HYBRIDIZATION AND MISMATCH DISCRIMINATION USING OLIGONUCLEOTIDES CONJUGATED TO MINOR GROOVE BINDERS

• US 20090048427A1
• Filed: 07/14/2008
• Published: 02/19/2009
• Est. Priority Date: 04/03/1995
• Status: Active Grant

• Current Assignee: ELITechGroup B.V. (ELITech Group SAS)
• Original Assignee: Epoch Biosciences Inc. (Ngen, Inc.)
• Inventors: AFONINA, IRINA A., MEYER, RICH B. JR., KUTYAVIN, IGOR V., BELOUSOV, EVGENIY S., LUKHTANOV, EUGENY A., HEDGPETH, JOEL
• Granted Patent: US 7,794,945 B2
• Time in Patent Office: Days
• US Class Current: 530/322
• CPC Class Codes: C07D 207/34 with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
• CPC Class Codes: C07D 403/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 487/04 Ortho-condensed systems
• CPC Class Codes: C07H 19/073 with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C07H 19/173 with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C07H 19/23 Heterocyclic radicals containing two or more heterocyclic rings condensed among themselves or condensed with a common carbocyclic ring system, not provided for in groups C07H19/14 - C07H19/22
• 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 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm

Abstract

Conjugates between a minor groove binding molecule, such as the trimer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI₃), and an oligonucleotide form unusually stable hybrids with complementary target sequences, in which the tethered CDPI₃ group resides in the minor groove of the duplex. These conjugates can be used as probes and primers. Due to their unusually high binding affinity, conjugates as short as 8-mers can be used as amplification primers with high specificity and efficiency. MGB conjugation also increases the discriminatory power of short oligonucleotides, providing enhanced detection of nucleotide sequence mismatches by short oligonucleotides. The MGB-conjugated probes and primers described herein facilitate various analytic and diagnostic procedures, such as amplification reactions, PCR, detection of single-nucleotide polymorphisms, gene hunting, differential display, fluorescence energy transfer, hydrolyzable probe assays and others; by allowing the use of shorter oligonucleotides, which have higher specificity and better discriminatory power.

Claims

1. A method for hybridizing nucleic acids, comprising the steps of:

(a) providing a first nucleic acid and a second nucleic acid,

(b) incubating the nucleic acids under hybridization conditions, and

(c) identifying hybridized nucleic acids;

wherein at least one of the nucleic acids comprises a minor groove binder (MGB)-oligonucleotide conjugate;

wherein the minor groove binder is a molecule having a molecular weight of approximately 150 to approximately 2,000 Daltons that binds in a non-intercalating manner into the minor groove of a double-stranded nucleic acid with an association constant of greater than approximately 10³M<sup>−

1</sup>.

2. The method according to claim 1, wherein the MGB-oligonucleotide conjugate is a probe comprising a detectable label.

3. The method according to claim 2, wherein the detectable label is a fluorescent label.

4. The method according to claim 3, wherein the MGB-oligonucleotide conjugate further comprises an agent that quenches the emission of the fluorescent label.

5. The method according to claim 4, further comprising the step of altering the spatial relationship between the fluorescent label and the agent which quenches the emission of the fluorescent label.

6. The method according to claim 2, wherein the method further comprises the step of releasing label from the probe subsequent to hybridization.

7. The method according to claim 5, wherein the method further comprises the step of releasing label from the probe subsequent to hybridization.

8. The method according to claim 6, wherein release of label occurs as a result of exonuclease hydrolysis.

9. The method according to claim 7, wherein release of label occurs as a result of exonuclease hydrolysis.

10. The method according to claim 3, wherein more than one probe is used.

11. The method according to claim 10, wherein a first and second probe is used.

12. The method according to claim 11, wherein the first probe comprises a fluorescence donor and the second probe comprises a fluorescence acceptor, and further wherein the emission wavelengths of the fluorescence donor overlap the absorption wavelengths of the fluorescence acceptor.

