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-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; 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 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;   (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-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; 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 plurality of probes; wherein:   each probe of the plurality of probes comprises an affinity moiety linked to a tag moiety through a first linker;   each tag moiety comprises a first nucleic acid sequence; and   the affinity moiety selectively binds to a target in the sample;   (b) contacting the sample with at least one labeling agent comprising a second nucleic acid sequence linked to an optical label through a second linker, wherein the second nucleic acid sequence selectively hybridizes to the first nucleic acid sequence;   (c) obtaining an image of the sample comprising one or more optical signals corresponding to at least one optical label of the at least one labeling agent;   (d) at least one of chemically erasing and physically erasing optical signals arising from the at least one optical label from the sample; and   (e) repeating steps (b)-(d) with at least one additional labeling agent,   wherein the first and second linkers 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.    
     
     
       9. The method of  claim 8 , wherein at least one of chemically erasing and physically erasing optical signals arising from the at least one optical label from the sample comprises at least one member selected from the group consisting of:
 denaturing the at least one optical label;   displacing the at least one labeling agent comprising the at least one optical label from the sample;   cleaving the at least one optical label from the at least one labeling agent;   photobleaching the at least one optical label; and   quenching the at least one optical label.    
     
     
       10. The method of  claim 8 , wherein the target comprises a peptide or protein, and wherein the affinity moiety comprises an antibody.  
     
     
       11. The method of  claim 8 , wherein the target comprises a RNA, and wherein the affinity moiety comprises a nucleic acid sequence.  
     
     
       12. The method of  claim 11 , wherein the nucleic acid sequence of the affinity moiety comprises a DNA sequence.  
     
     
       13. The method of  claim 8 , wherein the first nucleic acid sequence is a DNA sequence.  
     
     
       14. The method of  claim 8 , wherein the first nucleic acid sequence comprises at least 10 nucleotides.  
     
     
       15. The method of  claim 14 , wherein the first nucleic acid sequence comprises at least 15 nucleotides.  
     
     
       16. The method of  claim 15 , wherein the first nucleic acid sequence comprises at least 20 nucleotides.  
     
     
       17. The method of  claim 16 , wherein the first nucleic acid sequence comprises at least 30 nucleotides.  
     
     
       18. The method of  claim 8 , wherein the second nucleic acid sequence is a DNA sequence.  
     
     
       19. The method of  claim 8 , wherein the second nucleic acid sequence comprises at least 10 nucleotides.  
     
     
       20. The method of  claim 19 , wherein the second nucleic acid sequence comprises at least 15 nucleotides.  
     
     
       21. The method of  claim 20 , wherein the second nucleic acid sequence comprises at least 20 nucleotides.  
     
     
       22. The method of  claim 21 , wherein the second nucleic acid sequence comprises at least 30 nucleotides.  
     
     
       23. The method of  claim 8 , wherein the optical label comprises a fluorescent moiety.  
     
     
       24. The method of  claim 8 , wherein the optical label comprises at least one fluorescent nucleotide.  
     
     
       25. The method of  claim 8 , wherein the plurality of probes comprises multiple different types of probes, and wherein each different type of probe comprises a different affinity moiety that selectively binds to a different target in the sample.  
     
     
       26. The method of  claim 25 , wherein the different types of probes comprise one or more types of probes that selectively bind to different proteins or peptides in the sample, and wherein each type of probe that selectively binds to a different protein or peptide in the sample comprises an affinity moiety comprising a different antibody.  
     
     
       27. The method of  claim 25 , wherein the different types of probes comprise one or more types of probes that selectively bind to different nucleic acid targets in the sample, and wherein each type of probe that selectively binds to a different RNA target in the sample comprises an affinity moiety comprising a different nucleic acid sequence.  
     
     
       28. The method of  claim 25 , wherein the different types of probes comprise:
 a first set of different types of probes that selectively bind to different proteins or peptides in the sample, wherein each probe type in the first set comprises an affinity moiety comprising a different antibody; and   a second set of different types of probes that selectively bind to different RNA targets in the sample, wherein each probe type in the second set comprises an affinity moiety comprising a different nucleic acid sequence.    
     
     
       29. The method of  claim 25 , wherein each different type of probe comprises a different first nucleic acid sequence.  
     
     
       30. The method of  claim 25 , wherein the at least one labeling agent comprises multiple different types of labeling agents, and wherein each different type of labeling agent comprises a second nucleic acid that selectively hybridizes to the first nucleic acid of only one of the different types of probes.  
     
