Compositions and method for multiplex biomarker profiling

Abstract

Provided herein are compositions and methods for identifying or quantitating one or more analytes in sample. The composition can comprise an affinity molecule reversibly conjugated to a label moiety via a double-stranded nucleic acid linker or via an adaptor molecule. The affinity molecule and the label moiety can be linked to different strands of the double-stranded nucleic acid linker. Compositions can be used in any biological assays for detection, identification and/or quantification of target molecules or analytes, including multiplex staining for molecular profiling of individual cells or cellular populations. For example, the compositions can be adapted for use in immunofluorescence, fluorescence in situ hybridization, immunohistochemistry, western blot, and the like.

What is claimed is: 
     
       1. A composition comprising a plurality of affinity molecules, wherein:
 (i) each member of the plurality binds a target, wherein each affinity molecule is conjugated via hybridized first and second nucleic acid strands to a label moiety, wherein detectable properties of the label moieties are distinguishable from one another, wherein the targets are distinguishable from one another, wherein the hybridized first nucleic acid strands are distinguishable from one another, wherein members of the plurality of affinity molecules are distinguishable from one another, wherein the affinity molecule is linked to the first nucleic acid strand via an adaptor molecule or via a linker selected from the group consisting of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, sulfo-SMCC linker, succinimidyl-6-hydrazino-nicotinamide (S-HyNic) linker, N-succinimidyl-4-formylbenzamide (S-4FB) linker, bis-aryl hydrazone bond, an amide bond, two amide bonds on a spacer for cross-linking two —NH 2  groups triazole bond from “click” reaction), a phosphodiester linkage, a phosphorothioate linkage, or a combination thereof, and wherein the label moiety is linked to the second nucleic acid strand via an adaptor molecule or via a linker selected from the group consisting of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, sulfo-SMCC linker, succinimidyl-6-hydrazinonicotinamide (S-HyNic) linker, N-succinimidyl-4-formylbenzamide (S-4FB) linker, bis-aryl hydrazone bond, an amide bond, two amide bonds on a spacer for cross-linking two —NH 2  groups triazole bond from “click” reaction a phosphodiester linkage a phosphorothioate linkage, or a combination thereof; or   (ii) each member of the plurality binding a target, wherein each affinity molecule is conjugated to a first single strand nucleic acid molecule that is specifically hybridized to a second single strand nucleic acid molecule, wherein the second single strand nucleic acid molecule is conjugated to a label moiety, such that each affinity molecule is conjugated via hybridized first and second nucleic acid strands to a label moiety, wherein detectable properties of the label moieties are distinguishable from one another, wherein the targets are distinguishable from one another, wherein the first single strand nucleic acid molecules are distinguishable from one another, wherein members of the plurality of affinity molecules are distinguishable from one another, and wherein the affinity molecule is linked to the first single strand nucleic acid molecule via an adaptor molecule or via a linker selected from the group consisting of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, sulfo-SMCC linker, succinimidyl-6-hydrazino-nicotinamide (S-HyNic) linker, N-succinimidyl-4-formylbenzamide (S-4FB) linker, bis-aryl hydrazone bond, an amide bond, two amide bonds on a spacer for cross-linking two —NH 2  groups, triazole bond (from “click” reaction), a phosphodiester linkage, a phosphorothioate linkage, or a combination thereof, and wherein the label is linked to the second single strand nucleic acid molecule via an adaptor molecule or via a linker selected from the group consisting of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, sulfo-SMCC linker, succinimidyl-6-hydrazino-nicotinamide (S-HyNic) linker, N-succinimidyl-4-formylbenzamide (S-4FB) linker, bis-aryl hydrazone bond, an amide bond, two amide bonds on a spacer for cross-linking two —NH 2  groups triazole bond from “click” reaction a phosphodiester linkage, a phosphorothioate linkage, or a combination thereof.   
     