13. The method according to claim 1 wherein the MGB-oligonucleotide conjugate is a primer comprising a free 3′

-hydroxyl group.

14. The method according to claim 13, further comprising the step of extending the primer with a polymerizing enzyme.

15. The method according to claim 14, wherein the polymerizing enzyme is a thermostable enzyme.

16. The method according to claim 13, wherein the MGB-oligonucleotide conjugate is a primer in an amplification reaction.

17. The method according to claim 16, wherein the amplification reaction is a polymerase chain reaction.

67. The method of hybridization according to claim 1, wherein the melting temperature of the hybridized nucleic acid is independent of base composition.

18. A method for primer extension, comprising the steps of:

(a) providing a sample containing a target sequence,

(b) providing one or more oligonucleotide primers complementary to regions of the target sequence,

(c) providing a polymerizing enzyme and nucleotide substrates, and

(d) incubating the sample, the oligonucleotide primers, the enzyme and the substrates under conditions favorable for polymerization;

wherein at least one of the primers comprises a MGB-oligonucleotide conjugate.

19. The method according to claim 18, wherein the method is an amplification reaction.

20. The method according to claim 19, wherein the amplification reaction is a polymerase chain reaction.

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

(a) providing a polynucleotide comprising a target sequence,

(b) providing at least two MGB-oligonucleotide conjugates, wherein one of the at least two MGB-oligonucleotide conjugates has a sequence that is perfectly complementary to the target sequence and at least one other of the MGB-oligonucleotide conjugates has a single-nucleotide mismatch with the target sequence;

(c) separately incubating each of the MGB-oligonucleotide conjugates with the polynucleotide under hybridization conditions; and

(d) determining the hybridization strength between each of the MGB-oligonucleotide conjugates and the polynucleotide.

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

(a) providing a MGB-oligonucleotide conjugate of defined sequence,

(b) providing at least two polynucleotides, each of which comprises a target sequence, wherein one of the polynucleotides has a target sequence that is perfectly complementary to the MGB-oligonucleotide conjugate and at least one other of the polynucleotides has a target sequence having a single-nucleotide mismatch with the MGB-oligonucleotide conjugate;

(c) separately incubating each of the polynucleotides with the MGB-oligonucleotide conjugate under hybridization conditions; and

(d) determining the hybridization strength between each of the polynucleotides and the MGB-oligonucleotide conjugate.

23. A method of ligating two or more oligonucleotides, each of which is hybridized to adjacent sites on a target nucleic acid, comprising the steps of:

(a) providing a sample containing a target sequence,

(b) providing at least two oligonucleotides which are complementary to adjacent sites on the target sequence,

(c) incubating the sample and the oligonucleotides under conditions favorable for ligation, and

(d) identifying ligated nucleic acids;

wherein at least one of the oligonucleotides comprises a MGB-oligonucleotide conjugate.

24. An oligonucleotide probe comprising a 5′

-end, a 3′

-end and one or more detectable labels, wherein the probe is a MGB-oligonucleotide conjugate.

25. The probe according to claim 24 wherein the detectable label is a fluorescent label.

26. The probe according to claim 25 wherein the label is a fluorescein.

27. The probe according to claim 25 wherein the label is a cyanine.

28. The probe according to claim 25 wherein the label is a rhodamine.

29. The probe according to claim 24 wherein the MGB is located at the oligonucleotide 5′

end.

30. The probe according to claim 24 wherein the MGB is located at the oligonucleotide 3′

end.

31. The probe according to claim 24 wherein the label is located at the oligonucleotide 5′

end.

32. The probe according to claim 24 wherein the label is located at the oligonucleotide 3′

end.

33. The probe according to claim 24 wherein the MGB is selected from the group consisting of a trimer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI₃) and a pentamer of N-methylpyrrole-4-carbox-2-amide (MPC₅).

