FLUORESCENT PROBES FOR DNA DETECTION BY HYBRIDIZATION WITH IMPROVED SENSITIVITY AND LOW BACKGROUND

• US 20080293586A1
• Filed: 03/12/2008
• Published: 11/27/2008
• Est. Priority Date: 10/28/2003
• Status: Active Grant

• Current Assignee: Elitechgroup MDX LLC
• Original Assignee: Epoch Biosciences Inc. (Ngen, Inc.)
• Inventors: Lukhtanov, Eugene, Lokhov, Sergey
• Granted Patent: US 7,759,126 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Minor groove binder-oligonucleotide probes are provided along with methods for their use wherein the probes have an attached fluorophore which, in an unhybridized form exhibits very low background signal.

Claims

1. An oligonucleotide-probe comprising an oligonucleotide portion having a 3′

-end and a 5′

-end, a minor groove binder moiety attached to at least one of said nucleotide units through a linking group which covalently binds the minor groove binder moiety to the oligonucleotide, and a fluorophore and quencher, said probe having the formula;

2. An oligonucleotide probe of claim 1, wherein W provides spacing between MB, Fl and [A-B]_n such that in an unhybridized form, the fluorescence of Fl in said oligonucleotide probe is less than 30% of unquenched fluorescence

3. An oligonucleotide probe of claim 1, wherein W is a member selected from the group consisting of:

4. An oligonucleotide probe of claim 1, wherein n is an integer of from 6 to 18, and said [A-B]_n portion comprises at least three consecutive guanine nucleotides wherein at least one of the guanine nucleotide bases is replaced by PPG.

5. An oligonucleotide probe of claim 4, wherein said [A-B]_n portion is a DNA, a RNA, chimera, a PNA or a locked nucleic acid.

6. An oligonucleotide probe of claim 1, wherein n is an integer of from 6 to 18, said probe being complementary to a target sequence having at least 30% adenine and thymine bases, wherein said probe contains at least one modified base sufficient to provide an increase of stability of duplex formation of at least 3°

C., relative to a probe without said at least one modified base.

7. An oligonucleotide probe of claim 6, wherein said [A-B]_n portion is a DNA, a RNA, chimera, a PNA or a locked nucleic acid.

8. An oligonucleotide probe of claim 6, wherein said probe is complementary to a target sequence having at least 50% adenine and thymine bases, wherein said probe contains sufficient modified bases to provide an increase of stability of duplex formation of at least 5°

C., relative to a probe without said modified bases.

9. An oligonucleotide probe of claim 1, wherein W and K have formulae IVb and IVc, respectively.

10. A method for continuous monitoring of polynucleotide amplification, comprising:

(a) combining a sample containing a target sequence, with one or more oligonucleotide primers complementary to regions of the target sequence, a polymerizing enzyme, nucleotide substrates, and an oligonucleotide conjugate having a formula;

11. A method of claim 10, wherein said MB portion is selected from the group consisting of CC1065 analogs, lexitropsins, distamycin, netropsin, berenil, duocarmycin, pentamidine, 4,6-diamino-2-phenylindole and pyrrolo[2,1-c][1,4]benzodiazepine analogs.

12. A method of claim 10, wherein said Fl portion is a fluorophore having an emission wavelengths of from about 400 nm to about 800 nm, said fluorophore being selected from the group consisting of coumarins, resorufins, xanthenes, benzoxanthenes, cyanines and bodipy analogs.

13. A method of claim 10, wherein said Q portion is a member selected from the group consisting of mono azo and bis azo dyes.

14. A method of claim 10, wherein said ODN portion of said conjugate is from 8-25 nucleotides in length.

15. A method of claim 10, wherein said ODN portion of said conjugate is from 8-15 nucleotides in length and K is a linker having a length of from 10-50 main chain atoms selected from the group consisting of C, O, N, S, P and Si.

16. A method of claim 10, wherein W and K have formulae IVb and IVc, respectively.

17. A method for monitoring gene expression comprising:

(a) providing an array of oligonucleotide probes of different sequences,

(b) incubating a population of polynucleotides with the array under hybridization conditions, and

(c) determining to which of the oligonucleotide probes in the array the population hybridizes;

wherein one or more of the oligonucleotide probes is an oligonucleotide conjugate having the formula;

18. A method of claim 17, wherein said MB portion is selected from the group consisting of CC1065 analogs, lexitropsins, distamycin, netropsin, berenil, duocarmycin, pentamidine, 4,6-diamino-2-phenylindole and pyrrolo[2,1-c][1,4]benzodiazepine analogs, said Fl portion is a fluorophore having an emission wavelength of from about 400 nm to about 800 nm, said fluorophore being selected from the group consisting of coumarins, resorufins, xanthenes, benzoxanthenes, cyanines and bodipy analogs, and said Q portion is a member selected from the group consisting of mono azo and bis azo dyes.