     
       2. A composition comprising a solid support comprising a sample for analysis for the presence of analytes, and a plurality of affinity molecules, each member of the plurality of affinity molecules conjugated to a first single-strand nucleic acid, wherein each member of the plurality of affinity molecules is bound to a different analyte in the sample, and wherein each first single-strand nucleic acid has a different nucleotide sequence, wherein members of the plurality of affinity molecules are distinguishable from one another, and wherein the affinity molecule is linked to the first single-strand nucleic acid via an adaptor molecule or via a linker selected from the group consisting of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, sulfo-SMCC linker, succinimidyl- 6 -hydrazino-nicotinamide (S-HyNic) linker, N-succinimidyl-4-formylbenzamide (S-4FB) linker, bis-aryl hydrazone bond, an amide bond, two amide bonds on a spacer for cross-linking two —NH 2  groups, triazole bond (from “click” reaction), a phosphodiester linkage, a phosphorothioate linkage, or a combination thereof. 
     
     
       3. The composition of  claim 2 , wherein the composition further comprises a plurality of second single strand nucleic acid molecules, each conjugated to a label moiety, wherein each second single strand nucleic acid molecule specifically hybridizes to a first single strand nucleic acid molecule conjugated to a member of the plurality of affinity molecules, such that at least a subset of the plurality of affinity molecules bound to the analytes in the sample is specifically associated with a plurality of label moieties, wherein detectable properties of the label moieties are distinguishable from one another, wherein members of the plurality of second single stand nucleic acid molecules are distinguishable from one another, and wherein the label moiety is linked to the second single strand nucleic acid molecule via an adaptor molecule or via a linker selected from the group consisting of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, sulfo-SMCC linker, succinimidyl-6-hydrazino-nicotinamide (S-HyNic) linker, N-succinimidyl-4-formylbenzamide (S-4FB) linker, bis-aryl hydrazone bond, an amide bond, two amide bonds on a spacer for cross-linking two —NH 2  groups, triazole bond (from “click” reaction), a phosphodiester linkage, a phosphorothioate linkage, or a combination thereof. 
     
     
       4. A method of analyzing a sample for a plurality of analytes, the method comprising, in order:
 (a) contacting the sample with a plurality of affinity molecules under conditions that permit specific analyte binding by the affinity molecules, wherein each affinity molecule specifically binds a different member of the plurality of analytes, and wherein each affinity molecule is conjugated to a first single strand nucleic acid, such that members of the plurality of affinity molecules become bound to members of the plurality of analytes present in the sample, wherein the first single strand nucleic acids are distinguishable from each other, wherein members of the plurality of affinity molecules are distinguishable from one another, and wherein the affinity molecule is linked to the first single strand nucleic acid via an adaptor molecule or via a linker 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, sulfo-SMCC linker, succinimidyl-6-hydrazino-nicotinamide (S-HyNic) linker, N-succinimidyl-4-formylbenzamide (S-4FB) linker, bis-aryl hydrazone bond, an amide bond, two amide bonds on a spacer for cross-linking two —NH 2  groups triazole bond from “click” reaction), a phosphodiester linkage, a phosphorothioate linkage, or a combination thereof;   (b) contacting the sample, under conditions that permit specific nucleic acid hybridization, with a first set of second single strand nucleic acid molecules, each conjugated to a label moiety, wherein each second single strand nucleic acid molecule specifically hybridizes to a first single strand nucleic acid molecule conjugated to a member of the plurality of affinity molecules, such that at least a subset of the plurality of affinity molecules bound to members of the plurality of analytes in the sample becomes specifically associated with a plurality of label moieties, wherein detectable properties of the label moieties are distinguishable from one another, wherein members of the first set of second single strand nucleic acid molecules are distinguishable from one another, and wherein the label moiety is linked to the second single strand nucleic acid molecule via an adaptor molecule or via a linker selected from the group consisting of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, sulfo-SMCC linker, succinimidyl-6-hydrazino-nicotinamide (S-HyNic) linker, N-succinimidyl-4-formylbenzamide (S-4FB) linker, bis-aryl hydrazone bond, an amide bond, two amide bonds on a spacer for cross-linking two —NH, groups, triazole bond (from “click” reaction), a phosphodiester linkage, a phosphorothioate linkage, or a combination thereof; and   (c) detecting signal from label moieties associated with affinity molecules bound to the sample, thereby detecting the presence or amount of at least a subset of the plurality of analytes.   
     