34. The probe according to claim 25 comprising multiple fluorescent labels.

35. The probe according to claim 34 wherein the emission wavelengths of one of the fluorescent labels overlaps the absorption wavelengths of another of the fluorescent labels.

36. The probe according to claim 25 further comprising a quenching agent which quenches the fluorescence emission of the fluorescent label.

37. The probe according to claim 36 wherein the fluorescent label is a fluorescein.

38. The probe according to claim 37 wherein the quenching agent is tetramethylrhodamine.

44. The probe according to claim 24, wherein inosine is substituted for guanosine.

45. The probe according to claim 24, wherein 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one is substituted for guanine.

46. The probe according to claim 24, wherein 4-amino-1H-pyrazolo[3,4-d]pyrimidine is substituted for adenine.

47. The probe according to claim 24, wherein 1H-pyrazolo[3,4-d]pyrimidin-4(5H)-6(7H)-dione is substituted for adenine.

52. A composition comprising the probe according to claim 24.

54. The probe according to claim 24, wherein the probe is less than 20 nucleotides in length.

57. A kit for hybridization analysis comprising one or more probes according to claim 24.

58. A kit for use in a hydrolyzable probe assay comprising one or more probes according to claim 24.

59. A kit for use in single nucleotide mismatch detection comprising one or more probes according to claim 24.

61. A kit for use in nucleotide sequence analysis comprising one or more of the probes of claim 24.

39. A MGB-oligonucleotide conjugate for use as a primer comprising a 5′

end and a 3′

end, wherein the 3′

end is extendible by a polymerizing enzyme.

40. The primer according to claim 39, wherein the 3′

end comprises a free 3′

hydroxyl group.

41. The primer according to claim 39, wherein the primer has been extended by a polymerizing enzyme.

42. The primer according to claim 41, wherein the primer has been extended during the synthesis of a cDNA molecule.

43. The primer according to claim 41, wherein the primer has been extended during an amplification reaction.

48. The primer according to claim 39, wherein inosine is substituted for guanosine.

49. The primer according to claim 39, wherein 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one is substituted for guanine.

50. The primer according to claim 39, wherein 4-amino-1H-pyrazolo[3,4-d]pyrimidine is substituted for adenine.

51. The primer according to claim 39, wherein 1H-pyrazolo[3,4-d]pyrimidin-4(5H)-6(7H)-dione is substituted for adenine.

53. A composition comprising the primer according to claim 39.

55. The primer according to claim 39, wherein the primer is less than 20 nucleotides in length.

56. A kit for amplification comprising one or more primers according to claim 39.

60. A kit for use in single nucleotide mismatch detection comprising one or more primers according to claim 39.

62. A kit for use in nucleotide sequence analysis comprising one or more of the primers of claim 39.

63. A method for primer-dependent nucleotide sequence analysis wherein a MGB-oligonucleotide conjugate is used as a primer.

64. A method for determining the sequence of a polynucleotide comprising the steps of:

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

(b) incubating the polynucleotide and the array under hybridization conditions, and

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

wherein one or more of the oligonucleotide probes comprises a MGB-oligonucleotide conjugate.

65. A method for examining gene expression comprising the steps of:

(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 comprises a MGB-oligonucleotide conjugate.

66. A method for identifying one or more mutations in a gene of interest comprising the steps of:

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

(b) incubating a polynucleotide sample with the array under hybridization conditions, and

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

wherein one or more of the oligonucleotide probes comprises a MGB-oligonucleotide conjugate.

68. The method of hybridization according to claim 66 wherein the melting temperature of the hybridized nucleic acid is dependent primarily on the length of the hybridized nucleic acid.

69. A MGB-oligonucleotide conjugate that hybridizes to a target nucleic acid to form a hybrid, wherein the melting temperature of the hybrid is independent of base composition.

70. A method for cDNA synthesis, wherein a MGB-oligonucleotide conjugate is used as a primer.