP conjugated- streptavidin in a buffer. The streptavidin-HRP solution may comprise 5 pg / mL HRP conjugated streptavidin in PBT.

[0076] Use of CDP is described further in US Patent Publication No. US20220026433A1, which is incorporated herein by reference in its entirety.

[0077] In a second aspect, provided herein is a method for characterizing the proximity of a first protein and a second protein in a fixed tissue, the method comprising: (a) contacting the tissue with an HRP -labeled first antibody that specifically binds to the first protein; (b) contacting the tissue with an alkyne-signal amplification probe comprising a cleavable linker, a fluorophore-tyramide, and a tyramide signal amplification (TSA) buffer; (c) contacting the tissue with an HRP blocking solution; (d) contacting the tissue with a HRP-labeled second antibody that specifically binds to the second protein; (e) contacting the tissue with a cleavable detection probe (CDP) and the TSA buffer; (f) visualizing the fluorescence of the fixed tissue; (g) contacting the tissue with a click chemistry buffer; (h) contacting the tissue with a tris(2-carboxyethyl)phosphine (TCEP) solution; (i) contacting the tissue with a l,3,5-triaza-7-phosphaadamantane (PTA) solution; and (j) visualizing the fluorescence of the fixed tissue.

[0078] In steps (a) and (d), the HRP-labeled antibody may be provided, for example, by contacting the tissue with a biotin-conjugated first antibody that specifically binds to the first protein, and then contacting the tissue with a streptavidin HRP solution. Contacting the tissue with the biotin- conjugated first antibody may be done for about 45 minutes at between about 0°C and about 100°C. In exemplary embodiments, this is done at about room temperature (RT) (e.g. between about 20- 22°C). Contacting the tissue with the streptavidin HRP solution may be done for about 30 minutesat about RT. Step (b) may be done for about 10 minutes at between about 0°C and about 100°C or at about RT. About 1 pl of the fluorophore-tyramide and about 1 pl of the alkyne-signal amplification probe with a concentration of between about 0.05 mM and about 5mM, or at about 0.5 mM) may be provided in the TSA buffer (about 1 pL of about 0.3 % hydrogen peroxide diluted in about 200 pL of about between about 0.01M and about IM, or about 0.1M boric acid PBT solution). Step (c) may be done for about 30 minutes at between about 0°C and about 100°C, or about RT. Step (d) may be done for about 30 minutes at between about 0°C and about 100°C , or about RT. Step (e) may be done for about 10 minutes at between about 0°C and about 100°C, or about RT. Steps (f) and (j) may be done using fluorescence microscopy. Step (g) may be done for about 30 minutes about between about 0°C and about 100°C, or about RT. Step (h) may be done with between about 0.01M and 1 M, or about 0.1 M TCEP solution in PBT, and it may be tuned to pH9 with between about 0. IM and about 10M, or about IM sodium hydroxide. Step (i) may be done for about 30 minutes at between about 0°C and about 100°C, or about 40°C.

[0079] The method may further comprise (k) contacting the tissue with a fluorophore bleaching solution; and (1) repeating steps (a)-(k) at least one time; wherein each time, at least one of the first antibody and the second antibody binds to a different first protein and a different second protein. Step (k) may be done for about 10 minutes at between about 0°C and about 100°C, or about 40°C.

[0080] Between steps (a) and (b), (b) and (c), (c) and (d), (d) and (e), (e) and (f), (g) and (h), (h) and (i), and (i) and (j), the tissue may be rinsed. Rinsing may be done 1-5 times in PBT. Rinsing may be done 3 times.

[0081] TSA is a horseradish peroxidase (HRP)-catalyzed deposition of tyramide on and near a target biomolecule in situ. In the presence of low concentrations of H2O2, HRP converts the phenolic portion of a labeled tyramide substrate into a reactive form that can covalently bind to a residue (e.g. tyrosine, tryptophan, phenylalanine) on proteins at or near the HRP, resulting in a high local density of covalently bound, labeled tyramide substrate. Once the TSA is complete, the HRP can be deactivated by application of an HRP blocking solution.

[0082] The antibodies may be prepared by: (1) Mix about 1 pL of the antibody and a primary antibody labeling solution (for every one pg of the antibody, add 5 pL of the primary antibody labeling solution) in 5 pL of PBS and let sit for five minutes at room temperature. (2) Add the same amount of blocking solution to the antibody solution. Mix and let sit for five minutes at room temperature. (3) Dilute the antibody solution with antibody -blocking buffer to the desired workingconcentration of the antibody.

[0083] The tissue may be a formalin-fixed paraffin-embedded (FFPE) tissue sample. The alkyne- signal amplification probe, and the CDP may be any of those described herein.

[0084] In a third aspect, provided herein is a kit comprising: a first antibody that specifically binds to a first protein, wherein the first antibody is conjugated to an alkyne probe through a disulfide linker; a second antibody that specifically binds to a second protein, wherein the second antibody is conjugated to a tetrazine probe having an azide-based cleavable linker or an allyl-based cleavable linker; a trans-cyclooctene (TCO)-labeled HRP; and a cleavable detection probe (CDP).

[0085] The tetrazine probe may comprise formula III(III), wherein is a linker, wherein Z is tetrazine, and

[0086] The tetrazine probe may comprises formula (IV)

[0087] The alkyne probe may comprise formula V(V).

[0088] The CDP may be any of the CDPs described herein.

[0089] The kit may further comprise at least one of a click chemistry buffer, a TCEP solution, and a PT A solution.

[0090] In a fourth aspect, provided herein is a method for characterizing the proximity of a first protein and a second protein in a fixed tissue, the method comprising: (a) contacting the tissue with (i) a first antibody that specifically binds to a first protein, wherein the first antibody is conjugated to an alkyne probe through a disulfide linker; and (ii) a second antibody that specifically binds to a second protein, wherein the second antibody is conjugated to a tetrazine probe having an azide- based cleavable linker; (b) contacting the tissue with a click chemistry buffer, (c) contacting the tissue with a TCEP solution; (d) contacting the tissue with a PTA solution; (e) contacting the tissue with a trans-cyclooctene (TCO) labeled HRP, a cleavable detection probe (CDP), and a tyramide signal amplification (TSA) buffer; (f) visualizing the fluorescence of the fixed tissue; (g) contacting the tissue with the TCEP solution; (h) contacting the tissue with the PTA solution; and (i) visualizing the fluorescence of the fixed tissue.

[0091] The method may further comprise: (j) contacting the tissue with a fluorophore bleaching solution; and (k) repeating steps (a)-(j ) at least one time; wherein each time, at least one of the first antibody and the second antibody binds to a different first protein and a different second protein.

[0092] In a fifth aspect, provided herein is an alkyne-signal amplification probe comprising formula la

[0093] The terms “antibody” and “antibody molecule” are used herein interchangeably and refer to immunoglobulin molecules or other molecules which comprise an antigen binding domain.Antibodies include whole antibodies (e.g., IgG, IgA, IgE, IgM, or IgD), monoclonal antibodies, chimeric antibodies, humanized antibodies, and antibody fragments, including single chain variable fragments (ScFv), single domain antibodies, and antigen-binding fragments, genetically engineered antibodies, among others, as long as the characteristic properties (e.g. ability to bind to the protein of interest or variant) are retained.

