Simultaneous detection of target protein and target nucleic acids in a single cell

Abstract

Methods and reagents for detection and analysis of nucleic acids are provided. The methods employ proximity extension assays for detection of a target nucleic acids of interest, e.g., a target RNA. The method can additionally be used in multiplex assays with a protein proximity extension assay to detect protein.

1 . A method of detecting a target nucleic acid in a sample, the method comprising
 (a) incubating in a reaction mixture:
 i) a sample comprising a target nucleic acid; and 
 ii) a pair of proximity probes comprising a first and second probe, where: 
 a first probe comprises a target binding (TB) segment that hybridizes to a first target (T) segment of the target nucleic acid, and an interacting (I) segment at the 3′ end of the probe, wherein the I segment is complementary to an I segment at the 3′ end of the second probe; and 
 a second probe comprises a TB segment that hybridizes to a second, non-overlapping T segment of the target nucleic acid that is in close proximity to the first T segment, and an I segment at the 3′ end, wherein the 3′ sequence is complementary to the I segment of the first probe, wherein 
   the reaction mixture is incubated under conditions in which the TB segment of the first probe hybridizes to the first T segment of the target nucleic acid and the TB segment of the second probe hybridizes to the second T segment of the target nucleic acid, thereby allowing the I segment of the first probe to hybridize to the I segment of the second probe to form a duplex comprising the I segments of the first and second probe;   (b) adding a DNA polymerase and maintaining the reaction mixture under conditions in which the first and/or second probe is extended to obtain a first extended product;   (c) amplifying the extended product, or a subregion thereof in an amplification reaction mixture comprising a pair of amplification primers that amplify the first extended product, or subregion thereof;   (d) detecting the amplicon obtained in (c).   
     
     
         2 . The method of  claim 1 , wherein the sample is RNA. 
     
     
         3 . The method of  claim 1 , wherein the sample is a single cell. 
     
     
         4 . The method of  claim 1 , wherein the detecting step comprises a quantitative amplification reaction. 
     
     
         5 . The method of  claim 4 , wherein the quantitative amplification reaction is qPCR. 
     
     
         6 . The method of  claim 1 , wherein one of the members of the proximity pair is blocked at the 3′ end so that only one probe is extended in step (b). 
     
     
         7 . The method of  claim 1 , wherein the DNA polymerase has 3′ exonuclease activity. 
     
     
         8 . The method of  claim 1 , wherein the amplification of step (c) is performed with a DNA polymerase different from the DNA polymerase employed in the extension reaction of step (b). 
     
     
         9 . The method of  claim 8 , wherein the DNA polymerase in amplification reaction is a thermostable polymerase. 
     
     
         10 . The method of  claim 1 , further comprising detecting a target protein in the sample, wherein the method further comprises:
 incubating the sample in the reaction mixture of (a) with a pair of protein-detecting proximity probes comprising a first and a second protein detecting proximity probe where:
 the first protein-detecting probe comprises a first antibody that binds to the target protein joined to a first polynucleotide that comprises an I segment at the 3′ end that is complementary to an I segment on the 3′ end of the second probe; and 
 the second protein-detecting probe comprises a second antibody that binds to the target protein joined to a second polynucleotide that comprises an I segment complementary to the I segment at the 3′ end of the first polynucleotide; 
   wherein binding of the first antibody to the target protein and binding of the second antibody to the target protein allows the I segment of the first protein proximity probe to hybridize to the I segment of the second protein proximity probe to form a duplex which is extended in step (b) to provide a second extended product;   amplifying the second extended product, or subregion thereof, in the amplificiation reaction of (c) using a set of primers that amplify the second extended product or subregion thereof; and   detecting the amount of amplicon from amplification of the second extended product or subregion thereof.   
     
     
         11 . The method of  claim 10 , wherein the detecting step comprises a quantitative amplification reaction. 
     
     
         12 . The method of  claim 11 , wherein the quantitative amplification reaction is qPCR.