Nucleic acid mediated electron transfer

• US 5,591,578 A
• Filed: 12/10/1993
• Issued: 01/07/1997
• Est. Priority Date: 12/10/1993
• Status: Expired due to Term

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Meade, Thomas J., Fraser, Scott E., Kayyem, Jon F.
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US08/166,036
• Time in Patent Office: 1,124 Days
• Field of Search: 435/6, 435/91.2, 935/77, 935/78, 536/23.1
• 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 the 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. Hybridization assays employing these complexes are disclosed.

Claims

1. A single-stranded nucleic acid containing one or multiple electron donor moieties and one or multiple electron acceptor moieties, wherein said electron donor and acceptor moieties are transition metal complexes covalently attached to the 2'"'"' or 3'"'"' position of a ribose of the ribose-phosphate backbone of said nucleic acid, said transition metal selected from the group consisting of Cd, Mg, Cu, Co, Pd, Zn, Fe and Ru, and wherein electron transfer can occur between said electron donor and acceptor moieties when said single stranded nucleic acid is hybridized to a target sequence.

3. A single-stranded nucleic acid according to claim 1 or 2 wherein said attachment is at the 3'"'"' position of said ribose.

4. A single-stranded nuclei;

acid according to claim 1 or 2 wherein said attachment is at the 2'"'"' position of said ribose.

2. A single-stranded nucleic acid containing one or multiple electron donor moieties and one or multiple electron acceptor moieties, wherein one of the electron donor or acceptor moieties is an electrode and the other is a transition metal complex covalently attached to the 2'"'"' or 3'"'"' position of a ribose of the ribose-phosphate backbone of said nucleic acid, wherein said transition metal is selected from the group consisting of Cd, Mg, Cu, Co, Pd, Zn, Fe and Ru, and wherein electron transfer can occur between said electron donor and acceptor moieties when said single stranded nucleic acid hybridizes to a target sequence.

5. A composition comprising a first single stranded nucleic acid containing one or multiple electron donor moieties and a second single stranded nucleic acid containing one or multiple electron acceptor moieties, wherein said electron donor and acceptor moieties are transition metal complexes covalently attached to the 2'"'"' or 3'"'"' position of a ribose of the ribose-phosphate backbone of said first and second single stranded nucleic acids, wherein said transition metal is selected from the group consisting of Cd, Mg, Cu, Co, Pd, Zn, Fe and Ru, and wherein electron transfer can occur between said electron donor and said electron acceptor moieties when said first and second single stranded nucleic acids are adjacently hybridized to a target domain.

7. A composition according to claim 5 or 6 wherein said attachment is at the 2'"'"' position of said ribose.

8. A composition according to claim 5 or 6 wherein said linkage is at the 3'"'"' position of said ribose.

6. A composition comprising a first single stranded nucleic acid containing one or multiple electron donor moieties and a second single stranded nucleic acid containing one or multiple electron acceptor moieties, wherein one of the electron transfer moieties is an electrode and the other is a transition metal complex covalently attached to the 2'"'"' or 3'"'"' position of a ribose of the ribose-phosphate backbone, wherein said transition metal is selected from the group consisting of Cd, Mg, Cu, Co, Pd, Zn, Fe and Ru, and wherein electron transfer can occur between said electron donor and said electron acceptor moieties when said single stranded nucleic acids are adjacently hybridized to a target sequence.

9. A method of detecting a target sequence in a nucleic acid sample comprising:

a) hybridizing a single stranded nucleic acid containing one or multiple electron donor moieties and one or multiple electron acceptor moieties to said target sequence to form a hybridization complex, wherein said electron donor and electron acceptor moieties are transition metal complexes covalently attached to the 2'"'"' or 3'"'"' position of a ribose of the ribose-phosphate backbone of said nucleic acids, wherein said transition metal is selected from the group consisting of Cd, Mg, Cu, Co, Pd, Zn, Fe and Ru; and

b) detecting electron transfer between said electron donor and acceptor moieties in the hybridization complex as an indicator of the presence or absence of said target sequence.

10. A method of detecting a target sequence in a nucleic acid sample comprising:

a) hybridizing a single stranded nucleic acid containing one or multiple electron donor moieties and one or multiple electron acceptor moieties to said target sequence to form a hybridization complex, wherein one of said electron donor or acceptor moieties is an electrode and the other is a transition metal complex covalently attached to the 2'"'"' or 3'"'"' position of a ribose of the ribose-phosphate backbone of said nucleic acid, wherein said transition metal is selected from the group consisting of Cd, Mg, Cu, Co, Pd, Zn, Fe and Ru; and

b) detecting electron transfer between said electron donor moieties and said electron acceptor moieties in the hybridization complex as an indicator of the presence or absence of said target sequence.

