Methods for multiplex imaging using labeled nucleic acid imaging agents

Abstract

The present application provides certain advantageous ways of conducting multiplexed imaging.

What is claimed is: 
     
         1 . A method to test a sample for the presence of one or more targets comprising
 (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a nucleic acid strand and wherein target-specific binding partners of different specificity are linked to different nucleic acid strands,   (2) optionally removing unbound target-specific binding partners,   (3) contacting the sample with a nonlinear amplifier strand having complementarity to a nucleic acid strand, wherein the nucleic acid strand is either a docking strand or a primer strand,   (4) optionally removing unbound nonlinear amplifier strands,   (5) in either one or two steps amplifying the docking strand with rolling circle amplification and contacting the sample with labeled imager strands having complementarity to the docking strand or amplified strand,   (6) imaging the sample to detect bound labeled imager strands,   (7) removing the bound labeled imager strands, and   (8) optionally repeating steps (1)-(8), or any subset thereof.   
     
     
         2 . The method of  claim 1 , wherein a polymerase is used for rolling circle amplification. 
     
     
         3 . The method of  claim 1 , wherein the nonlinear amplifier strand is combined with the target-specific binding partner linked to a nucleic acid strand before contacting the sample. 
     
     
         4 . The method of  claim 1 , wherein amplifying the docking strand with rolling circle amplification occurs separately from contacting the sample with labeled imager strands having complementarity to the docking strand or amplified strand. 
     
     
         5 . The method of  claim 1 , wherein amplifying the docking strand with rolling circle amplification occurs in the same step as contacting the sample with labeled imager strands having complementarity to the docking strand and wherein the imager strand optionally comprises a 3′ modification to prevent amplification of the imager strand. 
     
     
         6 . The method of  claim 1 , wherein the imager strand is a circular imager strand for rolling circle amplification. 
     
     
         7 . The method of  claim 1 , wherein the imager strand or amplifier strand comprises at least two regions that are complementary to the docking strand. 
     
     
         8 . The method of  claim 1 , wherein the labeled imager strands are linear strands. 
     
     
         9 . The method of  claim 1 , wherein the nonlinear amplifier strand is a circular strand. 
     
     
         10 . The method of  claim 1 , wherein the nonlinear amplifier strand becomes circular after ligation. 
     
     
         11 . A method to test a sample for the presence of one or more targets comprising
 (1) contacting the sample with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a nucleic acid strand, directly or indirectly, and wherein target-specific binding partners of different specificity are linked to different nucleic acid strands,   (2) optionally removing unbound target-specific binding partners,   (3) wherein the nucleic acid strand is a docking strand or a primer strand and if the nucleic acid is
 (a) a docking strand, optionally increasing the number of docking strands associated with each target-specific binding partner, or 
 (b) a primer strand, optionally associating more than one docking strand with the primer strand, 
   (4) contacting the sample with labeled imager strands capable of binding a docking strand, directly or indirectly,   (5) optionally removing unbound labeled imager strands,   (6) imaging the sample to detect bound labeled imager strands, and   (7) optionally extinguishing signal from the bound labeled imager strand;   (8) optionally repeating steps (1)-(7) or any subset thereof.   
     
     
         12 . The method of  claim 11 , wherein after step (6) and after optionally performing step (7) the method further comprises increasing the number of docking strands associated with each target-specific binding partner and repeating steps (4), optionally (5), (6), and optionally (7). 
     
     
         13 . The method of  claim 11 , wherein the method comprises at step (3) increasing the number of docking strands associated with each target-specific binding partner or associating more than one docking strand with the primer strand. 
     
     
         14 . The method of  claim 11 , where the increase in the number of docking strands associated with each target-specific binding partner is achieved using an enzyme and wherein the enzyme is optionally a polymerase. 
     
     
         15 . The method of  claim 11 , wherein enzymatically cleaving, modifying, or degrading unbound labeled imager strands is achieved by using an enzyme and wherein the enzyme is optionally a glycosylase, a restriction endonuclease, a nicking endonuclease, an RNase, and an enzyme that cleaves at a non-natural nucleotide. 
     
     
         16 . A composition comprising:
 a sample bound to more than one target-specific binding partners, each binding partner bound to a nucleic acid strand and at least one docking strand stably bound to a labeled imager strand, directly or indirectly, wherein the nucleic acid strand is a docking strand or a primer strand if the nucleic acid is
 (a) a docking strand, increasing the number of docking strands associated with each target-specific binding partner, or 
 (b) a primer strand, associating more than one docking strand with the primer strand. 
   
     
     
         17 . The composition of  claim 16 , wherein the docking strand bound to a labeled imager strand, directly or indirectly, comprises at least 90% binding for 30 minutes. 
     
     
         18 . The composition of  claim 16 , wherein the
 (a) docking strand or   (b) docking strand and any intermediate strand is 80 nucleotides or less, 70, 60, 50, 40, or 30 nucleotides or less.   
     
     
         19 . The composition of  claim 16 , wherein the imager strand is 60 nucleotides or less. 
     
     
         20 . A composition comprising
 (1) a label,   (2) a first nucleic acid domain, a second nucleic acid domain, and a third nucleic acid domain, wherein each nucleic acid domain is from 1 to 9 nucleotides long,   (3) a first linking moiety linking the first nucleic acid domain and the second nucleic acid domain and   (4) a second linking moiety linking the second nucleic acid domain and the third nucleic acid domain,   wherein both linking moieties are independently chosen from (a) an abasic site with an intact phosphodiester backbone, (b) a linker cleavable by a nucleic acid glycosylase, (c) non-natural nucleotides, or (d) restriction site or a nicking site.   
     
     
         21 . The composition of  claim 20 , wherein additional nucleic acid domains are linked by additional linking moieties. 
     
     
         22 . The composition of  claim 20 , wherein at least one linking moiety is an abasic site (apyrimidinic) with an intact phosphodiester backbone. 
     
     
         23 . The composition of  claim 20 , wherein at least one linking moiety is susceptible to cleavage from Endonuclease VIII. 
     
     
         24 . The composition of  claim 20 , wherein the nucleic acid domains comprise DNA. 
     
     
         25 . The composition of  claim 20 , wherein the nucleic acid domains comprise RNA. 
     
     
         26 . The composition of  claim 20 , wherein at least one linking moiety comprises at least one non-natural nucleotide. 
     
     
         27 . The method of  claim 1 , wherein the imager strand and/or the intermediate strand comprises at least one U capable of cleavage by USER. 
     
     
         28 . The method of  claim 1 , wherein the imager strand and/or intermediate strand comprises at least one abasic site. 
     
     
         29 . The method of  claim 1 , wherein at least two targets are imaged using at least two labels in the same imaging step. 
     
     
         30 . The method of  claim 1 , wherein at least two targets are imaged using at least two labels, the signal extinguished, and then at least one more target is imaged using at least one of the same labels, wherein the imaging steps may be performed in either order.