High-throughput and highly multiplexed imaging with programmable nucleic acid probes

Abstract

The present invention provides, inter alia, methods and compositions for imaging, at high spatial resolution, targets of interest.

1 - 51 . (canceled) 
     
     
         52 . A composition for testing a sample for the presence of at least two targets, comprising:
 at least two target-specific binding partners, wherein each target-specific binding partner is linked to a docking strand, and wherein target-specific binding partners of different specificity are linked to different docking strands; and   at least two labeled imager strands comprising a signal emitting moiety, wherein each labeled imager strand is capable of binding to a respective docking strand linked to a different target-specific binding partner.   
     
     
         53 . The composition of  claim 52 , wherein the labeled imager strands are removable by enzymatic cleavage, modification, or degradation. 
     
     
         54 . The composition of  claim 53 , wherein the labeled imager strands comprise an enzymatically cleavable moiety. 
     
     
         55 . The composition of  claim 54 , wherein the labeled imager strands are labeled imager nucleic acid strands and the docking strands are docking nucleic acid strands. 
     
     
         56 . The composition of  claim 55 , wherein the labeled imager nucleic acid strands and the docking nucleic acid strands are complementary nucleic acids relative to each other. 
     
     
         57 . The composition of  claim 55 , wherein the enzymatically cleavable moiety is cleavable by a nucleic acid glycosylase. 
     
     
         58 . The composition of  claim 57 , wherein the nucleic acid glycosylase is a uracil-DNA glycosylase. 
     
     
         59 . The composition of  claim 55 , wherein the enzymatically cleavable moiety includes at least one deoxyuridine base cleavable by a uracil-specific excision reagent enzyme. 
     
     
         60 . The composition of  claim 55 , wherein the enzymatically cleavable moiety includes at least one uracil cleavable by a uracil-specific excision reagent enzyme. 
     
     
         61 . The composition of  claim 55 , wherein the enzymatically cleavable moiety includes at least one uracil that can be cleaved by uracil-DNA glycosylase alone or with Endonuclease VIII. 
     
     
         62 . The composition of  claim 55 , wherein the enzymatically cleavable moiety includes at least one abasic site that can be cleaved by endonuclease. 
     
     
         63 . The composition of  claim 55 , wherein the enzymatically cleavable moiety includes at least one restriction site cleavable by nicking enzyme or restriction enzyme. 
     
     
         64 . The composition of  claim 54 , wherein the enzymatically cleavable moiety includes at least one ribonucleotide cleavable by RNase. 
     
     
         65 . The composition of  claim 52 , wherein the labeled imager strands comprise a photocleavable moiety that can be cleaved photochemically by UV exposure. 
     
     
         66 . The composition of  claim 52 , wherein the signal-emitting moiety is linked to the imager strand via a cleavable linker. 
     
     
         67 . The composition of  claim 52 , wherein the signal-emitting moiety can be chemically or photochemically modified without removing the imager strand. 
     
     
         68 . The composition of  claim 52 , wherein the signal-emitting moiety is a fluorophore that can be bleached by chemical agents or photobleached. 
     
     
         69 . The composition of  claim 52 , wherein the labeled imager strands comprise a chemically cleavable moiety selected from allyl groups, azido groups, bridging phosphorothiolates, disulfide bonds, and ribose. 
     
     
         70 . The composition of  claim 52 , wherein the labeled imager and the respective docking strand can be stably bound to each other for at least 30, 35, 40, 45, 50, 55, or 60 minutes or at least 2 hours. 
     
     
         71 . The composition of  claim 70 , wherein the labeled imager and the respective docking strand can be stably bound to each other for at least 30 to 60 minutes, 30 to 120 minutes, 40 to 60 minutes, 40 to 120 minutes, or 60 to 120 minutes. 
     
     
         72 . The composition of  claim 52 , wherein the target-specific binding partner is an antibody or an antibody fragment. 
     
     
         73 . The composition of  claim 52 , wherein the target-specific binding partner is a ligand, a small molecule, an aptamer, a peptide or an oligonucleotide. 
     
     
         74 . The composition of  claim 52 , wherein the labeled imager strands are labeled in the same manner. 
     
     
         75 . The composition of  claim 52 , wherein the at least two labeled imager strands comprise a distinct label. 
     
     
         76 . The composition of  claim 52 , wherein the at least two targets are proteins. 
     
     
         77 . The composition of  claim 52 , wherein the sample is a cell or tissue sample, a cell lysate or a tissue lysate, or a bodily fluid. 
     
     
         78 . The composition of  claim 52 , wherein the labeled imager strands are branched imager strands. 
     
     
         79 . The composition of  claim 52 , wherein the labeled imager strands and/or the docking strands have a toehold sequence, a molecular beacon, a hemiduplex, a hairpin loop, a dendrimeric structure or a polymeric structure, 2D nanostructure, or 3D nanostructure, and optionally wherein the docking strand is part of a multi-strand complex. 
     
     
         80 . A kit for testing a sample for the presence of at least two targets, comprising:
 at least two target-specific binding partners, wherein each target-specific binding partner is linked to a docking strand, and wherein target-specific binding partners of different specificity are linked to different docking strands; and   at least two imager strands, capable of binding to a respective docking strand linked to a different target-specific binding partner and comprising a signal emitting moiety and a cleavable moiety; and   a reagent for cleaving the cleavable moiety or modifying or removing the signal emitting moiety.   
     
     
         81 . The kit of  claim 80 , wherein the reagent comprises one or more chosen from uracil-specific excision reagent enzyme, uracil-DNA glycosylase, Endonuclease VIII, TCEP, hydroxide, imidazole, Pd-based reagents, phosphorous-based reagents, silver-based reagents, reducing agents, and nucleophiles.