Compositions and methods 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:
 (1) contacting a sample being tested for the presence of one or more analytes with one or more affinity molecules, wherein each of the affinity molecules is linked to an encoding molecule,   wherein the encoding molecule is linked to the affinity 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 —NH2 groups, triazole bond (from “click” reaction), a phosphodiester linkage, a phosphorothioate linkage, and a combination thereof,   and wherein affinity molecules of different specificity are linked to different encoding molecules to produce one or more analytes bound to one or more affinity molecules;   (2) optionally removing unbound affinity molecules;   (3) contacting the sample with labeled nucleic acid strands conjugated to a label moiety that bind to encoding molecules to produce label moieties bound to encoding molecules;   wherein each of the labeled nucleic acid strands are linked to the label moiety 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 —NH2 groups, triazole bond (from “click” reaction), a phosphodiester linkage, a phosphorothioate linkage, and a combination thereof,   (4) optionally removing unbound labeled nucleic acid strands;   (5) imaging the sample to detect the label moieties bound to encoding molecules;   (6) removing the label moieties from the encoding molecules, wherein the labeled nucleic acid strands are removed from the encoding molecules by chemical, physical, or enzymatic means; and   (7) repeating at least some of steps (3)-(6) at least once with a labeled nucleic acid strand having a unique composition relative to at least one other labeled nucleic acid strand of step (3).    
     
     
       9. The method of claim 8, wherein the label moieties are removed by enzymatic means.  
     
     
       10. The method of claim 9, wherein the encoding molecules are first nucleic acid strands and the labeled nucleic acid strands are second nucleic acid strands, and wherein the first nucleic acid strands and the second nucleic acid strands are complementary nucleic acids relative to each other.  
     
     
       11. The method of claim 10, wherein the labeled nucleic acid strands are cleaved by a single-stranded nuclease.  
     
     
       12. The method of claim 10, wherein the label moieties are removed by at least one restriction endonuclease.  
     
     
       13. The method of claim 8, wherein the label moieties are removed by cleavage at one or more ribonucleotide by RNase.  
     
     
       14. The method of claim 8, wherein the label moieties are capable of being quenched by photo-bleaching or cleavage.  
     
     
       15. The method of claim 8, wherein removing the label moieties comprises:
 a. cleavage and photo-bleaching; or   b. cleavage and chemical modification.    
     
     
       16. The method of claim 10, wherein the sample is contacted with more than one affinity molecule in (1).  
     
     
       17. The method of claim 10, wherein the one or more affinity molecules comprise an antibody or portion thereof.  
     
     
       18. The method of claim 10, wherein the one or more affinity molecules is a ligand, an aptamer, a peptide, or an oligonucleotide.  
     
     
       19. The method of claim 10, wherein the labeled nucleic acid strands are labeled identically.  
     
     
       20. The method of claim 10, wherein the labeled nucleic acid strands comprise a distinguishable label moiety.  
     
     
       21. The method of claim 8, wherein the label moieties are fluorescent molecules.  
     
     
       22. The method of claim 8, wherein the one or more analytes are proteins and/or the sample is a cell or tissue sample.  
     
     
       23. The method of claim 10, wherein the sample is imaged in step (5) using fluorescence microscopy.  
     
     
       24. The method of claim 8, wherein the label moieties are removed by strand displacement.  
     
     
       25. The method of claim 24, wherein the strand displacement is performed using a displacement nucleic acid complementary to the labeled nucleic acid strands or the encoding molecules.  
     
     
       26. The method of claim 25, wherein the sample is contacted with more than one affinity molecule in (1).  
     
     
       27. The method of claim 25, wherein the one or more affinity molecules comprise an antibody or portion thereof.  
     
     
       28. The method of claim 25, wherein the one or more affinity molecules comprise a ligand, an aptamer, a peptide, or an oligonucleotide.  
     
     
       29. The method of claim 25, wherein the labeled nucleic acid strands are labeled identically relative to each other.  
     
     
       30. The method of claim 25, wherein each of the labeled nucleic acid strands comprises a distinguishable label moiety.  
     
     
       31. The method of claim 25, wherein the label moieties are fluorescent molecules.  
     
     
       32. The method of claim 25, wherein the one or more analytes are proteins and/or the sample is a cell or tissue sample.  
     
     
       33. The method of claim 25, wherein the sample is imaged in step (5) using fluorescence microscopy.