[0094] The term antibody includes “antibody fragments” or “antibody-derived fragments” and “antigen binding fragments” which comprise an antigen binding domain and displays antigen binding function, for example, Fab, Fab', F(ab')2, scFv, Fv, dsFv, ds-scFv, Fd, mini bodies, monobodies, and multimers thereof and bispecific antibody fragments. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv), (see for instance Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context.

[0095] As mentioned above, fragments may comprise a heavy chain variable region (VH domain) and light chain variable region (VL). Fragments may comprise one or more of the heavy chain complementarity determining regions (CDRHs) of the antibodies or of the VH domains, and one or more of the light chain complementarity determining regions (CDRLs), or VL domains to form the antigen binding site.

[0096] The terms “complementarity determining region” and “CDR” refer to part of the variablechains in immunoglobulins (antibodies) and T cell receptors, generated by B-cells and T-cells respectively, where these molecules bind to their specific antigen. As the most variable parts of the molecules, CDRs are crucial to the diversity of antigen specificities generated by lymphocytes. There are three CDRs (CDR1, CDR2 and CDR3), arranged non-consecutively, on the amino acid sequence of a variable domain of an antigen binding site. Since the antigen binding sites are typically composed of two variable domains (on two different polypeptide chains, heavy and light chain), there are six CDRs for each antigen binding site that can collectively come into contact with the antigen. A single whole antibody molecule has two antigen binding sites and therefore contains twelve CDRs. For further example, sixty CDRs can be found on a pentameric IgM molecule.

[0097] Within the variable domain, CDR1 and CDR2 may be found in the variable (V) region of a polypeptide chain, and CDR3 includes some of V, and all of diversity (D, heavy chains only) and joining (J) regions. Since most sequence variation associated with immunoglobulins and T cell receptors is found in the CDRs, these regions are sometimes referred to as hypervariable regions. Among these, CDR3 shows the greatest variability as it is encoded by a recombination of VJ in the case of a light chain region and VDJ in the case of heavy chain regions. The tertiary structure of an antibody is important to analyze and design new antibodies.

[0098] The terms “specifically binds”, or “binding specificity” are used alternatively and in relation to an antibody, refers to the ability of the antibody to form one or more noncovalent bonds with an epitope or antigen via the antibody variable domains. Specificity can be characterized by an antibody-antigen affinity, e.g. as characterized by a dissociation constant (KD) of <100 nM, 10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nanomolar (nM) (e.g. 10XM or less, e.g. from 10XM to 1013M, e.g., from 109M to 1013M).

[0099] As used herein, the terms “protein” or “polypeptide” or “peptide” may be used interchangeably to refer to a polymer of amino acids. Typically, a “polypeptide” or “protein” is defined as a longer polymer of amino acids, of a length typically of greater than 50, 60, 70, 80, 90, or 100 amino acids. A “peptide” is defined as a short polymer of amino acids, of a length typically of 50, 40, 30, 20 or less amino acids.

[0100] A “protein” as contemplated herein typically comprises a polymer of naturally or non-naturally occurring amino acids (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine, and valine). The proteins contemplated herein may be further modified in vitro or in vivo to include non-amino acid moieties. These modifications may include but are not limited to acylation (e.g., O-acylation (esters), N-acylation (amides), S- acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues), formylation lipoylation (e.g., attachment of a lipoate, a C8 functional group), myristoylation (e.g., attachment of myristate, a C14 saturated acid), palmitoylation (e.g., attachment of palmitate, a C16 saturated acid), alkylation (e.g., the addition of an alkyl group, such as an methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g., the addition of an isoprenoid group such as farnesol or geranylgeraniol), amidation at C- terminus, glycosylation (e.g., the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein). Distinct from glycation, which is regarded as a nonenzymatic attachment of sugars, polysialylation (e.g., the addition of polysialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e.g., the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine).

[0101] The terms “polynucleotide,” “polynucleotide sequence,” “nucleic acid,” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. A “polynucleotide” may refer to a polydeoxyribonucleotide (containing 2-deoxy-D-ribose), a polyribonucleotide (containing D- ribose), and to any other type of polynucleotide that is an N glycoside of a purine or pyrimidine base. There is no intended distinction in length between the terms “nucleic acid”, “oligonucleotide” and “polynucleotide”, and these terms will be used interchangeably. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA. For use in the present methods, an oligonucleotide also can comprise nucleotide analogs in which the base, sugar, or phosphate backbone is modified as well as non-purine or non-pyrimidine nucleotide analogs. These phrases also refer to DNA or RNA of genomic, natural, or synthetic origin (which may be single-stranded or double-stranded and may represent the sense or the antisense strand).

[0102] As used herein, the term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and DNA / RNA hybrids. Polynucleotides may be single- stranded or double-stranded. Nucleic acids include, but are not limited to: pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, synthetic RNA, genomic RNA (geRNA), guide RNA, tracRNA, crRNA, sgRNA, plus strand RNA (RNA(+)), minus strand RNA (RNA(-)), synthetic RNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA.

[0103] Miscellaneous

[0104] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.”

[0105] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

[0106] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of’ those certain elements.

[0107] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same.In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”

[0108] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0109] In those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together ). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0110] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicatedotherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.

[0111] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any nonclaimed element as essential to the practice of the invention.

[0112] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0113] The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims.EXAMPLES

[0114] Example 1

[0115] in-situ Protein-Protein Interaction

[0116] Prior to this disclosure, the only method to accurately identify PPI in situ was through proximity ligation assay (PLA). PLA utilizes a set of antibodies with attached oligonucleotide probes targeting two proteins of interest, which can hybridize to circularizable probes to generate a circular DNA by DNA ligase when the two oligonucleotide strands are in proximity by binding to the same protein complex. The subsequently added linear connector oligonucleotides can hybridize with the two proximity probes to form a circular structure covalently joined by enzymatic DNA ligation. Using the oligonucleotide on one of the probes as a primer, rolling-circle amplification (RCA) is initiated to elongate the oligonucleotide.Fluorescence-tagged oligonucleotides complementary to the probe sequence are then added to label the RCA product, and the signal representing the protein complex can be captured by microscopy (FIG. 22.).

[0117] PLA enables localized detection of individual, endogenous interacting protein complexes in fixed tissues with improved accuracy. Additionally, it has been implemented to enhance sensitivity, specificity, and target range in other techniques for localized protein detection, including western blotting 39, flow cytometry 40, and sandwich enzyme-linked immunosorbent assay (ELISA)39, 40. Multiplexed characterization of PLA can be achieved by using unique DNA sequences for each probe decoded in parallel. Despite that, when the number of different probes increases, the risk of DNA mis-hybridization between different probes or endogenous oligonucleotides increases as well. Except for the possible false positive signals, the generation of non-circular ligation products, which will result in false negative signals, is another potential drawback. In addition, cost and assay time are two major issues in PLA. Since steps in PLA involve enzymatic reactions like ligation and enzymatic polymerization, the use of enzymes is necessary. The cost of enzymes is a hurdle; the storage of the enzymes has to be carefully handled, and a long assay time is needed to ensure the enzyme is fully functioning. To address such issues, a whole new approach to characterize PPIs in situ using cleavable fluorescent tyramide (CFT) and click chemistry is proposed and demonstrated in the following sections.