     
       31. The method of  claim 30 , wherein within each step (b), each of the multiple different types of labeling agents comprises a different optical label.  
     
     
       32. The method of  claim 25 , wherein the plurality of probes comprises at least 3 different types of probes.  
     
     
       33. The method of  claim 32 , wherein the plurality of probes comprises at least 10 different types of probes.  
     
     
       34. The method of  claim 31 , wherein within each step (b), the at least one labeling agent comprises at least 3 different types of labeling agents.  
     
     
       35. The method of  claim 31 , wherein within each step (b), the at least one labeling agent comprises at least 5 different types of labeling agents.  
     
     
       36. The method of  claim 8 , further comprising detecting at least one target in the sample based on the one or more optical signals of the obtained image.  
     
     
       37. The method of  claim 25 , further comprising detecting multiple different targets in the sample based on one or more optical signals of the obtained image.  
     
     
       38. The method of  claim 25 , further comprising, by repeating steps (b)-(d), obtaining multiple images of the sample each comprising one or more optical signals associated with the different types of probes, and detecting multiple different targets in the sample based on the optical signals of the multiple images of the sample.  
     
     
       39. A kit, comprising:
 a first composition comprising a plurality of different types of probes, wherein each different type of probe comprises:   a different affinity moiety; and   linked to the different affinity moiety through a first linker, a different tag moiety comprising a first nucleic acid sequence that is different from first nucleic acids of other types of probes among the plurality of different types of probes; and   a second composition comprising a plurality of different types of labeling agents, wherein each different type of labeling agent comprises:   a second nucleic acid sequence that is different from second nucleic acid sequences of other types of labeling agents among the plurality of different types of labeling agents, and hybridizes to only one of the first nucleic acid sequences of the different types of probes; and   linked to the second nucleic acid sequence through a second linker, an optical label,   wherein the first and second linkers 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.    
     
     
       40. The kit of  claim 39 , wherein the different types of probes comprise one or more types of probes that each selectively bind to a different peptide or protein, and each comprise an affinity moiety comprising a different antibody.  
     
     
       41. The kit of  claim 39 , wherein the different types of probes comprise one or more types of probes that each selectively bind to a different RNA, and each comprise an affinity moiety comprising a different nucleic acid sequence.  
     
     
       42. The kit of  claim 41 , wherein the nucleic acid sequence of the affinity moiety comprises a DNA sequence.  
     
     
       43. The kit of  claim 39 , wherein the first nucleic acid sequence is a DNA sequence.  
     
     
       44. The kit of  claim 39 , wherein the first nucleic acid sequence comprises at least 10 nucleotides.  
     
     
       45. The kit of  claim 44 , wherein the first nucleic acid sequence comprises at least 15 nucleotides.  
     
     
       46. The kit of  claim 45 , wherein the first nucleic acid sequence comprises at least 20 nucleotides.  
     
     
       47. The kit of  claim 46 , wherein the first nucleic acid sequence comprises at least 30 nucleotides.  
     
     
       48. The kit of  claim 39 , wherein the second nucleic acid sequence is a DNA sequence.  
     
     
       49. The kit of  claim 39 , wherein the second nucleic acid sequence comprises at least 10 nucleotides.  
     
     
       50. The kit of  claim 49 , wherein the second nucleic acid sequence comprises at least 15 nucleotides.  
     
     
       51. The kit of  claim 50 , wherein the second nucleic acid sequence comprises at least 20 nucleotides.  
     
     
       52. The kit of  claim 51 , wherein the second nucleic acid sequence comprises at least 30 nucleotides.  
     
     
       53. The kit of  claim 39 , wherein the optical label comprises a fluorescent moiety.  
     
     
       54. The kit of  claim 39 , wherein the optical label comprises at least one fluorescent nucleotide.  
     
     
       55. The kit of  claim 39 , wherein the different types of probes comprise:
 a first set of different types of probes that selectively bind to different proteins or peptides in the sample, wherein each probe type in the first set comprises an affinity moiety comprising a different antibody; and   a second set of different types of probes that selectively bind to different nucleic acid targets in the sample, wherein each probe type in the second set comprises an affinity moiety comprising a different nucleic acid sequence.    
     
     
       56. The kit of  claim 39 , wherein the plurality of different types of probes comprises at least 3 different types of probes.  
     
     
       57. The kit of  claim 46 , wherein the plurality of different types of probes comprises at least 10 different types of probes.