19. A method of claim 18, wherein said conjugates are attached to a solid support.

20. A method of claim 17, wherein W and K have the formulae IVb and IVc, respectively.

21. A method for discriminating between polynucleotides which differ by a single nucleotide, the method comprising:

(a) separately incubating each of at least two polynucleotides with an oligonucleotide conjugate having the formula;

22. A method of claim 21, wherein said MB portion is selected from the group consisting of CC1065 analogs, lexitropsins, distamycin, netropsin, berenil, duocarmycin, pentamidine, 4,6-diamino-2-phenylindole and pyrrolo[2,1-c][1,4]benzodiazepine analogs, said Fl portion is a fluorophore having an emission wavelength of from about 400 nm to about 800 nm, said fluorophore being selected from the group consisting of coumarins, resorufins, xanthenes, benzoxanthenes, cyanines and bodipy analogs, and said Q portion is a member selected from the group consisting of mono azo and bis azo dyes.

23. A method of claim 21, wherein W and K have the formulae IVb and IVc, respectively.

24. A method for detecting a target sequence in a polynucleotide, wherein the polynucleotide is present in a mixture of other polynucleotides, and wherein one or more of the other polynucleotides in the mixture comprise sequences that are related but not identical to the target sequence, the method comprising:

(a) contacting the mixture of polynucleotides with an oligonucleotide conjugate having the formula;

25. A method of claim 24, wherein said MB portion is selected from the group consisting of CC1065 analogs, lexitropsins, distamycin, netropsin, berenil, duocarmycin, pentamidine, 4,6-diamino-2-phenylindole and pyrrolo[2,1-c][1,4]benzodiazepine analogs, said Fl portion is a fluorophore having an emission wavelength of from about 400 nm to about 800 nm, said fluorophore being selected from the group consisting of coumarins, resorufins, xanthenes, benzoxanthenes, cyanines and bodipy analogs, and said Q portion is a member selected from the group consisting of mono azo and bis azo dyes.

26. A method of claim 24, wherein W and K have the formulae IVb and IVc, respectively.

27. A method for distinguishing between wild-type, mutant and heterozygous target polynucleotides, said method comprising:

(a) contacting a sample containing a target polynucleotide with two probes wherein a first probe is specific for said wild-type target polynucleotide and a second probe is specific for said mutant target polynucleotide, each of said probes having a formula;

28. A method of claim 27, wherein the melting temperatures (T_m) for each hybrid produced between said first and second probes and their respective targets are within about 5°

C. of each other.

29. A method of claim 27, wherein the ODN portion of each of said probes is an oligonucleotide or modified oligonucleotide having from 8 to 18 bases or modified bases.

30. A method of claim 27, wherein the ODN portion of each of said probes is an oligonucleotide or modified oligonucleotide having from 10 to 15 bases or modified bases.

31. A method of claim 27, wherein the fluorophore portions of each of said probes are selected from the group consisting of 5-FAM™

, 6-FAM™

, TET™

, JOE™

, HEX™

, VIC™

, NED™

, TAMRA™

, ROX™ and

YY™

.