[0118] Novel Approach to Characterize PPIs Using CFT and Click Chemistry

[0119] The central concept of applying CFT to characterize PPI is through click chemistry between the azido group, which is the cleavable site of the CFT, and the alkyne. Theoretically, if the azido group undergoes click chemistry with an alkyne, the formerly cleavable CFT should become non-cleavable. Thus, to identify if the two proteins are in the same complex or not, a set of two probes with a CFT and an alkyne-tyramide may be deposited to the two target proteins respectively, using the approach for reiterative immunohistochemistry developed in references 52- 57 . When the two target proteins are physically close enough, the two probes will have the capability to perform click chemistry between each other to form a five-member ring crosslinking the two probes together (FIG. 1). After the formation of the five-member ring, the azido group of the CFT can no longer perform the Staudinger reaction to remove the fluorescence signal under the presence of TCEP or PTA. This means that after applying the signal removal condition, if the fluorescence signal still shows up under fluorescence microscopy, the two protein targets are inproximity and have a certain level of interaction.

[0120] The chemical structural design of the alkyne probe starts with a tyramide residue since the whole probe needs to be introduced to the target protein with tyramide signal amplification (TSA). The other end of the probe is designed to be an alkyne to perform click chemistry with the azido group of the CFT. To increase the length of the alkyne probe and incorporate multiple disulfide bonds into the structure at the same time, the synthesis of the probe was designed to conjugate the two ends together using a click reaction. With the modification of disulfide bonds on both the tyramide end and alkyne end, tetrazine and trans-cyclooctene (TCO) are conjugated to the tyramide and the alkyne during the process. TCO and tetrazine can then crosslink to give the final structure of the alkyne probe (FIG. 2).

[0121] The long linear structure of the alkyne probe with many free rotation bonds helps increase the probe's flexibility and theoretically increases the odds of the alkyne probe getting into the distance close enough to the CFT for click chemistry. Disulfide bonds can react with TCEP, so by incorporating them, the alkyne probe may be cleaved to remove the alkyne end of the probe from the tyramide end attached to the target, just like the CFTs when the cleavage condition is applied. Such a design aims to increase the multiplexity of the PPI assay with the same concept as CFT. By removing the probe at the end of one target complex, the same sample can go through another cycle of analysis targeting different proteins of interest. The synthetic route of the alkyne probe is shown in FIG. 3. The chemical structure of the alkyne probe was designed with a tyramine residue to allow its introduction to the target protein using tyramide signal amplification (TSA). The other end of the probe was designed as an alkyne to enable click chemistry with the azido group of the cleavable fluorescent tyramide (CFT). To increase the length of the alkyne probe and incorporate multiple disulfide bonds, the synthesis strategy involved conjugating the two ends using a click reaction. During the process, tetrazine and trans-cyclooctene (TCO) were conjugated to the tyramine and alkyne ends, respectively, enabling crosslinking and forming the final alkyne probe structure.

[0122] The synthesis process started with a commercially available tetrazine-conjugated disulfide compound and NHS ester (compound 1) for further conjugation. The tetrazine group was then linked to tyramine, generating compound 2, which facilitated its deposition onto the target protein via TSA. A separate conjugation reaction was performed to obtain the alkyne moiety, beginning with a commercially sourced molecule featuring an alkyne functional group, a PEG4linker, and a disulfide bond, ending in an NHS ester (compound 3). This molecule was then conjugated to trans-cyclooctene (TCO), which had another disulfide bond with an amino-terminal group, resulting in compound 4, forming the TCO-alkyne complex. The final alkyne probe was assembled by crosslinking the tetrazine-tyramide and TCO-alkyne components, incorporating three disulfide bonds within the final structure.

[0123] The reaction between tetrazine and TCO proceeded via catalyst-free click chemistry, which was carried out by mixing the two under slightly basic conditions. The reaction progress was monitored visually, as the TCO solution appeared light violet and became colorless upon complete reaction with tetrazine. Once the solution turned transparent, the synthesis of the alkyne probe was considered complete. Due to the small reaction scale and high efficiency, no purification was performed. The final concentration of the alkyne probe solution was adjusted to 1 mM, assuming a 100% reaction yield for TCO-tetrazine crosslinking in a 1 : 1 molar ratio.

[0124] After obtaining the alkyne probe, a human tonsil FFPE tissue was prepared to examine the feasibility and multiplexity of the concept characterizing PPI with CFT and click chemistry with the workflow in FIG. 4. Three different combinations of antibodies were selected to deposit the CFT and the alkyne probe, respectively. Proteins known to have physical interaction (histone H3 and histone H4) or spatially separated (H3S10P and Na+ / K+-ATPAse, CD79a and HLADRA1) were included in the testing. Ideally, distinct results will be observed between different sets of target proteins. The staining signal should be removed normally after applying the click chemistry and the cleavage conditions for the set of proteins that are not close to each other. On the contrary, fluorescence signals at the position where the two proteins are close to each other should remain the same after all the steps. The same CFT (Cy3-N3-Tyr) was used for all three sets of target proteins for a better comparison and quantification of the signal intensity as well as the cleavage efficiency when different sets of antibodies were stained.

[0125] Since the alkyne probe does not generate visible signals under microscopy, it was deposited together with the Cy5-Tyramide for visible signals by TSA to track the locations of the alkyne probes. For the first set of antibodies, histone H3 and histone H4 were selected since they are known to form tetramers during nucleosome assembly and should be in close proximity under normal conditions. Histone H4 was stained with the alkyne probe and Cy5-Tyramide for visualization (52-57). After HRP deactivation, histone H3 was stained with Cy3-Na-Tyr. After taking the staining images, the click chemistry condition was applied by applying copper(II)sulfate solution for thirty minutes at room temperature. The cleavage conditions for the CFTs were applied to the tissue right after click chemistry, and the images were captured again using fluorescence microscopy. As expected, the fluorescence signals were not removed by TCEP and PTA (FIG. 5), the fluorescence images and the intensity profile both show that the Cy3 staining did not decrease after all the steps. Such a result proves the formation of the crosslinking five- member ring between the CFT and alkyne probe. With the azido group of the CFT involved in the click chemistry reaction, the ability of the CFT to undergo the Staudinger reaction is hindered, resulting in remaining signals after the cleavage reaction.

[0126] The disulfide bonds in the alkyne probes must be efficiently removed during the signal removal process to guarantee the multiplexity of the assay. To verify the cleavage of the disulfide bond, right after the testing with the first set of antibodies targeting histone H3 and H4. Secondary antibodies conjugated with HRP were added to the tissue, followed by TSA buffer to deposit Cy3-N3-Tyr on the histone proteins again. The images were taken, and the slide underwent another round of click chemistry and cleavage conditions. If the disulfide bonds can’t be removed entirely by TCEP and PTA, the click chemistry will happen, and the staining signal will become non-cleavable. On the contrary, the removable staining signal represents an effective cleavage of the disulfide bonds. As expected, the Cy3 signal was removed after the incubation of the reducing reagents, showing that the alkyne probe cleavage is efficient (FIG. 6).

