Multiplexed Signal Amplified FISH Via Splinted Ligation Amplification and Sequencing

Abstract

The present invention relates to a method for amplifying at least one target RNA in a fixed and, optionally, expanded biological sample. In an embodiment of the invention, the method comprises incubating the fixed biological sample with a pair of polynucleotides complementary to non-overlapping and proximal sequences of a target RNA, wherein the polynucleotide pair hybridizes to the target RNA; ligating the polynucleotide pair using a ligase; and amplifying the ligation product. The invention further provides methods for detecting and optionally quantifying and/or sequencing the amplification product. As the method comprises hybridizing polynucleotide pairs to a target RNA in a fixed biological sample, the target RNA can be hybridized in situ.

What is claimed is: 
     
         1 . A method for amplifying one or more target RNAs in a fixed biological sample comprising:
 (a) incubating the biological sample with a pair of polynucleotides complementary to non-overlapping and proximal sequences of a target RNA wherein the polynucleotides hybridize to the target RNA;   (b) ligating the polynucleotide pair using a ligase; and   (c) amplifying the ligation product.   
     
     
         2 . The method of  claim 1 , wherein the fixed biological sample is an expandable biological sample. 
     
     
         3 . The method of  claim 2 , wherein prior to step (a) the sample is:
 (i) contacted with a small molecule linker or a nucleic acid adaptor comprising a binding moiety and an anchor, wherein the binding moiety binds to target nucleic acids in the sample; and wherein the anchor comprises a polymerizable moiety;   (ii) permeated with a composition comprising precursors of a swellable material; and   (iii) polymerization of the precursors of the swellable material is initiated to form a swellable material, wherein the swellable material is bound to the small molecule linker or a nucleic acid adaptor to form a sample-swellable material complex, thereby providing an expandable sample.   
     
     
         4 . The method of  claim 3 , further comprising expanding the sample. 
     
     
         5 . The method of  claim 3 , wherein expanding the sample comprises adding an aqueous solvent or liquid to cause the sample-swellable material complex to swell, thereby physically expanding the complex. 
     
     
         6 . The method of  claim 5 , wherein the biological sample is expanded prior to step (c) or post step (c). 
     
     
         7 . The method of  claim 3 , wherein the swellable material is a hydrogel. 
     
     
         8 . The method of  claim 1 , wherein the pair of polynucleotides is hybridized to the target RNA in situ. 
     
     
         9 . The method of  claim 1 , further comprising the step of sequencing the amplified ligation product within the fixed biological sample. 
     
     
         10 . A method for detecting one or more target RNAs in a fixed biological sample comprising:
 (a) incubating the biological sample with a pair of polynucleotides complementary to non-overlapping and proximal sequences of a target RNA wherein the polynucleotides hybridize to the target RNA;   (b) ligating the polynucleotide pair using a ligase;   (c) amplifying the ligation product;   (d) detecting the amplified product.   
     
     
         11 . The method of  claim 10 , wherein the fixed biological sample is an expandable sample. 
     
     
         12 . The method of  claim 11 , wherein prior to step (a) the sample is
 (i) contacted with a small molecule linker or a nucleic acid adaptor comprising a binding moiety and an anchor, wherein the binding moiety binds to target nucleic acids in the sample; and wherein the anchor comprises a polymerizable moiety;   (ii) permeated with a composition comprising precursors of a swellable material; and   (iii) polymerization of the precursors of the swellable material is initiated to form a swellable material, wherein the swellable material is bound to the small molecule linker or a nucleic acid adaptor to form a sample-swellable material complex, thereby providing an expandable sample.   
     
     
         13 . The method of  claim 12 , further comprising expanding the sample. 
     
     
         14 . The method of  claim 13 , wherein expanding the sample comprises adding an aqueous solvent or liquid to cause the sample-swellable material complex to swell, thereby physically expanding the complex. 
     
     
         15 . The method of  claim 14 , wherein the biological sample is expanded prior to step (c) or post step (c). 
     
     
         16 . The method of  claim 12 , wherein the swellable material is a hydrogel. 
     
     
         17 . The method of  claim 10 , wherein the pair of polynucleotides is hybridized to the target RNA in situ. 
     
     
         18 . The method of  claim 10 , further comprising the step of localizing the amplified product within the fixed biological sample. 
     
     
         19 . The method of  claim 10 , further comprising the step of sequencing the amplified ligation product within the fixed biological sample.