Multiplexed signal amplification

Abstract

Provided herein, in some embodiments, are methods and compositions for highly multiplexed in situ signal amplification via hairpin-mediated concatemerization.

1 - 17 . (canceled) 
     
     
         18 . A multiplexed target detection method, comprising:
 (a) combining a sample containing a plurality of protein or peptide targets with a plurality of primary binding partners, each of which binds specifically to a protein or peptide target and is linked to a probe strand, and producing a first reaction mixture comprising protein or peptide bound to primary binding partners;   (b) combining the first reaction mixture produced in step (a) with dNTPs, strand-displacing polymerase, and a plurality of catalytic molecules, each catalytic molecule comprising, 5′ to 3′, a first domain, a second domain, and a third domain, wherein the first domain is bound to the second domain, and the third domain is an unpaired 3′ toehold domain complementary to the probe strand of one of the primary binding partners, and producing a second reaction mixture comprising nucleic acid concatemers bound to primary binding partners; and   (c) combining the second reaction mixture produced in step (b) with a plurality of signal strands, each signal strand linked to a different detectable molecule and comprising a domain complementary to the probe strand of one of the primary binding partners, and producing concatemers labeled by a plurality of signal strands.   
     
     
         19 . A multiplexed target detection method, comprising:
 (a) combining a sample containing a plurality of protein or peptide targets with a plurality of primary binding partners, each of which binds specifically to a protein or peptide target and is linked to a bridge strand, and producing a first reaction mixture comprising protein or peptide bound to primary binding partners;   (b) combining the first reaction mixture with concatemers bound to probe strands produced by combining in a second reaction mixture dNTPs, strand-displacing polymerase, a plurality of probe strands, and a plurality of catalytic molecules, wherein each probe strand comprises (i) an unpaired 5′ target domain complementary to the bridge strand of one of the primary binding partners and (ii) an unpaired 3′ primer domain, and wherein each catalytic molecule comprises, 5′ to 3′, a first domain, a second domain, and a third domain, wherein the first domain is bound to the second domain, and the third domain is an unpaired 3′ toehold domain complementary to one of the probe strands, and producing a third reaction mixture comprising nucleic acid concatemers bound to primary binding partners; and   (c) combining the third reaction mixture produced in step (b) with a plurality of signal strands, each signal strand linked to a different detectable molecule and comprising a domain complementary to the bridge strand of one of the primary binding partners, and producing concatemers labeled by a plurality of signal strands.   
     
     
         20 . A multiplexed target detection method, comprising:
 (a) combining a sample containing a plurality of protein or peptide targets with a plurality of primary binding partners, each of which binds specifically to a protein or peptide target and is linked to a bridge strand, and producing a first reaction mixture comprising protein or peptide bound to primary binding partners;   (b) combining the first reaction mixture with a plurality of probe strands, wherein each probe strand comprises (i) an unpaired 5′ target domain complementary to the bridge strand of one of the primary binding partners and (ii) an unpaired 3′ primer domain, and producing a second reaction mixture comprising primary binding partners bound to probe strands;   (c) combining the second reaction mixture with dNTPs, strand-displacing polymerase, and a plurality of catalytic molecules, wherein each catalytic molecule comprising, 5′ to 3′, a first domain, a second domain, and a third domain wherein the first domain is bound to the second domain, and the third domain is an unpaired 3′ toehold domain complementary to one of the probe strands, and producing a third reaction mixture comprising nucleic acid concatemers bound to primary binding partners; and   (d) combining the third reaction mixture produced in step (b) with a plurality of signal strands, each signal strand linked to a different detectable molecule and comprising a domain complementary to the bridge strand of one of the primary binding partners, and producing concatemers labeled by a plurality of signal strands.   
     
     
         21 . The method of  claim 18 , wherein the primary binding partners are antibodies. 
     
     
         22 . The method of  claim 18 , wherein the catalytic molecules and/or probe strands are comprised of DNA and/or RNA. 
     
     
         23 . The method of  claim 18 , wherein the first domain of each catalytic molecule is bound to the second domain of the same catalytic molecule, wherein the second domain of each catalytic molecule comprises a sequence identical to the third domain of the same catalytic molecule, and/or wherein the first domain each catalytic molecule comprises a sequence wholly complementary to the second domain of the same catalytic molecule. 
     
     
         24 . The method of  claim 18 , wherein each catalytic molecule further comprises a stopper molecule or modification that terminates polymerization located between the first and second domains of the same catalytic molecule. 
     
     
         25 . The method of  claim 24 , wherein the stopper molecule or modification that terminates polymerization is selected from a triethylene glycol (TEG), 18-atom hexa-ethylene glycol, adenylation, azide, digoxigenin, cholesteryl-TEG, 3-cyanovinylcarbazole (CNVK), iso-dG and iso-dC, wherein the stopper molecule is guanine and the catalytic molecule is comprised of adenine, thymine and cytosine, or wherein the stopper molecule is cytosine and the catalytic molecule is comprised of adenine, thymine and guanine. 
     
     
         26 . The method of  claim 18 , wherein each catalytic molecule is a catalytic hairpin molecule further comprising a loop domain located between the first and second domains. 
     
     
         27 . The method of  claim 18 , wherein the detectable molecule of the signal strands is a fluorophore. 
     
     
         28 . The method of  claim 18 , wherein the strand-displacing polymerase is selected from phi29 DNA polymerases, Bst DNA polymerases, and Bsu DNA polymerase, large fragment. 
     
     
         29 . The method of  claim 18 , wherein the sample is a cell sample or a tissue sample. 
     
     
         30 .- 33 . (canceled) 
     
     
         34 . The method of  claim 18 , further comprising:
 (d) imaging the labeled concatemers.   
     
     
         35 . The method of  claim 19 , further comprising:
 (d) imaging the labeled concatemers.   
     
     
         36 . The method of  claim 19 , wherein the primary binding partners are antibodies. 
     
     
         37 . The method of  claim 19 , wherein each catalytic molecule further comprises a stopper molecule or modification that terminates polymerization located between the first and second domains of the same catalytic molecule. 
     
     
         38 . The method of  claim 20 , further comprising:
 (d) imaging the labeled concatemers.   
     
     
         39 . The method of  claim 20 , wherein the primary binding partners are antibodies. 
     
     
         40 . The method of  claim 20 , wherein each catalytic molecule further comprises a stopper molecule or modification that terminates polymerization located between the first and second domains of the same catalytic molecule. 
     
     
         41 . The method of  claim 19 , wherein the detectable molecule of the signal strands is a fluorophore.