[0127] Another set of antibodies targeting Na / K+-ATPAse and phosphorylated histone H3 were also selected and stained with the same set of CFT and alkyne probes. Na+ / K+-ATPAse is known to have a membranous expression, while modified histone has its expression in the nucleoplasm. As the two proteins are spatially separated, applying the click chemistry condition will not cause a click reaction to occur between the CFT and alkyne probes. Thus, after the cleavage condition is applied after the click chemistry reaction, the staining signal from CFTs should still be removable. H3S10P was stained with Cy5-Tyramide and the alkyne probe for this set of antibodies, followed by Na+ / K+-ATPAse stained with Cy3-N3-Tyr. Click chemistry condition was then applied, as well as the cleavage condition. The result showed that the staining signal on the membrane could be effectively removed by TCEP and PTA (FIG. 7), indicating that click chemistry did not happen between the alkyne probes inside the nuclei and the CFTs on the cellular membrane.

[0128] With two sets of antibodies having already stained and characterized the PPIbetween the targets consecutively on the same slide, one more set of antibodies was selected to better showcase the assay's multiplexity. CD79a and HLA-DRA1 antibodies were applied to the tissue and stained with an alkyne probe and Cy3-N3-Tyr, respectively. The clear and distinct staining patterns of the two proteins can be captured. At the same time, the click chemistry showed no apparent interaction between CD79a and HLA-DRA1 in normal human tonsils since the fluorescence signal can be removed by applying cleavage conditions after the click chemistry (FIG. 8). The staining and click chemistry results suggest that the previous two other sets of antibodies do not affect the following experiments.

[0129] Comparing the results from the three sets of antibodies, it is clear that the ability of the fluorescence signal to be removed in the click chemistry PPI assay is highly related to the subcellular expression location of the selected protein targets. For histone H3 and H4, the staining signal of Cy3-N3-Tyr and Cy5 (representing the alkyne probes) is highly overlapped (FIG. 9A). On the other hand, the Cy3 and Cy5 signal are spatially separated in the zoomed-in images of protein sets H3S10P / Na' / K -ATPAse and CD79a / HLADRAl (FIG. 9B, 9C). The average cleavage efficiency of roughly thirty thousand identified cells was calculated to further confirm the relationship between the cleavage efficiency of the CFT and whether the selected proteins are in proximity (FIG. 9D). Based on the evidence, the cleavage efficiency can act as an indicator of highly possible interaction between the target proteins in this assay.

[0130] The CFT and click chemistry-based PPI assay provides robust evidence to reveal whether the two select target proteins are in proximity or not to have a high possibility of having a certain level of interaction between the two. One huge advantage of the assay is that it shows possible interactions between the two targets and simultaneously shows the expression of the target proteins. Compared to PLA, which shows fluorescence signals as spots when the two proteins are in close contact with each other, the click chemistry approach can provide more information with better sensitivity while investigating PPI in situ. Additionally, the CFT and click chemi stry-based assay can be combined with reiterative immunofluorescence for multiplexed protein staining, with no extra steps needed. The newly proposed PPI assay possesses a higher multiplexity than PLA and can theoretically characterize PPI between multiple targets simultaneously in one cycle of protein staining. The testing so far has only been done by applying the alkyne probe and the CFT with one specific fluorophore and by including more CFTs to stain different target proteins after, just like the multicycle staining. Then, applying the click chemistry and cleavage conditions to thetissue, characterization between the protein with alkyne probe deposited to all other target proteins with different CFTs can be achieved. Such an approach can significantly reduce the time to screen different PPIs, including the same protein, and possess great potential in various applications.

[0131] Protocol of PPI Characterization Using CFT and Click Chemistry

[0132] Deparaffinization and Antigen Retrieval of FFPE tissues:

[0133] The tissue preparation and pretreatment for click chemistry PPI assay, including deparaffinization, rehydration, heat-induced antigen retrieval, endogenous HRP deactivation, and blocking, is the same as reiterative immunofluorescence addressed in references 52-57 (Examples 1-2).

[0134] PPI characterization with click chemistry on FFPE tissues:

[0135] 1. Select two primary antibodies for the PPI assay.

[0136] 2 Mix 1 pL of the first antibody and primary antibody labeling solution (for every one pg of the antibody, add 5 pL of the primary antibody labeling solution) in 5 pL of PBS and let sit for five minutes at room temperature.

[0137] 3. Add the same amount of blocking solution to the antibody solution. Mix and let sit for five minutes at room temperature.

[0138] 4. Dilute the antibody solution with antibody-blocking buffer to the desired working concentration of the antibody.

[0139] 5. Incubate the FFPE tissue with the antibody solution at a working concentration for forty-five minutes under room temperature. Rinse the slide with phosphate buffered saline with Tween (PBT) three times after incubation.

[0140] 6. Incubate the FFPE tissue with streptavidin HRP solution (5 pg / mL diluted in PBT) for thirty minutes under room temperature. Rinse the slide with PBT three times after incubation.

[0141] 7. Incubate the FFPE tissue with fluorophore-tyramide and alkyne probe inTSA buffer (1 pL of fluorophore-tyramide and 1 pL of alkyne probe with a concentration of 0.5 mM, dilute 1 pL of 0.3 % hydrogen peroxide in 200 pL of 0.1M boric acid PBT solution) for 10 minutes under room temperature. Rinse the slide with PBT three times after incubation.

[0142] 8. For HRP blocking, apply the HRP blocking solution to the FFPE tissue and incubate for thirty minutes at room temperature. Rinse the slide with PBT three times after incubation.

[0143] 9. Repeat step 2. to step 6. with the second selected antibody.

[0144] 10. Incubate the FFPE tissue with CFT in TSA buffer (1 pL of CFT with a concentration of 0.5 mM, dilute 1 pL of 0.3 % hydrogen peroxide in 200 pL of 0.1M boric acid PBT solution) for 10 minutes under room temperature. Rinse the slide with PBT three times after incubation.

[0145] 11. Mount the cover glass with Prolong Diamond Antifade Reagent and capture the staining image with fluorescence microscopy.

[0146] 12. Incubate the slide with the click chemistry buffer containing 0.6 mMCuSO4, three mM THPTA in PBS, and four mM of sodium ascorbate (freshly made and added to CuSO4 immediately before adding to slide) for 30 minutes under room temperature. Rinse the slide with PBT three times after incubation.

[0147] 13. Prepare 0.1 M TCEP solution in PBT and tune the pH with IM sodium hydroxide to pH 9. Incubate the tissue with the TCEP solution for 30 minutes in the oven at 40 °C. Rinse the slide with PBT three times after incubation.

[0148] 14. Incubate the tissue with 0.1M PTA solution in PBT for 30 minutes in the oven at 40 °C. Rinse the slide with PBT three times after incubation.

