TARGETED TRANSPOSITION FOR USE IN EPIGENETIC STUDIES

• US 20160115474A1
• Filed: 05/22/2014
• Published: 04/28/2016
• Est. Priority Date: 11/22/2011
• Status: Active Grant

• Current Assignee: Active Motif, Inc.
• Original Assignee: Active Motif, Inc.
• Inventors: Fernandez, Joseph, Jelinek, Mary Anne, Egan, Brian Stanley
• Granted Patent: US 10,689,643 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1068 Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/159 Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin

Abstract

Disclosed herein are compositions, methods and kits useful for epigenetic analysis based on the use of transposons that are targeted to specific regions of chromatin based on DNA-DNA interactions, protein-protein interactions, RNA-RNA interactions, and nucleic acid-protein interactions.

Claims

1. A method of making a nucleic acid sequence library or libraries comprising:

a. extracting chromatin from a sample;

b. adding at least one protein-oligonucleotide conjugate comprising an extraction moiety to said chromatin;

c. allowing said protein conjugates to locate at its/their target proteins and/or target DNA-binding sites and/or noncoding RNA-binding sites in said chromatin

d. tagging the nucleic acid in said chromatin fragments with said conjugate by inducing an intermolecular reaction between said oligonucleotide and said nucleic acid; and

e. extracting the nucleic acids tagged using the extraction moiety.

2. A method according to claim 1 wherein the protein-oligonucleotide conjugate comprises transposase and the intermolecular reaction is transposition.

7. A method according to claim 1, wherein the extraction moiety is a biotin molecule.

12. A method according to claim 1, wherein the chromatin has been crosslinked to proteins in vivo.

13. A method according to claim 1, wherein the extracted tagged DNA is sequenced.

14. A kit for practicing the method according to claim 1, wherein said kit contains reagents, protein-transposome complex and/or oligonucleotide-transposome construct, and instructions for use.

3. A method of making a nucleic acid sequence library or libraries comprising:

a. extracting and optionally fragmenting chromatin from a sample;

b. adding at least one oligonucleotide-transposome construct comprising an extraction moiety to said chromatin;

c. allowing said oligonucleotide-transposome construct to locate at its/their target DNA and/or noncoding RNA-binding sites in said chromatin;

d. tagging the nucleic acid in said chromatin fragments with said construct by inducing an intermolecular reaction between said oligonucleotide and said nucleic acid; and

e. extracting the nucleic acids tagged using the extraction moiety.

4. A method according to claim 3 wherein the oligonucleotide-transposome construct comprises transposase and the intermolecular reaction is transposition.

5. The oligonucleotide of claim 3, wherein the oligonucleotide targets non-coding RNA.

6. The oligonucleotide of claim 3, wherein the oligonucleotide contains peptide nucleic acid that targets G-quadruplex structures.

18. A method according to claim 3, wherein the extraction moiety is a biotin molecule.

19. A method according to claim 3, wherein the chromatin has been crosslinked to proteins in vivo.

20. A method according to claim 3, wherein the extracted tagged DNA is sequenced.

8. A protein-transposome complex, comprising a protein that binds a protein-binding partner, methylated DNA, non-coding RNA, or DNA-binding site.

10. A protein-transposome complex according to claim 8, wherein the protein is selected from the group consisting of one or more of bZIP domain, DNA-binding domain, helix-loop-helix, helix-turn-helix, MG-box, leucine zipper, lexitropsin, nucleic acid simulations, zinc finger, histone methylases, recruitment proteins, Swi6 or chromodomain.

11. A protein-transposome complex according to claim 8, wherein the protein is MBD2.

15. The transposome of claim 8, wherein the transposon is Tn5 or TS-Tn5.

9. An oligonucleotide-transposome construct, comprising an oligonucleotide that hybridizes to non-coding RNA or G-quadruplex structures.

16. (canceled)

17. (canceled)