Direct oligonucleotide synthesis on cells and biomolecules

Abstract

The invention is directed to methods for synthesizing oligonucleotides direction on biomolecules or cells living or fixed. In some embodiments, template-free enzymatic synthesis is implemented under biological conditions with successive cycles of (i) enzymatic addition of a 3′-O-blocked nucleoside triphosphate and (ii) enzymatic deblocking of the incorporated nucleotide to regenerate a free 3′ hydroxyl. The invention has applications in single-cell cDNA library construction and analysis.

1 . A method of synthesizing on a viable cell an oligonucleotide with a predetermined sequence, the method comprising the steps of:
 a) providing an initiator with a free 3′-hydroxyl attached to a cell surface molecule of the cell or anchored in the cell surface membrane of the cell; and   b) repeating under biological conditions for a plurality of cycles the steps of
 (i) contacting the initiator or elongated fragments having free 3′-O-hydroxyls with a 3′-O-blocked nucleoside triphosphate and a template-independent DNA polymerase so that the initiator or elongated fragments are elongated by incorporation of a 3′-O-blocked nucleoside triphosphate to form 3′-O-blocked elongated fragments, and 
 (ii) deblocking the elongated fragments to form elongated fragments having free 3′-hydroxyls, thereby synthesizing the oligonucleotide of predetermined sequence. 
   
     
     
         2 . The method of  claim 1 , wherein said 3′-O-blocked nucleoside triphosphate is a 3′-phosphate-nucleoside triphosphate and said step of deblocking is carried out by treating said 3′-O-blocked elongated fragments with a 3′-phosphatase activity. 
     
     
         3 . The method of  claim 2 , wherein said 3′-phophatase activity is provided by T4 polynucleotide kinase, recombinant shrimp alkaline phosphatase, or a calf intestinal alkaline phosphatase. 
     
     
         4 . The method of  claim 1 . wherein said 3′-O-blocked nucleoside triphosphate is a 3′-ester-blocked nucleoside triphosphat; and said step of deblocking is carried out by treating said 3′-O-ester-blocked elongated fragments with an esterase activity. 
     
     
         5 . The method of  claim 1 , wherein said biological conditions comprise buffered physiological salts at a pH in the range of from 6.8 to 7.8 and a temperature in the range of from 15° C. to 41° C. 
     
     
         6 . The method of  claim 1 , wherein said viable cells are mammalian cells. 
     
     
         7 . The method of  claim 1 , wherein said initiator comprises an oligonucleotide having a lipophilic anchor covalently attached to a 5′ end, wherein the lipophilic anchor inserts stably into a cell surface membrane of said viable cell. 
     
     
         8 . A method of generating a cDNA library with cell-specific oligonucleotide barcodes, the method comprising the steps of:
 (a) synthesizing under biological conditions a unique oligonucleotide barcode on the cell surface membrane of each cell in a population of cells to form a population of barcoded cells;   (b) isolating each barcoded cell in a reactor;   (c) lysing barcoded cells in each reactor; and   (d) performing reverse-transcriptase polymerase chain reaction (RT-PCR) in each reactor to produce a cDNA library with cell-specific oligonucleotide barcodes.   
     
     
         9 . The method of  claim 8 , wherein said step of synthesizing comprises (a) attaching initiators to said cell surface membrane of each of said cells of said population, and (b) repeating cycles of
 (i) contacting under biological conditions the initiators or elongated fragments having free 3′-O-hydroxyls with a 3′-O-blocked nucleoside triphosphate and a template-independent DNA polymerase so that the initiators or elongated fragments are elongated by incorporation of a 3′-O-blocked nucleoside triphosphate to form 3′-O-blocked elongated fragments, and   (ii) deblocking the elongated fragments to form elongated fragments having free 3′-hydroxyls.   
     
     
         10 . The method of  claim 9 , wherein each of said cycles further comprises splitting said population of said cells among separate reaction mixtures in which said initiators or elongated fragments are elongated by a different kind of nucleoside triphosphate to form said elongated fragments after which said cells of the separate reaction mixtures are combined. 
     
     
         11 . The method of  claim 8 , wherein said reactors are micelles of a water-in-oil emulsion. 
     
     
         12 . The method of  claim 11 , wherein said micelles are generated by a microfluidics device. 
     