32. A method of claim 27, wherein the ODN portion of each of said probes contains at least one modified base.

33. A method of claim 32, wherein each modified base is independently selected from the group consisting of 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one, 4-amino-1H-pyrazolo[3,4-d]pyrimidine, 1H-pyrazolo[5,4-d]pyrimidin-4(5H)-6(7H)-dione, 6-4 amino-3-prop-1-ynyl-5-hydropyrazolo[3,4-d]pyrimidine-4-one, 6-amino-3-(3-hydroxyprop-1-yny)1-5-hydropyrazolo[3,4-d]pyrimidine-4-one, 6-amino-3-(3-aminoprop-1-ynyl)-5-hydropyrazolo[3,4-d]pyrimidine-4-one, 4-amino-3-(prop-1-ynyl)pyrazolo[3,4-d]pyrimidine, 4-amino-3-(3-hydroxyprop-1-ynyl)pyrazolo[3,4-d]pyrimidine, 4-amino-3-(3-aminoprop-1-ynyl)pyrazolo[3,4-d]pyrimidine, 3-prop-1-ynyl-4,6-diaminopyrazolo[3,4-d]pyrimidine, 2-(4,6-diaminopyrazolo[3,4-d]pyrimidin-3-yl)ethyn-1-ol, 3-(2-aminoethynyl)pyrazolo[3,4-d]pyrimidine-4,6-diamine, 5-prop-1-ynyl-1,3-dihydropyrimidine-2,4-dione, 5-(3-hydroxyprop-1-ynyl)-1,3-dihydropyrimidine-2,4-dione, 6-amino-5-prop-1-ynyl-3-dihydropyrimidine-2-one, 6-amino-5-(3-hydroxyprop-1-yny)-1,3-dihydropyrimidine-2-one, 6-amino-5-(3-aminoprop-1-yny)-1,3-dihydropyrimidine-2-one, 5-[4-amino-3-(3-methoxyprop-1-ynyl)pyrazol[3,4-d]pyrimidinyl]-2-(hydroxymethyl)oxolan-3-ol, 6-amino-1-[4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-3-(3-methoxyprop-1-ynyl)-5-hydropyrazolo[3,4-d]pyrimidin-4-one, 4-(4,6-Diamino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-but-3-yn-1-ol, 6-Amino-3-(4-hydroxy-but-1-ynyl)-1,5-dihydro-pyrazolo[3,4-d]pyrimidin-4-one, 5-(4-hydroxy-but-1-ynyl)-1H-pyrimidine-2,4-dione, 3-iodo-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine, 3-bromo-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine, 3-chloro-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine, 3-Iodo-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine, 3-Bromo-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine and 3-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine.

34. A method of claim 27, wherein said sample is further contacted with a set of primers under amplification conditions and each of said primers contains from one to ten modified bases selected from the group consisting of 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one, 4-amino-1H-pyrazolo[3,4-d]pyrimidine, 1H-pyrazolo[5,4-d]pyrimidin-4(5H)-6(7H)-dione, 6-amino-3-prop-1-ynyl-5-hydropyrazolo[3,4-d]pyrimidine-4-one, 6-amino-3-(3-hydroxyprop-1-yny)1-5-hydropyrazolo[3,4-d]pyrimidine-4-one, 6-amino-3-(3-aminoprop-1-ynyl)-5-hydropyrazolo[3,4-d]pyrimidine-4-one, 4-amino-3-(prop-1-ynyl)pyrazolo[3,4-d]pyrimidine, 4-amino-3-(3-hydroxyprop-1-ynyl)pyrazolo[3,4-d]pyrimidine, 4-amino-3-(3-aminoprop-1-ynyl)pyrazolo[3,4-d]pyrimidine, 3-prop-1-ynyl-4,6-diaminopyrazolo[3,4-d]pyrimidine, 2-(4,6-diaminopyrazolo[3,4-d]pyrimidin-3-yl)ethyn-1-ol, 3-(2-aminoethynyl)pyrazolo[3,4-d]pyrimidine-4,6-diamine, 5-prop-1-ynyl-1,3-dihydropyrimidine-2,4-dione, 5-(3-hydroxyprop-1-ynyl)-1,3-dihydropyrimidine-2,4-dione, 6-amino-5-prop-1-ynyl-3-dihydropyrimidine-2-one, 6-amino-5-(3-hydroxyprop-1-yny)-1,3-dihydropyrimidine-2-one, 6-amino-5-(3-aminoprop-1-yny)-1,3-dihydropyrimidine-2-one, 5-[4-amino-3-(3-methoxyprop-1-ynyl)pyrazol[3,4-d]pyrimidinyl]-2-(hydroxymethyl)oxolan-3-ol, 6-amino-1-[4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-3-(3-methoxyprop-1-ynyl)-5-hydropyrazolo[3,4-d]pyrimidin-4-one, 4-(4,6-Diamino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-but-3-yn-1-ol, 6-Amino-3-(4-hydroxy-but-1-ynyl)-1,5-dihydro-pyrazolo[3,4-d]pyrimidin-4-one, 5-(4-hydroxy-but-1-ynyl)-1H-pyrimidine-2,4-dione, 3-iodo-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine, 3-bromo-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine, 3-chloro-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine, 3-Iodo-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine, 3-Bromo-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine and 3-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine.