Catalytic de-crosslinking of samples for in situ analysis

Abstract

The present disclosure relates in some aspects to methods and compositions for in situ analysis involving catalytic de-crosslinking of biological samples.

1 . A method for sample analysis, comprising:
 a) providing a biological sample immobilized on a substrate, wherein the biological sample is fixed;   b) contacting the biological sample with a catalyst that catalyzes de-crosslinking of molecular crosslinks in the biological sample, wherein the catalyst is a compound of formula(I),   
       
         
           
           
               
               
           
         
       
       or a salt zwitterion, or solvate thereof, wherein:
 A is selected from the group consisting of —COOH, —P(═O)(OH) 2 , and S(═O) 2 OH; 
 X 1 , X 2 , X 3 , and X 4  are each independently selected from the group consisting of: CH, CR a , and N; 
 each occurrence of R a  is independently selected from the group consisting of C 1-6  alkyl, C 1-6  haloalkyl, C 1-6  alkoxy, —NO 2 , —NR′R″, and —C(═O)NR′R″; and 
 each occurrence of R′ and R″ is independently selected from the group consisting of H and C 1-6  alkyl which is optionally substituted with 
 
       
         
           
           
               
               
           
         
       
       wherein n1 is an integer from 12 to 16;
 c) contacting the biological sample with a labelling agent that directly or indirectly binds to a protein analyte at a location in the biological sample, wherein the labelling agent is conjugated to a reporter oligonucleotide comprising a reporter sequence that identifies the labelling agent; and 
 d) detecting an optical signal associated with the labelling agent or a product thereof, thereby detecting the protein analyte at the location in the biological sample. 
 
     
     
         2 . The method of  claim 1 , wherein the molecular crosslinks are products of one or more crosslinking agents. 
     
     
         3 . The method of  claim 2 , wherein the one or more crosslinking agents comprise an aldehyde, optionally wherein the crosslinking agent comprises formaldehyde. 
     
     
         4 - 11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein the catalyst comprises one or more compounds selected from the group consisting of 
       
         
           
           
               
               
           
         
       
       or a salt, zwitterion, or solvate thereof. 
     
     
         13 . The method of  claim 1 , wherein the catalyst comprises 
       
         
           
           
               
               
           
         
       
       or a salt, zwitterion, or solvate thereof. 
     
     
         14 . The method of  claim 1 , wherein the catalyst comprises 
       
         
           
           
               
               
           
         
       
       or a salt, zwitterion, or solvate thereof. 
     
     
         15 . The method of  claim 1 , wherein the catalyst comprises 
       
         
           
           
               
               
           
         
       
       or a salt, zwitterion, or solvate thereof. 
     
     
         16 . The method of  claim 1 , wherein the catalyst comprises 
       
         
           
           
               
               
           
         
       
       or a combination thereof, or a salt, zwitterion, or solvate thereof. 
     
     
         17 - 23 . (canceled) 
     
     
         24 . The method of  claim 1 , wherein the biological sample is a tissue section. 
     
     
         25 - 58 . (canceled) 
     
     
         59 . The method of  claim 1 , wherein a solution or a suspension comprising the catalyst and a buffer is contacted with the biological sample and the solution or suspension comprises sodium dodecyl sulfate (SDS), urea, and/or a proteinase. 
     
     
         60 - 70 . (canceled) 
     
     
         71 . The method of  claim 1 , wherein the labelling agent comprises a binding moiety, wherein the binding moiety comprises an antibody or epitope binding fragment thereof. 
     
     
         72 . The method of  claim 1 , wherein the labelling agent comprises a detectable label. 
     
     
         73 . The method of  claim 1 , wherein the reporter oligonucleotide comprises a barcode sequence. 
     
     
         74 - 87 . (canceled) 
     
     
         88 . The method of claim  4 , wherein the protein analyte is an intracellular protein, a membrane-bound protein, or an extracellular protein. 
     
     
         89 - 90 . (canceled) 
     
     
         91 . The method of  claim 1 , wherein the optical signal is detected in situ in the biological sample. 
     
     
         92 . The method of  claim 1 , wherein the optical signal is detected by imaging the biological sample. 
     
     
         93 . The method of  claim 92 , wherein the imaging comprises fluorescent microscopy. 
     
     
         94 - 119 . (canceled) 
     
     
         120 . The method of  claim 1 , wherein the biological sample is contacted with a probe that hybridizes to the reporter oligonucleotide of the labelling agent to identify the protein analyte associated with the labelling agent. 
     
     
         121 . The method of  claim 1 , wherein the labelling agent binds indirectly to an intermediate probe comprising one or more barcode sequences. 
     
     
         122 . The method of  claim 121 , wherein the biological sample is contacted with a detectably labeled tertiary probe that hybridizes to the one or more barcode sequences of the intermediate probe.