     
         13 . The method of  claim 9 , wherein said cDNAs from said reactors are combined and analyzed by high throughput DNA sequencing. 
     
     
         14 . A method of extending one or more native polynucleotides with a predetermined nucleotide sequence, comprising:
 providing the one or more native polynucleotides in a reaction mixture under TdT reaction conditions, the one or more native polynucleotides having free 3′-O-hydroxyls; and   extending the one or more native polynucleotides with the predetermined nucleotide sequence by repeated cycles of the steps
 (i) contacting the one or more native polynucleotides or elongated native polynucleotides having the free 3′-O-hydroxyls with a 3′-O-blocked nucleoside triphosphate and a TdT variant so that the one or more native polynucleotides or elongated native polynucleotides are elongated by incorporation of the 3′-O-blocked nucleoside triphosphate to form 3′-O-blocked elongated native polynucleotides, and 
 (ii) deblocking the elongated native polynucleotides to form elongated native polynucleotides having free 3′-O-hydroxyls, thereby synthesizing on the one or more native polynucleotides an oligonucleotide of the predetermined nucleotide sequence. 
   
     
     
         15 . The method of  claim 14 , wherein said predetermined nucleotide sequence comprises at least a plurality of different kinds of nucleotides. 
     
     
         16 . The method of  claim 15 , wherein said predetermined nucleotide sequence is unique for each native polynucleotide of the one or more native polynucleotides. 
     
     
         17 . The method of  claim 14 , wherein each of said cycles further comprises:
 prior to step (i), splitting said one or more native polynucleotides or said elongated native polynucleotides among two or more separate reaction mixtures, wherein   contacting with the 3′-O-blocked nucleoside triphosphate in step (i) comprises contacting said native polynucleotides or said elongated native polynucleotides with two or more different kinds of nucleoside triphosphate to form said elongated native polynucleotides: and   after step (i), combining said elongated native polynucleotides of the separate reaction mixtures.   
     
     
         18 . A method of generating cDNA libraries each with an oligonucleotide label, the method comprising the steps of:
 (a) capturing an mRNA by hybridizing the mRNA to capture oligonucleotides attached to one or more solid supports, wherein
 the capture oligonucleotides are complementary to segments of the mRNA, and 
 the capture oligonucleotides are attached to the one or more solid supports by 5′-ends and have 3′-ends with free 3′-hydroxyls; 
   (b) extending the 3′-ends of the capture oligonucleotides with a reverse transcriptase using the captured mRNAs as templates to form the cDNA libraries on the one or more solid supports; and   (c) synthesizing oligonucleotide labels on cDNAs of the one or more solid supports by template-free enzymatic synthesis.   
     
     
         19 . The method of  claim 18 , wherein:
 said step of capturing includes capturing the mRNA of a single cell on a bead to form said cDNA libraries that are cell-specific cDNA libraries; and   said oligonucleotide labels are unique cell-specific oligonucleotide barcodes.   
     
     
         20 . The method of  claim 18 , wherein said step of synthesizing comprises synthesizing said unique cell-specific oligonucleotide barcodes is by a split and mix synthesis method. 
     
     
         21 . The method of  claim 18 , wherein:
 said one or more solid supports is a solid surface with said capture oligonucleotides attached thereto; and   said step of capturing includes capturing mRNA of a permeabilized tissue slice disposed on the solid surface to form a spatial cDNA library array that preserves a spatial distribution of the cDNAs of the permeabilized tissue slice.   
     
     
         22 . The method of  claim 21 , wherein said step of synthesizing includes synthesizing at each of a plurality of different predetermined positions on said spatial cDNA library array a unique position tag to form position tag-cDNA conjugates. 
     
     
         23 . The method of  claim 22 , further comprising:
 steps of releasing and sequencing said position tag-cDNA conjugates to determine the spatial distribution of said mRNAs in said permeabilized tissue slice.   
     
     
         24 . The method of  claim 21 , wherein said solid surface includes binding compounds attached thereto for capturing predetermined non-nucleic acid ligands. 
     
     
         25 . The method of  claim 24 , wherein said binding compounds comprise one or more kinds of antibodies each with a predetermined specificity for one of said predetermined non-nucleic acid ligands, each different kind of antibody having attached a releasable oligonucleotide barcode from which the antibody can be identified.