[0149] 15. Mount the cover glass with Prolong Diamond Antifade Reagent and capture the cleavage images.

[0150] 16. Chemical bleaching of the dye was done by incubating the fluorophore bleaching solution from Spatomics for 10 minutes in the oven at 40 °C to remove the fluorescence signal that cannot be removed due to click chemistry.

[0151] 17. To investigate PPI between the other two target proteins, repeat step 1. to step 16.

[0152] Example 2

[0153] Diagnostic Applications of PPI Characterization Using CFT and Click Chemistry

[0154] According to the Centers for Disease Control and Prevention, cancer is one of the leading causes of death in the United States, with over a million new cases reported each year. In the past few decades, tremendous efforts have been devoted to the field, and significant progress has been made. In cancer treatment, the revolution has been made by gradually shifting the use ofchemotherapy or radiation into immunotherapies. Ever since the first clinical success of blocking Cytotoxic T-lymphocyte associated protein 4 (CTLA-4)41, 42and Programmed Death 1 (PD-1)43'44was published, immune-checkpoint blockade (ICB) has drawn significant focus from the research field. Numerous new targets and drugs have been explored, though most are still in the early development stages; the FDA has already approved some for clinical use. Despite cases showing positive clinical benefits and durable responses, ICB drugs actually suffer from low response rates from the majority of patients. On average, only around 30% of response rate can be found in most of the cancers43; the highly heterogeneous tumor immune microenvironment (TIME) and the lack of proper diagnostic testing to maximize personalized treatment for the best efficacy are two of the many factors affecting the treatment.

[0155] Take PD-1 as an example; it is known for its role in controlling autoimmune response by down-regulating the function of T lymphocytes45, 46. When PD-L1, a protein found in several types of tumors, binds to PD-1, the immune response will be suppressed to help the tumor cells escape from the immune system47. The ICB drugs targeting either PD-1 or PD-L1 can bind to the protein to suppress the interaction between the two proteins, resulting in the reactivation of the immune response to fight against cancer cells48. Recent studies suggest that TIME plays a vital role in whether the ICB treatment will have a positive response or not. Tumors presenting a population of immune cells but lacking cytotoxic lymphocytes (CTLs) in the core are classified as infiltrated-excluded (I- E) TIMEs. This type of TIME is often associated with epithelial cancers such as melanoma, colorectal cancer, and pancreatic ductal adenocarcinoma. Tumors identified as I-E TIME have usually been considered poorly immunogenic due to the lack of immune cells in the core. On the other hand, tumors with a high population of infiltrated CTLs and leukocytes in the core with tumor cells expressing PD-L1 are classified as Infiltrated-inflamed (I— I) TIMEs. This type of TIME is considered immunologically active and more suitable for ICB treatment. Because the diversity of different TIMEs influences the response to therapy, the ability to understand and identify the TIME quickly and accurately can help improve the response rate of the treatment or even discover new therapeutic targets.

[0156] Diagnostic testing can be the key to solving the problem of why more patients are experiencing minimal or no response from the same treatment, and some patients have pronounced results. In clinical practice, the prescription criteria did not include the expression of PD-L1 on tumor cells for some ICB drugs. While other drugs do require confirmed PD-L1 overexpressionon tumor cells, even in certain cancers found to be more reactive to PD-1 inhibitors, such as nonsmall cell lung cancer and renal cell carcinoma, the response rate is still less than 50%. Such a result suggests that PD-L1 expression does not guarantee the response of the treatment, and a more accurate criterion is needed. Moreover, there are thousands of ongoing clinical trials related to cancer immunotherapy. This means in the near future, how physicians select the most suitable treatment for each patient will become a much more severe issue. The current challenges all emphasize the need for a proper diagnosis and a more personalized treatment to maximize the efficacy of cancer immunotherapy.

[0157] The click chemistry PPI assay has the potential to solve the issue of the lack of diagnostic tools for cancer immunotherapy since the assay has the multiplexity to characterize multiple sets of protein complexes on the same sample, which is especially important due to the fact that the amount of patient biopsy samples is minimal. The ability to identify which set of ICB drug targets and their receptors have actual interactions with each other in each tumor being tested. Based on PPI results instead of protein expression, hopefully, the selection of ICB drug can be more accurate, and the overall response rate for the treatment can be increased. More importantly, the assay can be carried out in standard clinical and laboratory settings, which significantly increases its accessibility.

[0158] A demonstration of applying the click chemistry PPI assay for cancer diagnostic applications is shown below. A cancer panel slide with FFPE tissues of breast cancer (invasive ductal carcinoma), colon cancer (adenocarcinoma), liver cancer (hepatocellular carcinoma), lung cancer (squamous cell carcinoma), prostate cancer (adenocarcinoma), and control tissues for each cancer and human tonsil were prepared. Multiplexed PPI characterization of five sets of different proteins, including PD-1 / PD-L1, CD3 / CD4, CD45 / HLADRA1, histone H3 / histone H4, and Ku80 / Vimentin, was done on the slide to showcase the feasibility and potential of the technique in clinical practices.

[0159] The first is PD-1 and PD-L1, the most common immune checkpoint targets in ICB treatment, with PD-1 stained with the alkyne probe and Cy5 while PD-L1 labeled with Cy3-Ns- Tyr. The two proteins show distinct staining signals on the tonsil control tissue, suggesting the two antibodies work properly (FIG. 10A). The expression of the two proteins in cancer tissues varies. PD-1 and PD-L1 have higher expression in the breast cancer tissue than in the control tissue (FIG. 10B, 10C.). In contrast, the two proteins are better expressed in the control colon tissue than in thecolon cancer tissue (FIG. 10D, 10E). As for liver, lung, and prostate cancer, the PD-1 and PD-L1 expressions mainly overlap (FIG.1 OF- 1 OK).

[0160] Surprisingly, after applying the click chemistry and cleavage conditions, the fluorescence signal was removed not only in the tonsil control tissue but also in the cancer tissues where the staining pattern of the two proteins overlapped (FIG. 11). Such a result indicates the chance of PD-1 and PD-L1 interacting with each other is low in these five cancer tissues and the control tissue. This may be why the average response rate is lower than 50% for PD-1 / PD-L1 ICB treatment even when the drug was prescribed based on PD-L1 expression level in the tumor. A higher expression of PD-L1 does not guarantee the interaction between PD-1 and PD-L1 since cancer cells can escape from the immune system through several different pathways. Such a conclusion again emphasizes the importance of proper diagnosis before ICB treatment for personalized medication and maximized efficacy.

[0161] On the same slide, CD3 and CD4 are stained with the alkyne probe and Cy3-N3- Tyr, respectively. As shown in the images, the expression of these two proteins is highly overlapped as CD4 is known to act as a coreceptor of T-cell receptors49. The click chemistry and cleavage results also show that the two proteins are in proximity and are likely to interact with each other as the fluorescence signal is not removable (FIG. 12).