     
       5. The method of  claim 4 , further comprising the steps of:
 (d) erasing the signal from the label moieties conjugated to the first set of second single strand nucleic acid molecules;   (e) contacting the sample, under conditions that permit specific nucleic acid hybridization, with a second set of second single strand nucleic acid molecules, each conjugated to a label, wherein each second single strand nucleic acid molecule specifically hybridizes to a first single strand nucleic acid molecule conjugated to a member of the plurality of affinity molecules, such that a subset of the plurality of affinity molecules bound to members of the plurality of analytes in the sample becomes specifically associated with a plurality of label moieties, wherein members of the second set of second single strand nucleic acid molecules are distinguishable from one another and from members of the first subset of single strand nucleic acid molecules of step (b), and wherein the label moiety is linked to the second single strand nucleic acid molecule via an adaptor molecule or via a linker selected from the group consisting of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, sulfo-SMCC linker, succinimidyl-6-hydrazino-nicotinamide (S-HyNic) linker, N-succinimidyl-4-formylbenzamide (S-4FB) linker, bis-aryl hydrazone bond, an amide bond, two amide bonds on a spacer for cross-linking two —NH 2  groups, triazole bond (from “click” reaction), a phosphodiester linkage, a phosphorothioate linkage, or a combination thereof;   (f) detecting signal from label moieties associated with affinity molecules bound to the sample, thereby detecting the presence or amount of the subset of the plurality of analytes; and   (g) optionally repeating steps (d)-(f) with a further set of second single strand nucleic acid molecules.   
     
     
       6. A method of analyzing a sample for a plurality of analytes, the method comprising, in order:
 (a) contacting the sample with a first plurality of affinity molecules under conditions that permit specific analyte binding by the affinity molecules, wherein each affinity molecule specifically binds a different member of the plurality of analytes, wherein each affinity molecule is conjugated to a first single strand nucleic acid molecule that is specifically hybridized to a second single strand nucleic acid molecule, wherein the second single strand nucleic acid molecule is conjugated to a label moiety, such that each affinity molecule is conjugated via hybridized first and second nucleic acid strands to a label moiety and members of the plurality of different affinity molecules become bound to members of the plurality of analytes present in the sample, wherein detectable properties of the label moieties are distinguishable from one another, wherein the first single strand nucleic acids are distinguishable from each other, and wherein the affinity molecule is linked to the first single strand nucleic acid molecule via an adaptor molecule or via a linker selected from the group consisting of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, sulfo-SMCC linker, succinimidyl-6-hydrazinonicotinamide (S-HyNic) linker, N-succinimidyl-4-formylbenzamide (S-4FB) linker, bis-aryl hydrazone bond, an amide bond, two amide bonds on a spacer for cross-linking two —NH 2  groups triazole bond from “click” reaction), a phosphodiester linkage, a phosphorothioate linkage, or a combination thereof, and wherein the label is linked to the second single strand nucleic acid molecule via an adaptor molecule or via a linker selected from the group consisting of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, sulfo-SMCC linker, succinimidyl-6-hydrazinonicotinamide (S-HyNic) linker, N-succinimidyl-4-formylbenzamide (S-4FB) linker, bis-aryl hydrazone bond, an amide bond, two amide bonds on a spacer for cross-linking two —NH 2  groups, triazole bond (from “click” reaction), a phosphodiester linkage, a phosphorothioate linkage, or a combination thereof; and   (b) detecting signal from label moieties associated with first plurality of affinity molecules bound to the sample, thereby detecting the presence or amount of the plurality of analytes.   
     
