Polar dyes

• US 20060229441A1
• Filed: 03/09/2006
• Published: 10/12/2006
• Est. Priority Date: 03/09/2005
• Status: Active Grant

• Current Assignee: Cepheid (Danaher Corporation)
• Original Assignee: Cepheid (Danaher Corporation)
• Inventors: Gall, Alexander A.
• Granted Patent: US 7,799,926 B2
• Time in Patent Office: Days
• US Class Current: 534/653
• CPC Class Codes: C09B 11/24 Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
• CPC Class Codes: C09B 23/0066 the polymethine chain being part of a carbocyclic ring,(e.g. benzene, naphtalene, cyclohexene, cyclobutenene-quadratic acid)
• CPC Class Codes: C09B 23/083 five >CH- groups
• CPC Class Codes: C09B 29/00 Monoazo dyes prepared by diazotising and coupling
• CPC Class Codes: C09B 43/00 Preparation of azo dyes from other azo compounds
• CPC Class Codes: C09B 56/005 Azo-nitro dyes
• CPC Class Codes: C09B 57/00 Other synthetic dyes of known constitution
• CPC Class Codes: C09B 57/02 Coumarine dyes
• CPC Class Codes: C09K 11/06 containing organic luminescent materials
• CPC Class Codes: C09K 2211/1014 bridged by heteroatoms, e.g. N, P, Si or B
• CPC Class Codes: C09K 2211/1022 bridged by heteroatoms, e.g. N, P, Si or B
• CPC Class Codes: C09K 2211/1029 containing one nitrogen atom as the heteroatom
• CPC Class Codes: C09K 2211/1048 with oxygen
• CPC Class Codes: C09K 2211/1088 containing oxygen as the only heteroatom
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention relates to novel polar fluorescent and quenchers dyes, and minor groove binder with enhanced polarity. The present invention further relates to methods of preparing oligonucleotide probes labeled with polar arsonate dyes under the condition of automated synthesis and method of using such probes.

Claims

1. A polar fluorescent dye comprising a fluorescent compound substituted with one or more arsonate groups, the fluorescent compound having an excitation between 340 nm and 1100 nm and fluorescence emission maximum between 440 and 1150 nm.

2. The polar fluorescent dye of claim 1 comprising a compound of Formula (I):

3. The polar fluorescent dye of claim 1 comprising a compound of Formula (II):

4. The polar fluorescent dye of claim 1 comprising a compound of Formula (III):

6. The polar fluorescent dye of claim 1 comprising a compound of Formula (V):

7. The polar fluorescent dye of claim 1 comprising a compound of Formula (VI):

8. The polar fluorescent dye of claim 1 comprising a compound of Formula (VII):

9. The polar fluorescent dye of claim 1 comprising a compound of Formula (VIII):

5. A compound of Formula (IV)

10. A compound of Formula (IX):

11. (canceled)

12. An oligonucleotide conjugate comprising an oligonucleotide substituted with a dye having an arsonate group.

13. The oligonucleotide of claim 12 wherein the dye is a fluorescent dye having an excitation between 340 nm and 1100 nm and fluorescence emission maximum between 440 and 1150 nm.

14. The oligonucleotide of claim 12 wherein the dye is a non-fluorescent quencher being adapted to absorb fluorescence emitted from a reporting fluorophore prior to the binding of the oligonucleotide to a target.

15. The oligonucleotide of claim 12 wherein the dye is a compound of Formula (I), Formula (II), Formula (III), Formula (V), Formula (VI), Formula (VII), or Formula (VIII).

16. The oligonucleotide of claim 12 further comprising an MGB group.

17. The oligonucleotide of claim 16 wherein the MGB group is substituted with a polar arsonate group.

18. The oligonucleotide conjugate of claim 12 wherein the oligonucleotide is a probe selected from molecular beacons, Scorpion®

probes, sunrise probes, conformationally assisted probes and TaqMan™

probes.

19. A method of directly detecting a target nucleic acid, comprising:

providing a dye-labeled oligonucleotide probe comprising an oligonucleotide conjugated to one or more dye compounds selected from the group consisting of Formula (I), Formula (II), Formula (III), Formula (V), Formula (VI), Formula (VII), and Formula (VIII), the oligonucleotide further optionally comprising an MGB optionally substituted with one or more polar arsonate groups,

adding the dye-labeled oligonucleotide probe to a biological sample, wherein the probe is complementary or substantially complementary to the target nucleic acid;

allowing the probe to hybridize with the target nucleic acid under an appropriate condition and for sufficient amount of time; and

detecting fluorescence emitted from the dye.

20. A method of preparing an oligonucleotide in an automated synthesis, using a dye reagent of any of Formulae (I)-(VIII), wherein R_x is a phosphoramidite group, the method comprising:

performing automated synthesis of oligonucleotide using phosphoramidite method to provide a dye-labeled oligonucleotide;

deprotecting —

As(═

O)(OR⁶)(OR⁷) or —

As(OR⁶)₂(OR⁷)₂ of the dye-labeled oligonucleotide to provide a dye-labeled oligonucleotide having negatively charged polar group selected from —

AsO₃²⁻

or —

AsO₃H⁻

; and

isolating the dye-labeled oligonucleotide from the reaction medium.

21. The method of claim 20 further comprising a step of conjugating an MGB in the oligonucleotide.

22. The method of claim 21 wherein the MGB is substituted with an arsonate group.

23. A method of detecting amplification of a target sequence in real life using a TaqMan assay, the method comprising the steps of:

providing a dye-labeled oligonucleotide probe comprising an oligonucleotide conjugated to a quencher dye of Formula (IV) and a fluorescent dye of any of the Formulae (I-III) and (V-VIII), each dye having one or more polar arsonate group, wherein the probe is in a conformation allowing the fluorescent dye to be quenched by the quencher dye;

adding the dye-labeled oligonucleotide probe to a PCR reaction medium;

running the PCR temperature cycles to allow the probe to hybridize with the target and get cleaved by Taq polymerase when the target is present; and

reading the fluorescence signal from the cleaved dye in the real time.

24. A method of detecting amplification of a target sequence in real life using a Molecular Beacon assay, the method comprising the steps of:

providing a dye-labeled oligonucleotide probe comprising an oligonucleotide conjugated to a quencher dye of Formula (IV) and a fluorescent dye of any of the Formulae (I-III) and (V-VIII), each dye having one or more polar arsonate group, wherein the probe is in a conformation allowing the fluorescent dye to be quenched by the quencher dye;

adding the dye-labeled oligonucleotide probe to a PCR reaction medium;

running the PCR temperature cycles to allow the probe to hybridize with the target thereby separating the fluorescent dye and the quencher dye when the target is present; and

reading the fluorescence signal from the unquenched fluorescent dye in the real time.

25. A method of detecting amplification of a target sequence in real life using a Scorpion Probe assay, the method comprising the steps of:

providing a dye-labeled oligonucleotide probe comprising a primer, an oligonucleotide conjugated to a quencher dye of Formula (IV) and a fluorescent dye of any of the Formulae (I-III) and (V-VIII), each dye having one or more polar arsonate group, wherein the probe is in a conformation allowing the fluorescent dye to be quenched by the quencher dye;

adding the dye-labeled oligonucleotide probe to a PCR reaction medium;

running the PCR temperature cycles to allow the primer to hybridize with a target DNA, thereby extending the probe on target DNA;

heat denaturing the extended probe which causes the quencher dye to disassociate;

cooling the extended probe to allow for an internal rearrangement which causes the fluorescent dye to fluorescence in a target specific manner; and

reading the fluorescence signal from the fluorescent dye in the real time.