[0162] CD45 and HLADRA1 are stained with the alkyne probe and Cy3-N3-CFT as the third set of proteins studied on the slide. With this set of proteins, the advantage of visualizing protein expression while characterizing PPI is that HLADRA1 shows different expressions in different tissues. For example, HLADRA1 is mainly expressed in the secondary follicle in the control tonsil tissue (FIG. 13 A). Meanwhile, in the breast and prostate, it shows much higher expressions in cancer tissues and low expressions in normal tissues (FIGS. 13B, C, J, K). Lastly, in colon, liver, and lung tissues, HLADRA1 shows high expressions in normal tissues but low in cancer tissues (FIG. 13D-I). Such trends of protein expressions are hard to observe when other in- situ PPI assays like PLA are used.

[0163] Not only is there a difference in the expression of the protein in different tissues, but also in the signal removal results after click chemistry. As shown in the results below, the cleavage efficiency of the CFT in tonsil and breast tissues is much lower than in other tissue types (FIG. 14). Although no evidence or research indicates a direct interaction between CD45 and HLADRA1 so far, these two proteins are sometimes found to be correlated with each other underdifferent conditions50, 51. It may not be accurate enough to say that there is interaction between these two proteins solely based on the result of the non-removable fluorescence signal and the images of protein staining. However, the two proteins are in proximity or co-expressed in the same cell in the tonsil and breast cancer tissue while further separate or not expressed in the other four cancer tissues.

[0164] The fourth (histone H3 and histone H4) and fifth set (Ku80 and Vimentin) of proteins stained on the slide are two sets of interacting and physically separated proteins. The results follow the expectation that the fluorescence signal is not removable for histone proteins but is removable for Vimentin (FIGS. 15, 16). These two sets of proteins also act as a control group for the credibility of the previous three sets of proteins and demonstrate the multiplexity of the assay.

[0165] In summary, PPI between five different pairs of proteins was characterized in a set of control and cancer tissues, including a common ICB treatment target PD-1 / PD-L1, two sets of immune cell markers CD3 / CD4 and CD45 / HLADRA1, and two sets of control group H3 / H4 and Ku80 / VIM. The results show that PD-1 and PD-L1 do not interact in the five cancer tissue samples. This explains the low overall response rate in ICB treatment under current prescription criteria and highlights the significance of personalized medication with better diagnosis. Moreover, by combining immunofluorescence and PPI assay, the expression of the protein targets can be characterized and studied simultaneously, which is beneficial to biological studies. The multiplexity of the click chemistry and CFT-based PPI assay is higher than any other in-situ PPI assays. Theoretically, it does not have an upper limit as long as the antigen integrity and the sample's morphology are well preserved.

[0166] One massive potential of this PPI assay is the ability to characterize interactions between different omics. Since the alkyne and fluorescence probes are both deposited to the target through TSA, the target is not limited to proteins only in that case. When multi-omics technologies become available in the near future, the interactions between transcriptomics, proteomics, and other biomolecules can be achieved to further advance the research to a higher level.

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[0168] Example 3

[0169] 1.1 Critical need for imaging protein interactome in FFPE tissues

[0170] Protein-protein interaction (PPI) plays key roles in various cellular processes, such as transcription, translation, signaling, development, and diseases1’4. With yeast two-hybrid screening, the verified human binary protein interactome analysis identifies at least 52,569 PPI involving 8275 varied proteins5. In biological systems, these PPI are often formed in molecularly and functionally distinct cells. Thus, comprehensive PPI profiling in single cells in situ is crucially required to advance our understanding of the spatial organization, expression regulation, and cellcell interactions in multicellular organisms.

[0171] Formalin-fixed paraffin-embedded (FFPE) tissues are the most common type of preserved clinical samples, and are extensively used for routine diagnosis and studies of disease mechanisms6. Clearly, imaging protein interactome in FFPE tissues is critically needed to advance our understanding of disease pathogenesis and enhance the diagnosis accuracy.

[0172] 1.2 Outstanding challenges in imaging protein interactome in FFPE tissues

[0173] Although different PPI profiling methods have been developed, four fundamental limitations still exist (Table 1). (i) Without single cell resolution and in situ analysis: To enablePPT analysis at the proteome level, different approaches have been developed, including yeast two- hybrid (Y2H), affinity purification mass spectrometry (AP-MS), cross-linking mass spectrometry (CL-MS), phage display, protein microarray, and proximity labeling (PL)7. However, all these methods are carried out on isolated and purified proteins. As a result, the information on the microenvironment of the PPI and their heterogeneity between different compartments or single cells are lost during analysis, (ii) Low multiplexing capacity: Imaging-based approaches have been explored to quantify PPI in their native cellular contexts at the subcellular resolution. These methods include proximity ligation8, hybridization chain reaction (HCR)9, fluorescence resonance energy transfer (FRET)10, and super-resolution microscopy (SRM)11. Nonetheless, due to the spectral overlap of common fluorophores and / or the limited number of highly specific and orthogonal oligonucleotide probes, these fluorescence microscopy methods can only quantify no more than 3 pairs of PPI in one specimen, (iii) Limitations on FFPE tissue profiling: Without signal amplification, FRET and SRM usually can’t detect PPI in highly autofluorescent FFPE tissues. And to amplify signals, proximity ligation and HCR require the formation of large complexes composed of varied biomolecules. In heavily fixed and crosslinked FFPE tissues, the generation of such large complexes could have low yield. Also, as it is challenging to isolate the proteins from crosslinked FFPE tissues, the PPI interactome analysis methods are not applicable to FFPE tissues, (iv) No hub protein interactome analysis: Hub proteins are the highly connected nodes, and tend to be essential and lethal in the PPI network. The imaging-based approaches only allow the pairwise PPI analysis. They can’t simultaneously quantify the large number of proteins interacting with the same hub protein. Thus, currently no methods allow comprehensive PPI profiling in single cells in situ.

[0174] Table 1. Technologies for PPI profiling

[0175] 2.1 Highly multiplexed single-cell in situ PPI profiling in FFPE tissues

[0176] Rationale. To enable highly multiplexed PPI imaging in FFPE tissues, we will develop clickable and cleavable probes (FIG. 17). In this approach, a pair of antibodies are applied to recognize their targets in their native cellular contexts. One antibody is tethered to alkyne through a disulfide linker; while the other antibody is conjugated with tetrazine through an azide- based cleavable linker. Only when the two protein targets are in close proximity, Cu catalyzed click chem will quench the azide-based cleavable linker. As a result, the tetrazine on the antibody will not be cleaved by the mild reducing reagent tris(2-carboxyethyl)phosphine (TCEP). Subsequently, trans-cyclooctene (TCO) labeled HRP will couple with the tetrazine in situ, similarly as we demonstrated in our recent publication12. This HRP will enzymatically deposit a large number of CFT molecules to fluorescently label the detected PPI. In contrast, the click chem will not occur when the two protein targets are not in sufficient close proximity. Consequently, tetrazine will be cleaved by TCEP and washed away. And no further fluorophore deposition will happen. As we demonstrated in our recent publication13 14, the fluorophores on CFT will be chemically cleaved by TCEP, which also simultaneously deactivates HRP to initiate the next PPI profiling cycle. Through reiterative analysis cycles, highly multiplexed PPI imaging can be achieved in single cells in situ in FFPE tissues. With HRP catalyzed enzymatic signal amplification and by avoiding the formation of large biomolecule complexes, this approach can sensitively detect PPI in highly autofluorescent FFPE tissues.