     
       7. The method of  claim 6 , further comprising the steps of:
 (c) erasing the signal from the label molecules conjugated to the first set of second single strand nucleic acid molecules;   (d) contacting the sample with a second plurality of affinity molecules under conditions that permit specific analyte binding by the affinity molecules wherein members of the second plurality of affinity molecules are distinguishable from one another and from members of the first plurality of affinity molecules of step (a);   (e) detecting signal from label moieties associated with second plurality of affinity molecules bound to the sample, thereby detecting the presence or amount of at least a subset of the plurality of analytes; and   (f) optionally repeating steps (c)-(e) with a further second set of the affinity molecules.   
     
     
       8. A method, comprising:
 (a) contacting a sample with a first encoding molecule comprising a first affinity molecule linked to a first nucleic acid strand via a first linker, wherein the first affinity molecule binds to a first analyte in the sample;   (b) contacting the sample with a first labeling molecule, wherein the first labeling molecule comprises a second nucleic acid strand linked to an enzyme via a second linker, wherein the second nucleic acid strand can hybridize to the first nucleic acid strand;   (c) contacting the sample with a first enzyme substrate to cause a reaction between the enzyme and the first enzyme substrate that generates a first reaction product in the sample; and   (d) measuring fluorescence emission from the first reaction product to detect the first analyte in the sample,   wherein the first and second linkers each comprise one or more members selected from the group consisting of a succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) moiety, a sulfo-SMCC moiety, a succinimidyl-6-hydrazino-nicotinamide (S-HyNic) moiety, a N-succinimidyl-4-formylbenzamide (S-4FB) moiety, a bis-aryl hydrazine bond, an amide bond, two amide bonds on a spacer for cross-linking two amino groups, a triazole bond, a phosphodiester moiety, or a phosphorothioate moiety.   
     
     
       9. The method of claim 8, wherein the enzyme comprises a peroxidase. 
     
     
       10. The method of claim 9, wherein the enzyme comprises horseradish peroxidase. 
     
     
       11. The method of claim 8, wherein the first enzyme substrate comprises a tyramide molecule conjugated to a first fluorescent moiety. 
     
     
       12. The method of claim 8, wherein the first affinity molecule comprises an antibody or a portion of an antibody. 
     
     
       13. The method of claim 8, wherein the first affinity molecule comprises a receptor. 
     
     
       14. The method of claim 8, wherein the first affinity molecule comprises at least one non-naturally occurring moiety. 
     
     
       15. The method of claim 14, wherein the non-naturally occurring moiety is selected from the group consisting of recombinant moieties, chimeric moieties, hybrid moieties, and truncated moieties. 
     
     
       16. The method of claim 8, wherein step (a) comprises contacting the sample with a composition comprising populations of different types of encoding molecules, and wherein each different type of encoding molecule comprises an affinity molecule that binds to a different type of analyte in the sample and is linked to a nucleic acid strand via a linker selected from the group consisting of a succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) moiety, a sulfo-SMCC moiety, a succinimidyl-6-hydrazino-nicotinamide (S-HyNic) moiety, a N-succinimidyl-4-formylbenzamide (S-4FB) moiety, a bis-aryl hydrazine bond, an amide bond, two amide bonds on a spacer for cross-linking two amino groups, a triazole bond, a phosphodiester moiety, or a phosphorothioate moiety. 
     
     
       17. The method of claim 16, wherein for each different type of encoding molecule, the nucleic acid strands comprise a common sequence of nucleotides. 
     
     
       18. The method of claim 17, wherein for each different type of encoding molecule, the nucleic acid strands comprise an identical sequence of nucleotides. 
     
     
       19. The method of claim 17, wherein the nucleic acid strands corresponding to each different type of encoding molecule are different from nucleic acid strands corresponding to other types of encoding molecules in the composition. 
     
     
       20. The method of claim 16, further comprising:
 (e) removing the first labeling molecule from the sample.   
     
     
       21. The method of claim 20, wherein the composition comprises a second encoding molecule that is a different type of encoding molecule than the first encoding molecule and comprises a second affinity molecule linked to a third nucleic acid strand via a third linker, and wherein the second affinity molecule binds to a second analyte in the sample that is different from the first analyte, the method further comprising:
 (f) contacting the sample with a second labeling molecule, wherein the second labeling molecule comprises a fourth nucleic acid strand linked to an enzyme via a fourth linker, wherein the fourth nucleic acid strand can hybridize to the third nucleic acid strand;   (g) contacting the sample with a second enzyme substrate to cause a reaction between the enzyme of the second labeling molecule and the second enzyme substrate that generates a second reaction product in the sample;   (h) removing the second labeling molecule from the sample; and   (i) measuring fluorescence emission from the second reaction product to detect the second analyte in the sample,   wherein the third and fourth linkers each comprise one or more members selected from the group consisting of a succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) moiety, a sulfo-SMCC moiety, a succinimidyl-6-hydrazino-nicotinamide (S-HyNic) moiety, a N-succinimidyl-4-formylbenzamide (S-4FB) moiety, a bis-aryl hydrazine bond, an amide bond, two amide bonds on a spacer for cross-linking two amino groups, a triazole bond, a phosphodiester moiety, or a phosphorothioate moiety.   
     
     
       22. The method of claim 21, wherein the second enzyme substrate comprises a tyramide molecule conjugated to a second fluorescent moiety different from the first fluorescent moiety. 
     
     
       23. The method of claim 22, further comprising repeating steps (f)-(h) with different types of labeling molecules and enzyme substrates, wherein each of the different types of labeling molecules comprises a nucleic acid strand linked to an enzyme that can hybridize to a nucleic acid strand of a different type of encoding molecule in the composition, and wherein each of the enzyme substrates comprises a tyramide molecule conjugated to a different type of fluorescent moiety. 
     
     
       24. The method of claim 23, wherein each different type of the enzyme substrates reacts with the enzyme of a different type of labeling molecule to generate a different type of reaction product in the sample, the method further comprising measuring fluorescence emission from one or more different types of reaction products than the first and second reaction products to detect one or more additional types of analytes in the sample other than the first and second analytes. 
     
     
       25. The method of claim 24, comprising measuring fluorescence emission from each of the different types of reaction products to detect each of the different types of analytes in the sample. 
     
     
       26. A method, comprising:
 contacting a sample with a composition comprising a plurality of different types of encoding species, wherein each type of encoding species selectively binds to a different type of analyte in the sample and comprises a unique oligonucleotide sequence;   (a) contacting the sample with a first labeling species that selectively associates with one type of encoding species in the sample and comprises an enzyme;   (b) contacting the sample with a first enzyme substrate that reacts with the enzyme to deposit a first reaction product in the sample;   (c) removing the first labeling species from the sample;   repeating steps (a)-(c) with additional labeling species, each of which selectively associates with a different type of encoding species in the sample and comprises an enzyme, and with additional enzyme substrates, each of which reacts with the enzyme to deposit a different type of reaction product in the sample; and   detecting the first reaction product to measure a first type of analyte in the sample,   wherein the encoding species and the first labeling species each comprise at least one member selected from the group consisting of a succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) moiety, a sulfo-SMCC moiety, a succinimidyl-6-hydrazino-nicotinamide (S-HyNic) moiety, a N-succinimidyl-4-formylbenzamide (S-4FB) moiety, a bis-aryl hydrazine bond, an amide bond, two amide bonds on a spacer for cross-linking two amino groups, a triazole bond, a phosphodiester moiety, and a phosphorothioate moiety.   
     
     
       27. The method of claim 26, further comprising detecting one or more of the different types of reaction products in the sample to measure multiple types of analytes in the sample. 
     
     
       28. The method of claim 26, wherein each type of encoding species selectively binds to a different type of protein analyte in the sample. 
     
     
       29. The method of claim 26, wherein each labeling species selectively hybridizes to the unique oligonucleotide sequence of one type of encoding species. 
     
