Method for determining a nucleic acid

• US 6,225,052 B1
• Filed: 11/21/1997
• Issued: 05/01/2001
• Est. Priority Date: 02/24/1997
• Status: Expired due to Fees

• Current Assignee: PNA Diagnostics APS, Georgia Tech Research Corporation (University System of Georgia)
• Original Assignee: Roche Diagnostics GmbH (Roche Holding AG), Georgia Tech Research Corporation (University System of Georgia)
• Inventors: Orum, Henrik, Koch, Troels, Armitage, Bruce A., Ly, Danith, Schuster, Gary B., Hansen, Henrik Frydenlund, Batz, Hans-Georg
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US08/975,894
• Time in Patent Office: 1,257 Days
• Field of Search: 435/6, 435/810, 436/501, 536/23.1, 536/24.1, 536/24.3, 536/24.31, 536/24.32, 536/24.33, 536/25.3
• 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: C07K 14/003 Peptide-nucleic acids (PNAs)
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

New electron transfer moiety labeled nucleic acid analogue probes are provided that can be used in methods for determining nucleic acids in a sample. The new probes can be prepared using novel monomer subunits in a chemical synthesis route. The nucleic acids can be determined by binding the probe molecules to the nucleic acid and inducing electron transfer within the complex formed. The occurrence of the electron transfer is determined as a measure of the nucleic acid.

Claims

1. A method for determining a nucleic acid in a sample comprising

binding to the nucleic acid a probe molecule having a polymeric backbone different from the natural sugar phosphate backbone via base mediated hydrogen bonding, wherein an electron acceptor or an electron donor or an electron acceptor and an electron donor is covalently bound to said probe molecule at a non-terminal subunit of said polymeric backbone not bearing a nucleobase,

inducing an electron transfer from said electron donor or to said elector acceptor and

determining the occurrence of the electronic transfer as a measure of said nucleic acid.

2. A method according to claim 1, characterized in that the electronic transfer is induced by photo inducement.

3. A method according to claim 1, characterized in that said electron donor is a nucleobase.

4. A method according to claim 1, characterized in that said electron donor is bound to said probe molecule.

5. A method according to claim 1, characterized in that said electron donor is bound to a further probe molecule bound to said nucleic acid.

6. A method according to claim 1, characterized in that said electron acceptor is bound to said probe molecule at a position which is different from the last nucleobase bearing subunit of said probe molecule.

7. A method according to claim 1, characterized in that said occurrence of electron transfer is determined by analyzing changes in the electronic or photometric status of said electron acceptor.

8. A method according to claim 1, characterized in that said occurrence of electron transfer is determined by analyzing changes in the electronic or photometric status of said electron donor.

9. A method according to claim 1, characterized in that said occurrence of electron transfer is determined by detecting a change in the molecular structure of said nucleic acid compared to the structure without the inducement of said electron transfer.

10. A method according to claim 1, characterized in that the electron donor is a guanine base.

11. A method according to claim 1, characterized in that the electron donor is an electrode.

12. A method according to claim 1, characterized in that the probe molecule is bound to a solid phase.

13. A method according to claim 10, characterized in that said change is the cleavage of a nucleobase from said backbone.

14. A method according to claim 1, characterized in that said backbone comprises at least one peptide bond.

15. The method of claim 1, wherein said electron acceptor is a molecule of the general formula IIIa or IIIb,

wherein X, Y, Z, Q, V, and W are independently selected from the atoms C, N, S, and O;

X, Y, Z, Q, V, and W are connected by either single or double bonds;

R₁-R₆ are independently selected from the group of —

H, —

O⁻

, —

OH, —

OR′

, —

SH, —

SR′

, —

NH₂, —

NO₂, —

SO₃⁻

, —

SO₂⁻

, —

CN, —

PO₃²⁻

, —

PO₂⁻

, —

COOH, —

CO—

R′

, —

COOR′

, —

CS—

R′

, —

CSO—

R′

, —

COO⁻

, —

N═

N—

, halogen, —

NHR′

, —

N(R′

R″

), hydrocarbyl, and heterocycle;

R′ and

R″

are independently selected from the same groups as R₁-R₆;

wherein at least one of X, Y, Z, Q, V, and W can together with any one of R₁-R₆ also be —

CO⁻

, —

SO⁻

or —

SO₂⁻

; and

at least one of R₁-R₆ can be a single or double bond.

16. The method of claim 1 wherein the polymeric backbone different from the natural sugar phosphate backbone is a peptide nucleic acid (PNA).

17. The method of claim 1 wherein one of the electron donor or electron acceptor is not a naturally occurring nucleobase.

18. The method of claim 1 wherein both the electron donor and electron acceptor are not naturally occurring nucleobases.

19. The method of claim 9 wherein said change is the cleavage of the backbone of the nucleic acid.