Compositions and methods for genomic dna and gene expression analysis in single cells

Abstract

Provided herein are compositions and methods to assess the genomic landscape of fixed cells using light activated oligonucleotides that can be directed to the nucleus, mitochondria, or cytoplasm of fixed cells and that, upon activation, can be extended for in situ copying of nuclear single-stranded DNA (i.e., open chromatin), open mitochondrial DNA, and/or cytoplasmic RNA into barcoded complementary DNA. These methods also provide for gene specific 3D chromatin structural niche analysis.

What is claimed is: 
     
         1 . A method for identifying the expressed RNAs in a cell, the method comprising:
 (a) introducing a population of oligonucleotide molecules into the cell, wherein each molecule comprises, from 5′ to 3′, an amplification segment, an index barcode segment, a hybridization segment, and a reversibly terminating nucleotide;   (b) incubating the cell under conditions to allow for the hybridization segments of the population of oligonucleotide molecules to anneal to expressed RNAs;   (c) activating at least a portion of the annealed oligonucleotide molecules to expose an extendable 3′ hydroxy group; and   (d) synthesizing cDNAs from the expressed RNAs by extending the activating oligonucleotide molecules from their extendable 3′ hydroxy groups.   
     
     
         2 . The method of  claim 1 , wherein the amplification segment is an RNA polymerase promoter. 
     
     
         3 . The method of  claim 1 , wherein the amplification segment is a primer binding site. 
     
     
         4 . The method of  claim 1 , wherein the amplification segment comprises between about seven and about fifty nucleotides. 
     
     
         5 . The method of  claim 1 , wherein the hybridization segment comprises a poly-T sequence. 
     
     
         6 . The method of  claim 1 , wherein the hybridization segments comprise one or more known nucleotide sequence. 
     
     
         7 . The method of  claim 6 , wherein each known nucleotide sequence is complementary to a target RNA sequence. 
     
     
         8 . The method of  claim 1 , wherein the hybridization segment comprises between about seven and about thirty nucleotides. 
     
     
         9 . The method of  claim 1 , wherein the population of oligonucleotide molecules further comprise a spacer segment positioned between the amplification segment and the index barcode segment. 
     
     
         10 . The method of  claim 1 , wherein the reversibly terminating nucleotide is a photoactivatable terminating nucleotide. 
     
     
         11 . The method of  claim 10 , wherein the photoactivatable terminating nucleotide comprises a structure of the formula: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein: 
         R 1  is O or S; 
         R 2  is hydrogen or hydroxy; 
         R 3  is alkyl (C≤6)  or substituted alkyl (C≤6) ; 
         R 4  is
 hydrogen, hydroxy, halo, amino, nitro, cyano, azido or mercapto; 
 alkyl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkyl-amino (C≤6) , amido (C≤6) , or a substituted version of any of these groups; 
 
         R 5 , R 6 , and R 7  are each independently:
 hydrogen, hydroxy, halo, amino, nitro, cyano, azido or mercapto; 
 alkyl (C≤6) , alkenyl (C≤6) , alkynyl (C≤6) , aryl (C≤6) , aralkyl (C≤8) , heteroaryl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkyl-amino (C≤6) , amido (C≤6) , or a substituted version of any of these groups; 
 a group of formula: 
 
       
       
         
           
           
               
               
           
         
         
           
             wherein
 X is 
  —O—, —S—, or —NH—; or 
  alkanediyl (C≤12) , alkenediyl (C≤12) , alkynediyl (C≤12) , or a substituted version of any of these groups; 
 Y is —O—, —NH—, alkanediyl (C≤12)  or substituted alkane-diyl (C≤12) ; 
 n is an integer from 0-6; and 
 m is an integer from 0-6; or 
 
           
           a -linker-reporter; 
         
         or a tautomer or optical isomer thereof. 
       
     
     
         12 . The method of  claim 11 , wherein R 7  is methoxy. 
     
     
         13 . The method of  claim 12 , wherein the photoactivatable terminating nucleotide comprises a structure of the formula: 
       
         
           
           
               
               
           
         
         wherein R 5  is a -linker-reporter. 
       
     
     
         14 . The method of  claim 13 , wherein the photoactivatable terminating nucleotide comprises a structure of the formula: 
       
         
           
           
               
               
           
         
       
     
     
         15 . The method of  claim 1 , wherein the cell is fixed. 
     
     
         16 . The method of  claim 1 , wherein activation comprises exposing the cytoplasm to ultraviolet light. 
     
     
         17 . The method of  claim 1 , wherein activation is performed throughout the cytoplasm. 
     
     
         18 . The method of  claim 1 , wherein activation is performed at a particular site within the cytoplasm. 
     
     
         19 . The method of  claim 18 , wherein the particular site is an axon or a dendrite. 
     
     
         20 . The method of  claim 1 , wherein synthesizing cDNAs comprises adding an RNA-dependent DNA polymerase. 
     
     
         21 . The method of  claim 1 , further comprising processing the synthesized cDNAs to generate double-stranded cDNAs comprising the index barcode segment and the amplification segment of the oligonucleotides. 
     
     
         22 . The method of  claim 21 , further comprising obtaining a sequence of at least a portion of the double-stranded cDNAs, thereby identifying the expressed RNAs. 
     
     
         23 . The method of  claim 1 , wherein the method is a multiplex method, wherein the method is (i) performed sequentially on two or more particular sites in the cell or (ii) performed sequentially on two or more cells in a sample.