Spatial sequencing of nucleic acids using DNA origami probes

• US 10,640,826 B2
• Filed: 02/01/2018
• Issued: 05/05/2020
• Est. Priority Date: 06/05/2012
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Church, George M., Terry, Richard C., Vigneault, Frederic
• Primary Examiner(s): Bhat, Narayan K
• Application Number:
  US15/886,163
• Publication Number:
  US 20180230533A1
• Time in Patent Office: 824 Days
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2563/185 Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals

Abstract

A method of sequencing nucleic acids is provided using DNA origami as a barcode for a nucleic acid probe.

Claims

1. A method for identifying one or more nucleotides of a single stranded nucleic acid molecule, comprising:

(a) providing said single stranded nucleic acid molecule comprising at least one nucleic acid probe coupled thereto, wherein said at least one nucleic acid probe includes a nucleic acid sequence complementary to said single stranded nucleic acid molecule and a spatially distinct nucleic acid structure, wherein said nucleic acid sequence is attached to said spatially distinct nucleic acid structure via a linker, wherein said spatially distinct nucleic acid structure is a folded configuration comprising a plurality of separate strands, and wherein said spatially distinct nucleic acid structure corresponds to one or more nucleotides of said at least one nucleic acid probe coupled to said single stranded nucleic acid molecule;

(b) detecting one or more signals from said spatially distinct nucleic acid structure; and

(c) using said one or more signals detected in (b) to identify said one or more nucleotides of said at least one nucleic acid probe, thereby identifying said one or more nucleotides of said single stranded nucleic acid molecule.

2. The method of claim 1, wherein in at least (a), said nucleic acid probe is coupled to an oligonucleotide molecule coupled to said single stranded nucleic acid molecule.

3. The method of claim 2, further comprising, prior to (a), bringing said single stranded nucleic acid molecule in contact with said oligonucleotide molecule under conditions sufficient to permit said oligonucleotide molecule to couple to said single stranded nucleic acid molecule.

4. The method of claim 3, further comprising, prior to (a), bringing said at least one nucleic acid probe in contact with said single stranded nucleic acid molecule comprising said oligonucleotide molecule coupled thereto, under conditions sufficient to permit said nucleic acid probe to couple to said single stranded nucleic acid molecule and said oligonucleotide molecule.

5. The method of claim 2, wherein said oligonucleotide molecule is a sequencing primer.

6. The method of claim 1, wherein said one or more signals are one or more optical signals.

7. The method of claim 1, wherein said at least one nucleic acid probe is a plurality of nucleic acid probes.

8. The method of claim 1, wherein said one or more nucleotides of said at least one nucleic acid probe are a plurality of nucleotides coupled to said single stranded nucleic acid molecule.

9. The method of claim 8, wherein said plurality of nucleotides comprises up to 100 nucleotides.

10. The method of claim 1, wherein said linker is a cleavable linker.

11. The method of claim 1, further comprising, prior to (b), straightening said single stranded nucleic acid molecule.

12. The method of claim 1, wherein said single stranded nucleic acid molecule is attached to a support.

13. The method of claim 1, wherein said folded configuration is a two-dimensional or three-dimensional structure.

14. The method of claim 1, wherein said one or more nucleotides of said at least one nucleic acid probe is one or more nucleotide analogs.

15. The method of claim 1, wherein said spatially distinct nucleic acid structure corresponds to a plurality of nucleotides of said at least one nucleic acid probe, which plurality of nucleotides is coupled to said single stranded nucleic acid molecule.

16. A composition for identifying one or more nucleotides of a single stranded nucleic acid molecule, comprising said single stranded nucleic acid molecule comprising at least one nucleic acid probe coupled thereto, wherein said at least one nucleic acid probe is coupled to an oligonucleotide molecule coupled to said single stranded nucleic acid molecule, wherein said at least one nucleic acid probe includes a spatially distinct nucleic acid structure, which spatially distinct nucleic acid structure is a folded configuration comprising a plurality of separate strands, and wherein said spatially distinct nucleic acid structure corresponds to one or more nucleotides of said nucleic acid probe coupled to said single stranded nucleic acid molecule.

17. The composition of claim 16, wherein said one or more nucleotides of said at least one nucleic acid probe are a plurality of nucleotides coupled to said single stranded nucleic acid molecule.

18. The composition of claim 16, wherein said one or more nucleotides of said at least one nucleic acid probe are part of a sequence of nucleotides coupled to said single stranded nucleic acid molecule, and wherein said spatially distinct nucleic acid structure is attached to said sequence of nucleotides via a linker.

19. The composition of claim 16, wherein said folded configuration is a two-dimensional or three-dimensional structure.