[0177] Results. To demonstrate the feasibility of this approach, we designed and synthesized cleavable and clickable probes conjugated antibodies (FIG. 17B), using the approach similar to the one in our recent publication15. To access whether these chemically modified antibodies can still recognize their targets, we stained protein H3S10P, Na / K ATPase (FIG. 18 A) and histone H3, H4 (FIG. 18C) in FFPE tissues. The staining results are consistent with the conventional immunohistochemistry results with unconjugated primary antibodies (FIG. 18B, 18D). These results suggest that the cleavable and clickable probes conjugated antibodies can still bind to their targets with high specificity and affinity. Applying these modified antibodies, PPI between H3 and H4 (Fig. 18E) are successfully detected on a human FFPE tissue. In the sequent cycle on the same tissue, PPI between H3S10P and Na / K ATPase is analyzed (FIG. 18F). As thesetwo proteins are in different cellular compartments (FIG. 18A, 18B), no detected PPI staining signal is observed. The results from these positive and negative control experiments indicate the feasibility of applying our approach for highly multiplexed imaging of PPI in FFPE tissues.

[0178] Experimental Design. To detect PPI at varied distances, cleavable linkers with different length are designed and synthesized. Poly (ethylene glycol) (PEG) linkers with 1-20 monomers or linkers with multiple cleavage sites as we recently developed12are applied to conjugate antibodies. To further enhance the cleavage efficiency and reduce the assay time, the azide-based linker is optimized. New linkers with strong electron-donating groups to both sides of the functional moiety -O-CH(Ns)- are explored (FIG. 19A). Oxygen is substituted to sulfur and its cleavage effectiveness is evaluated (FIG. 19A). As disulfide bond potentially reacts with thiol groups or other nucleophiles in cells, allyl-based cleavable linkers are designed and synthesized (FIG. 19B), which have been successfully applied in DNA sequence by synthesis16. Linkers with substituents on one end or on both ends of the allyl moiety are tested. Different cleavage conditions with varied reagent concentrations, pH, reaction time or reaction temperature, are evaluated.

[0179] 2.2 Single-cell in situ hub protein interactome profiling in FFPE tissues

[0180] Rationale. Recently, many hub protein interactomes have been explored by proximity labeling17, 18. However, as the labeled proteins are isolated and then quantified by mass spectrometry. All the current hub protein interactome profiles are not at the single cell resolution. And the information on PPI’s tissue location and their nearby cells is also lost. To enable singlecell in situ hub protein interactome profiling in FFPE tissues, clickable and cleavable oligonucleotides conjugated antibodies are developed. In this approach (FIG. 20), a library of antibodies tethered to varied oligonucleotide sequences through azide-based cleavable linkers are applied to recognize the potential hub protein interactome, which have been identified by proximity labeling. Subsequently, the hub protein is bound by HRP conjugated antibodies. As demonstrated and used in proximity labeling, peroxidase catalyzes the deposition of cleavable alkyne tyramide within the radius of 20 nm of the hub protein19. As a result, only the azide-based cleavable linkers in close proximity of the hub protein are quenched by the subsequent click chemistry reaction. The following chemical cleavage reaction only removes the oligonucleotides on the distal proteins and the unreacted quenchers. After post-fixation, reiterative cycles of PPI detection is carried out, including HRP conjugated oligo hybridization, staining by cleavable fluorescent tyramide, fluorophore cleavage and HRP deactivation. We have documented the sameprocedure for multiplexed RNA detection in FFPE tissues in our recent publication20. In this way, for the first time direct visualization of the hub protein interactome in single cells of FFPE tissues in situ is enabled.

[0181] Experimental Design. To prepare cleavable oligonucleotides conjugated antibodies, cleavable 4-formylbenzamide NHS ester is synthesized, similarly to cleavable fluorophore NHS esters as we prepared before15. Then, the oxime ligation reation21is applied to tether oligonucleotides to antibodies through the azide-based cleavable linker (FIG. 21). Up to 100 unique oligonucleotides from the 240,000 orthogonal sequences are selected, which have been established to minimize cross-hybridization22. The binding affinity and specificity of the conjugated antibodies revalidated by comparing with the unconjugated antibodies, as in FIG. 18.

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Claims

CLAIMS1. A kit comprising: an alkyne-signal amplification probe comprising formula I or a salt thereofwherein R1 - R5 are each independently selected from H, alkyl, alkenyl, alkynyl, halogen, carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronyl, aryl, and heteroaryl; wherein Linker comprises a cleavable linker; wherein Y is selected from hydrogen, a counter cation, and a moiety that can be cleaved by an enzyme, an ion, light, a base, or an acid; wherein m is an integer from 1 to 100; and a cleavable detection probe (CDP) comprising formula II or a salt thereofwherein X is O or S; wherein R1 - R4 are each independently selected from H, alkyl, alkenyl, alkynyl, halogen, carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronyl, aryl, and heteroaryl, wherein optionally R1 and R2 form a 3-10 member ring; wherein Y is selected from hydrogen, a counter cation and a moiety that can be cleaved by an enzyme, an ion, light, a base, or an acid; wherein TAG is a biologically detectable moiety comprising a fluorophore, a chromophore, or a metal isotope; wherein Linker 1 and Linker 2 each comprise a rigid chain or a flexible chain; and wherein n is an integer from 1 to 100.

2. The kit of claim 1, wherein the alkyne-signal amplification probe comprises: a terminal alkyne; and a terminal tyramide; and wherein the cleavable linker comprises at least one disulfide bond.

3. The kit of claim 1 or 2, wherein the alkyne-signal amplification probe comprises formula4. The kit of any one of claims 1-3, wherein the fluorophore of the CDP is Cy3 or Cy5.

5. The kit of any one of claims 1-4, further comprising a fluorophore-tyramide comprising a fluorophore and a tyramide.

6. The kit of claim 5, wherein the fluorophore of the CDP and the fluorophore of the fluorophore-tyramide are different.

7. The kit of claim 5 or 6, wherein the fluorophore of the fluorophore-tyramide is Cy5.

8. The kit of any one of claims 1-7, further comprising at least two antibodies, wherein each antibody is specific for a different protein, and wherein each antibody is conjugated to HRP or biotin.

9. The kit of any one of claims 1-8, further comprising a click chemistry buffer.

10. The kit of any one of claims 1-9, further comprising at least one of a tris(2- carboxyethyl)phosphine (TCEP) solution and a l,3,5-triaza-7-phosphaadamantane (PTA) solution.

