Methods for extending polynucleotides
Inventors
Godron, Xavier • Horgan, Adrian • Gariel, Sylvain • Jeddeloh, Jeffrey • Nicol, Robert • Ybert, Thomas
Assignees
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Publication Number
US-11993773-B2
Publication Date
2024-05-28
Expiration Date
Abstract
The invention is directed to methods for synthesizing oligonucleotides directly 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.
Core Innovation
The invention provides a method of generating cDNA libraries each having an oligonucleotide label by capturing mRNA on one or more solid supports using capture oligonucleotides. The capture oligonucleotides are complementary to segments of the mRNA, are attached to the solid supports by 5′-ends, and have 3′-ends with free 3′-O-hydroxyls. The method extends the 3′-ends of the capture oligonucleotides with a reverse transcriptase using the captured mRNAs as templates to form cDNA libraries on the solid supports.
After cDNA formation on the solid supports, the method synthesizes oligonucleotide labels on cDNAs by template-free enzymatic synthesis. The approach supports generating cell-specific or tissue-specific cDNA libraries by capturing the mRNA of a single cell or a single tissue on the solid supports, and using unique cell-specific or tissue-specific oligonucleotide barcodes as the oligonucleotide labels. Template-free barcode or label synthesis on cDNAs is positioned as an enabling feature for uniquely identifying the source cell or tissue.
In further embodiments, the solid supports include binding compounds attached thereto for capturing predetermined non-nucleic acid ligands, and the binding compounds include antibody species with predetermined specificity to predetermined non-nucleic acid ligands, with releasable oligonucleotide barcodes for identification. Related embodiments include monitoring protein expression using protein-specific antibodies linked to distinct initiators that can be extended by enzymatic synthesis, and determining spatial protein distributions in a tissue section using sequential antibody binding, barcode elongation cycles, enzymatic deblocking to add position tags, releasing position tag-antibody barcode conjugates, and sequencing them.
Claims Coverage
The independent claim recites a three-step workflow: mRNA capture on solid supports, reverse transcription extension on the solid supports, and template-free enzymatic synthesis of oligonucleotide labels on cDNAs on the solid supports. Dependent limitations address split-and-mix barcode synthesis, antibody-based binding compounds with releasable barcodes, barcoding of living or fixed/permeabilized cells, protein-expression monitoring, and spatial protein distribution determination using sequential antibody applications and barcode elongation/deblocking cycles.
Cell-specific and tissue-specific cDNA library generation with template-free oligonucleotide labels
A method of generating cDNA libraries each having an oligonucleotide label comprising capturing mRNA on one or more solid supports with capture oligonucleotides having 5′-end attachment and free 3′-O-hydroxyls at the 3′-ends, extending the 3′-ends with a reverse transcriptase using the captured mRNAs as templates to form the cDNA libraries on the solid supports, and synthesizing oligonucleotide labels on the cDNAs of the solid supports by template-free enzymatic synthesis, wherein the capturing includes capturing the mRNA of a single cell or single tissue to form cell-specific or tissue-specific cDNA libraries and the oligonucleotide labels are unique cell-specific or tissue-specific oligonucleotide barcodes.
Solid-support capture via binding compounds for predetermined non-nucleic acid ligands
The method further wherein the one or more solid supports include binding compounds attached thereto for capturing predetermined non-nucleic acid ligands.
Split-and-mix synthesis for unique cell-specific barcodes
The method wherein synthesizing the unique cell-specific oligonucleotide barcodes is done using a split-and-mix synthesis method.
Antibody-based binding compounds with releasable oligonucleotide barcodes
The method wherein the binding compounds are antibody species with predetermined specificity to predetermined non-nucleic acid ligands, and each antibody has an attached releasable oligonucleotide barcode enabling identification of the antibody.
Uniquely barcoding living cells and fixed/permeabilized cells
The method wherein uniquely barcoding the cells includes living cells, fixed cells, or fixed and permeabilized cells.
Monitoring protein expression using protein-specific antibodies linked to extendable initiators
The method wherein monitoring protein expression uses one or more protein-specific antibodies linked to distinct initiators that can be extended by enzymatic synthesis.
Spatial determination of protein distributions in tissue using sequential antibody types and barcode position tags
A method for spatially determining protein distributions in a tissue section by sequentially applying multiple antibody types with barcode initiators, cycling barcode elongation using a template-independent DNA polymerase with 3′-O-blocked nucleoside triphosphates followed by enzymatic deblocking to add position tags, releasing the resulting position tag-antibody barcode conjugates, and sequencing them.
The claim set centers on generating cell- and tissue-specific cDNA libraries on solid supports by reverse transcription from mRNA captured with 3′-OH-containing capture oligonucleotides, followed by template-free enzymatic synthesis of unique oligonucleotide barcodes or labels on the cDNAs. Dependent features specify split-and-mix barcode synthesis, antibody-based capture for predetermined non-nucleic acid ligands with releasable barcodes, applicability to living and fixed/permeabilized cells, protein-expression monitoring via protein-specific antibody-linked initiators, and spatial protein distribution analysis using sequential antibody applications with barcode elongation, enzymatic deblocking, releasing position tag-antibody barcode conjugates, and sequencing.
Stated Advantages
Generates cell-specific or tissue-specific cDNA libraries using unique cell-specific or tissue-specific oligonucleotide barcodes.
Enables template-free enzymatic synthesis of oligonucleotide labels on cDNAs of the solid supports.
Supports identification of antibodies via releasable oligonucleotide barcodes attached to antibody species.
Enables monitoring protein expression using protein-specific antibodies linked to distinct initiators that can be extended by enzymatic synthesis.
Enables spatial determination of protein distributions in a tissue section using position tags added during barcode elongation and deblocking cycles followed by releasing and sequencing.
Documented Applications
Single-cell cDNA library construction by capturing mRNA of a single cell on one or more solid supports and generating cell-specific cDNA libraries with unique oligonucleotide barcodes.
Tissue-specific cDNA library generation by capturing mRNA of a single tissue on one or more solid supports and generating tissue-specific cDNA libraries with unique oligonucleotide barcodes.
Uniquely barcoding cells including living cells and fixed or fixed and permeabilized cells.
Monitoring protein expression using one or more protein-specific antibodies linked to distinct initiators.
Spatial distribution analysis of proteins in a tissue section using sequential antibody binding, barcode elongation cycles with subsequent deblocking, release of position tag-antibody barcode conjugates, and sequencing.
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