Nucleic acid mediated electron transfer

• US 6,268,149 B1
• Filed: 12/06/1999
• Issued: 07/31/2001
• Est. Priority Date: 12/10/1993
• Status: Expired due to Term

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Kayyem, Jon Faiz, Meade, Thomas J., Fraser, Scott E.
• Primary Examiner(s): Zitomer, Stephanie
• Application Number:
  US09/454,498
• Time in Patent Office: 603 Days
• Field of Search: 435/6, 435/287.2, 435/288.7, 536/23.1, 536/24.3
• US Class Current: 435/6.11
• 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: C07H 23/00 Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12 esters with inorganic acids C07H11/00; metal salts, see parent compounds
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2537/101 Homogeneous assay format, e.g. one pot reaction
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels
• CPC Class Codes: C12Q 2563/137 Metal/ion, e.g. metal label
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)

Abstract

The present invention provides for the selective covalent modification of nucleic acids with redox active moieties such as transition metal complexes. Electron donor and electron acceptor moieties are covalently bound to the ribose-phosphate backbone of a nucleic acid at predetermined positions. The resulting complexes represent a series of new derivatives that are bimolecular templates capable of transferring electrons over very large distances at extremely fast rates. These complexes possess unique structural features which enable the use of an entirely new class of bioconductors and photoactive probes.

Claims

1. A method of detecting a mismatch in a target sequence comprising:

a) providing a hybridization complex comprising said target nucleic acid, which if present, is hybridized to at least a first nucleic acid comprising a mismatch with said target sequence, wherein said hybridization complex comprises an electrode and a covalently attached electron transfer moiety;

b) initiating electron transfer between said electrode and said electron transfer moiety under conditions that can distinguish between perfect matches and mismatches; and

c) determining whether a mismatch is present.

2. A method according to claim 1 wherein said hybridization complex comprises said target sequence and said first nucleic acid.

3. A method according to claim 1 wherein said hybridization complex comprises multiple electron transfer moieties.

4. A method according to claim 1 wherein said conditions include a temperature that will distinguish between perfect matches and mismatches.

5. A method according to claim 1 wherein said target sequence comprises said covalently attached electron transfer moiety.

6. A method according to claim 1 wherein said first nucleic acid comprises said covalently attached electron transfer moiety.

7. A method according to claim 1 wherein said first nucleic acid comprises said electrode.

8. A method of detecting a base pair mismatch in a target sequence comprising:

a) providing a first hybridization complex comprising said target nucleic acid, which if present, is hybridized to at least a first nucleic acid comprising a base pair mismatch with said target sequence, wherein said first hybridization complex comprises a first electrode and a covalently attached first electron transfer moiety;

b) detecting electron transfer between said first electrode and said first electron transfer moiety;

c) providing a second hybridization complex comprising said target nucleic acid, which if present, is hybridized to at least a second nucleic acid comprising a perfect match with said target sequence, wherein said second hybridization complex comprises a second electrode and a covalently attached second electron transfer moiety;

d) detecting electron transfer between said second electrode and said second electron transfer moiety; and

e) comparing the results of b) and d) to detect the presence of a mismatch in said target sequence.

9. A method according to claim 8 wherein said first hybridization complex comprises multiple electron transfer moieties.

10. A method according to claim 8 wherein said second hybridization complex comprises multiple electron transfer moieties.

11. A method of detecting a mismatch in a target sequence comprising:

a) providing a hybridization complex comprising said target nucleic acid, which if present, is hybridized to at least a first nucleic acid comprising a mismatch with said target sequence, wherein said hybridization complex comprises an electrode and a moiety capable of donating or accepting an electron;

b) initiating electron transfer between said electrode and said moiety capable of donating or accepting an electron under conditions that can distinguish between perfect matches and mismatches; and

c) determining whether a mismatch is present.

12. A method according to claim 11 wherein said hybridization complex comprises said target sequence and said first nucleic acid.

13. A method of detecting a mismatch in a target sequence comprising:

a) providing a hybridization complex comprising said target nucleic acid, which if present, is hybridized to at least a first nucleic acid comprising a mismatch with said target sequence, wherein said hybridization complex comprises a covalently attached electron donor moiety and a covalently attached electron acceptor moiety;

b) initiating electron transfer between said electron donor moiety and said electron acceptor moiety under conditions that can distinguish between perfect matches and mismatches; and

c) determining whether a mismatch is present.

14. A method according to claim 13 wherein said hybridization complex comprises said target sequence and said first nucleic acid.

15. A method of detecting a single point mutation in a target sequence comprising:

a) hybridizing a first single-stranded nucleic acid comprising an electrode to a first target domain of said target sequence;

b) hybridizing a second single-stranded nucleic acid comprising a covalently attached electron transfer moiety to a second target domain of said target sequence;

c) ligating said first and second single stranded nucleic acids together, wherein a single point mutation at the ligation point prevents ligation; and

d) detecting electron transfer between said electrode and said electron transfer moiety as an indication of the presence of a single point mutation in said target sequence.

16. A method of detecting a single point mutation in a target sequence comprising:

a) hybridizing a first single-stranded nucleic acid to a first target domain of said target sequence;

b) hybridizing a second single-stranded nucleic acid comprising a covalently attached electron transfer moiety to a second target domain of said target sequence;

c) ligating said first and second single stranded nucleic acids together to form a ligated probe, wherein a single point mutation at the ligation point prevents ligation;

d) forming a hybridization complex comprising said ligated probe, wherein said hybridization complex further comprises an electrode; and

e) detecting electron transfer between said electrode and said electron transfer moiety.

17. A method of detecting a mismatch in a target sequence comprising:

a) providing a hybridization complex comprising said target nucleic acid, which if present, is hybridized to at least a first nucleic acid comprising a mismatch with said target sequence, wherein said hybridization complex comprises an electrode and a moiety capable of donating or accepting an electron;

b) initiating electron transfer between said electrode and said moiety capable of donating or accepting an electron under conditions that can distinguish between perfect matches and mismatches; and

c) determining whether a mismatch is present.

18. A method according to claim 17 wherein said hybridization complex comprises said target sequence and said first nucleic acid.

19. A method according to claim 17 wherein said first nucleic acid comprises said electrode.