     
       30. The method of claim 26, wherein each labeling species comprises an oligonucleotide sequence that is complementary to only one of the unique oligonucleotide sequences of the different types of encoding species. 
     
     
       31. The method of claim 26, wherein the enzyme comprises a peroxidase. 
     
     
       32. The method of claim 26, wherein each type of enzyme substrate comprises a tyramide molecule conjugated to a different type of fluorescent moiety. 
     
     
       33. The method of claim 26, wherein for each different type of encoding species, the composition comprises a plurality of encoding molecules of the encoding species, and wherein each of the encoding molecules comprises an oligonucleotide comprising a common sequence of nucleotides. 
     
     
       34. A kit, comprising:
 a first composition comprising a plurality of different types of encoding molecules, each type of encoding molecule comprising a different affinity molecule linked to a different first nucleic acid strand via a first linker;   a second composition comprising a plurality of first labeling molecules, each first labeling molecule comprising a second nucleic acid strand linked to an enzyme via a second linker, wherein the second nucleic acid strand comprises a nucleotide sequence that is at least partially complementary to a nucleotide sequence of one of the types of first nucleic acid; and   a third composition comprising a plurality of first enzyme substrates, wherein each first enzyme substrate can react with the enzyme to deposit a first reaction product in the sample,   wherein the first and second linkers each comprise one or more members selected from the group consisting of a succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) moiety, a sulfo-SMCC moiety, a succinimidyl-6-hydrazino-nicotinamide (S-HyNic) moiety, a N-succinimidyl-4-formylbenzamide (S-4FB) moiety, a bis-aryl hydrazine bond, an amide bond, two amide bonds on a spacer for cross-linking two amino groups, a triazole bond, a phosphodiester moiety, or a phosphorothioate moiety.   
     
     
       35. The kit of claim 34, wherein one or more of the different types of affinity molecules comprise protein-binding moieties. 
     
     
       36. The kit of claim 34, wherein one or more of the different types of affinity molecules comprise antibodies or antibody portions. 
     
     
       37. The kit of claim 34, wherein one or more of the different types of affinity molecules comprises a receptor. 
     
     
       38. The kit of claim 34, wherein one or more of the different types of affinity molecules comprises a non-naturally occurring moiety. 
     
     
       39. The kit of claim 38, wherein the non-naturally occurring moiety is selected from the group consisting of recombinant moieties, chimeric moieties, hybrid moieties, and truncated moieties. 
     
     
       40. The kit of claim 34, wherein the enzyme comprises a peroxidase. 
     
     
       41. The kit of claim 40, wherein the enzyme comprises horseradish peroxidase. 
     
     
       42. The kit of claim 34, wherein each first enzyme substrate comprises a tyramide molecule conjugated to a first fluorescent moiety. 
     
     
       43. The kit of claim 42, further comprising a fourth composition comprising:
 a plurality of second labeling molecules, each second labeling molecule comprising a third nucleic acid strand linked to an enzyme via a third linker, wherein the third nucleic acid strand comprises a nucleotide sequence that is at least partially complementary to a nucleotide sequence of one of the types of first nucleic acid, and is different from the nucleotide sequence of the second nucleic acid strand,   wherein the third linker comprises one or more members selected from the group consisting of a succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) moiety, a sulfo-SMCC moiety, a succinimidyl-6-hydrazino-nicotinamide (S-HyNic) moiety, a N-succinimidyl-4-formylbenzamide (S-4FB) moiety, a bis-aryl hydrazine bond, an amide bond, two amide bonds on a spacer for cross-linking two amino groups, a triazole bond, a phosphodiester moiety, or a phosphorothioate moiety.   
     
     
       44. The kit of claim 43, further comprising a fifth composition comprising:
 a plurality of second enzyme substrates, wherein each second enzyme substrate can react with the enzymes of the second labeling molecules to deposit a second reaction product in the sample.   
     
     
       45. The kit of claim 44, wherein each second enzyme substrate comprises a tyramide molecule conjugated to a second fluorescent moiety that is different from the first fluorescent moiety.