11. The kit of any one of claims 1-10, further comprising a streptavidin-HRP solution.

12. A method for characterizing the proximity of a first protein and a second protein in a fixed tissue, the method comprising:(a) contacting the tissue with an HRP-labeled first antibody that specifically binds to the first protein;(b) contacting the tissue with an alkyne-signal amplification probe comprising a cleavable linker, a fluorophore-tyramide comprising a fluorophore and a tyramide, and a tyramide signal amplification (TSA) buffer;(c) contacting the tissue with an HRP blocking solution;(d) contacting the tissue with an HRP-labeled second antibody that specifically binds to the second protein;(e) contacting the tissue with a cleavable detection probe (CDP) and the TSA buffer;(f) visualizing the fluorescence of the fixed tissue;(g) contacting the tissue with a click chemistry buffer;(h) contacting the tissue with a tris(2-carboxyethyl)phosphine (TCEP) solution;(i) contacting the tissue with a l,3,5-triaza-7-phosphaadamantane (PTA) solution; and(j) visualizing the fluorescence of the fixed tissue; wherein the alkyne-signal amplification probe comprises formula I or a salt thereof(i),wherein R1 - R5 are each independently selected from H, alkyl, alkenyl, alkynyl, halogen, carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronyl, aryl, and heteroaryl; wherein Linker comprises a cleavable linker; wherein Y is selected from hydrogen, a counter cation, and a moiety that can be cleaved by an enzyme, an ion, light, a base, or an acid; and wherein m is an integer from 1 to 100; and wherein the CDP comprises formula II or a salt thereof(II), wherein X is O or S; wherein R1 - R4 are each independently selected from H, alkyl, alkenyl, alkynyl, halogen, carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronyl, aryl, and heteroaryl, wherein optionally R1 and R2 form a 3-10 member ring; wherein Y is selected from hydrogen, a counter cation and a moiety that can be cleaved by an enzyme, an ion, light, a base, or an acid; wherein TAG is a biologically detectable moiety comprising a fluorophore, a chromophore, or a metal isotope; wherein Linker 1 and Linker 2 each comprise a rigid chain or a flexible chain; and wherein n is an integer from 1 to 100.

13. The method of claim 12, further comprising:(k) contacting the tissue with a fluorophore bleaching solution; and(l) repeating steps (a)-(k) at least one time; wherein each time at least one of the first protein and the second protein binds to a different first protein and a different second protein, respectively.

14. The method of claim 12 or 13, wherein the fixed tissue is a formalin-fixed paraffin- embedded tissue.

15. The method of any one of claims 12-14 wherein the alkyne-signal amplification probe comprises: a terminal alkyne; a terminal tyramide; and wherein the cleavable linker comprises at least one disulfide bond.

16. The method of any one of claims 12-15, wherein the alkyne-signal amplification probe comprises formula la17. The method of any one of claims 12-16, wherein the fluorophore of the fluorophore- tyramide and the fluorophore of the CDP are different.

18. The method of any one of claims 12-17, wherein the fluorophore of the CDP is Cy3 or Cy5.

19. The method of any one of claims 12-18, wherein the fluorophore of the fluorophore- tyramide is Cy5.

20. A kit comprising:a first antibody that specifically binds to a first protein, wherein the first antibody is conjugated to an alkyne probe through a disulfide linker; a second antibody that specifically binds to a second protein, wherein the second antibody is conjugated to a tetrazine probe having an azide-based cleavable linker; a trans-cyclooctene (TCO)-labeled HRP; and a cleavable detection probe (CDP).

21. The kit of claim 20, wherein the tetrazine probe comprises formula IIIwherein L is a linker, wherein Z is tetrazine, and22. The kit of claim 20 or 21, wherein the tetrazine probe comprises formula (IV)(IV).

23. The kit of any one of claims 20-22, wherein the alkyne probe comprises formula V(V).

24. The kit of any one of claims 20-23, wherein the CDP comprises formula II or a salt thereofwherein X is O or S; wherein R1 - R4 are each independently selected from H, alkyl, alkenyl, alkynyl, halogen, carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronyl, aryl, and heteroaryl, wherein optionally R1 and R2 form a 3-10 member ring; wherein Y is selected from hydrogen, a counter cation and a moiety that can be cleaved by an enzyme, an ion, light, a base, or an acid; wherein TAG is a biologically detectable moiety comprising a fluorophore, a chromophore, or a metal isotope; wherein Linker 1 and Linker 2 each comprise a rigid chain or a flexible chain; and wherein n is an integer from 1 to 100.

25. The kit of claim 24, wherein the fluorophore is Cy3 or Cy5.

26. The kit of any one of claims 20-25, further comprising a click chemistry buffer.

27. The kit of any one of claims 20-27, further comprising at least one of a tris(2- carboxyethyl)phosphine (TCEP) solution and a l,3,5-triaza-7-phosphaadamantane (PTA)solution.

28. A method for characterizing the proximity of a first protein and a second protein in a fixed tissue, the method comprising:(a) contacting the tissue with(i) a first antibody that specifically binds to a first protein, wherein the first antibody is conjugated to an alkyne probe through a disulfide linker; and(ii) a second antibody that specifically binds to a second protein, wherein the second antibody is conjugated to a tetrazine probe having an azide-based cleavable linker;(b) contacting the tissue with a click chemistry buffer;(c) contacting the tissue with a tris(2-carboxyethyl)phosphine (TCEP) solution;(d) contacting the tissue with a l,3,5-triaza-7-phosphaadamantane (PTA) solution;(e) contacting the tissue with a trans-cyclooctene (TCO) labeled HRP, a cleavable detection probe (CDP), and a tyramide signal amplification (TSA) buffer;(f) visualizing the fluorescence of the fixed tissue;(g) contacting the tissue with the TCEP solution;(h) contacting the tissue with the PTA solution; and(i) visualizing the fluorescence of the fixed tissue.

29. The method of claim 28, further comprising:(j) contacting the tissue with a fluorophore bleaching solution; and(k) repeating steps (a)-(j ) at least one time; wherein each time, at least one of the first antibody and the second antibody binds to a different first protein and a different second protein.

30. The method of claim 28 or 29, wherein the fixed tissue is a formalin-fixed paraffin- embedded tissue.

31. The method of any one of claims 28-30, wherein the tetrazine probe comprises formula III(HI), wherein L is a linker, wherein Z is tetrazine, andwherein B is a reactive linker selected from, or32. The method of any one of claims 28-31, wherein the tetrazine probe comprises formula(IV)(IV).

33. The method of any one of claims 28-32, wherein the alkyne probe comprises formula V(V).

34. The method of any one of claims 28-33, wherein the CDP comprises formula II or a salt thereof(ii), wherein X is O or S; wherein R1 - R4 are each independently selected from H, alkyl, alkenyl, alkynyl, halogen, carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronyl, aryl, and heteroaryl, wherein optionally R1 and R2 form a 3-10 member ring; wherein Y is selected from hydrogen, a counter cation and a moiety that can be cleaved by an enzyme, an ion, light, a base, or an acid; wherein TAG is a biologically detectable moiety comprising a fluorophore, a chromophore, or a metal isotope; wherein Linker 1 and Linker 2 each comprise a rigid chain or a flexible chain; and wherein n is an integer from 1 to 100.

35. The method of claim 34, wherein the fluorophore is Cy3 or Cy5.

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