11. A method according to claim 9 or 10 wherein said linkage is at the 2'"'"' position of said ribose.

18. A method according to claim 9 or 10 wherein said attachment is at the 3'"'"' position of said ribose.

12. A method of detecting a target sequence in a nucleic acid wherein said target sequence comprises a first target domain and a second target domain adjacent to said first target domain, said method comprising:

a) hybridizing a first nucleic acid containing one or multiple electron donor moieties to said first target domain;

b) hybridizing a second nucleic acid containing one or multiple electron acceptor moieties to said second target domain, wherein said electron donor and electron acceptor moieties are transition metal complexes covalently attached to the 2'"'"' or 3'"'"' position of a ribose of the ribose-phosphate backbone of said nucleic acids, wherein said transition metal is selected from the group consisting of Cd, Mg, Cu, Co, Pd, Zn, Fe and Ru; and

c) detecting electron transfer between said electron donor and acceptor moieties while said first and second nucleic acids are hybridized to said first and second target domains as an indicator of the presence or absence of said target sequence in said nucleic acid sample.

14. A method according to claim 12 or 13 wherein said transition metal complex is attached at the 2'"'"' position of said ribose.

19. A method according to claim 12 or 13 wherein said attachment is at the 3'"'"' position of said ribose.

13. A method of detecting a target sequence in a nucleic acid wherein said target sequence comprises a first target domain and a second target domain adjacent to said first target domain, said method comprising:

a) hybridizing a first nucleic acid containing one or multiple electron donor moieties to said first target domain;

b) hybridizing a second nucleic acid containing one or multiple electron acceptor moieties to said second target domain, wherein one of said electron donor and acceptor moieties is an electrode and the other is a transition metal complex covalently attached to the 2'"'"' or 3'"'"' position of a ribose of the ribose-phosphate backbone of said nucleic acid, wherein said transition metal is selected from the group consisting of Cd, Mg, Cu, Co, Pd, Zn, Fe and Ru; and

c) detecting electron transfer between said electron donor and acceptor moieties while said first and second nucleic acids are hybridized to said first and second target domains as an indicator of the presence or absence of said target sequence in said nucleic acid sample.

15. A method of detecting a target sequence in a nucleic acid wherein said target sequence comprises a first target domain, a second target domain, and an intervening target domain between said first and said second target domains, said method comprising:

a) hybridizing a first nucleic acid containing one or multiple electron donor moieties to said first target domain;

b) hybridizing a second nucleic acid containing one or multiple electron acceptor moieties to said second target domain, wherein said electron donor and acceptor moieties are transition metal complexes covalently attached to the 2'"'"' or 3'"'"' position of a ribose of the ribose-phosphate backbone of said nucleic acid, wherein said transition metal is selected from the group consisting of Cd, Mg, Cu, Co, Pd, Zn, Fe and Ru;

c) hybridizing an intervening nucleic acid to said intervening target domain; and

d) detecting electron transfer between said electron donor and acceptor moieties while said first, second and intervening nucleic acids are hybridized to said first, second and intervening target domains as an indicator of the presence or absence of said target sequence in said nucleic acid sample.

17. A method according to claim 15 or 16 wherein said attachment is at the 2'"'"' position of said ribose.

20. A method according to claim 15 or 16 wherein said attachment is at the 3'"'"' position of said ribose.

16. A method of detecting a target sequence in a nucleic acid wherein said target sequence comprises a first target domain, a second target domain, and an intervening target domain between said first and said second target domains, said method comprising:

a) hybridizing a first nucleic acid containing one or multiple electron donor moieties to said first target domain;

b) hybridizing a second nucleic acid containing one or multiple electron acceptor moieties to said second target domain, wherein one of said electron donor and acceptor moieties is an electrode and the other is a transition metal complex covalently attached to the 2'"'"' or 3'"'"' position of a ribose of the ribose-phosphate backbone of said nucleic acid, wherein said transition metal is selected from the group consisting of Cd, Mg, Cu, Co, Pd, Zn, Fe and Ru;

c) hybridizing an intervening nucleic acid to said intervening target domain; and

d) detecting electron transfer between said electron donor moiety and said electron acceptor moiety while said first, second and intervening nucleic acids are hybridized to said first, second and intervening target domains as an indicator of the presence or absence of said target sequence in said nucleic acid sample.