Chemically modified nucleoside 5′-triphosphates for thermally initiated amplification of nucleic acid

• US 8,133,669 B2
• Filed: 05/21/2009
• Issued: 03/13/2012
• Est. Priority Date: 05/27/2008
• Status: Active Grant

• Current Assignee: TriLink BioTechnologies Incorporated
• Original Assignee: TriLink BioTechnologies Incorporated
• Inventors: Lebedev, Alexandre, Koukhareva, Inna
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US12/470,449
• Publication Number:
  US 20100003724A1
• Time in Patent Office: 1,027 Days
• Field of Search: 435/6, 435/91.1, 435/1, 435/91.2, 422/430, 422/68.1, 536/4.1, 536/23.1, 536/24.33, 536/26.6
• US Class Current: 435/6
• CPC Class Codes: C07H 19/00 Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2545/101 with an internal standard/control

Abstract

Provided herein are methods and compositions for nucleic acid replication. These methods involve the use of 3′-substituted nucleoside 5′-triphosphates or 3′-substituted terminated primers in nucleic acid replication reactions. In certain aspects, the methods are accomplished by use of 3′-substituted NTPs and/or 3′-substituted terminated primers which provide utility in nucleic acid replication. In preferred embodiments, the NTPs and/or primers are substituted at the 3′-position with particular heat labile chemical groups such as ethers, esters or carbonate esters.

Claims

1. A method of replicating nucleic acids, said method comprising:

replicating nucleic acid, wherein at least one NTP added to a replication reaction mixture comprises a thermally labile 3′

-substitution group and wherein said replication reaction mixture does not contain chemical reagents for removal of said thermally labile 3′

-substitution group.

2. The method of claim 1, wherein the 3′

-substituted NTP does not support, impairs or prevents nucleic acid polymerase extension in the reaction.

3. The method of claim 1, wherein the at least one 3′

-substituted NTP is a nonsubstrate NTP.

4. The method of claim 1, wherein the at least one 3′

-substituted NTP is a terminating NTP.

5. The method of claim 1, wherein the at least one 3′

-substituted NTP incorporates into an oligonucleotide primer resulting in a terminated primer.

6. The method of claim 1, wherein the 3′

-substitution group converts to an open 3′

-hydroxyl group during the nucleic acid replication.

7. The method of claim 1, wherein the 3′

-substitution group converts to an open 3′

-hydroxyl group during the initial denaturation step of the nucleic acid replication.

8. The method of claim 6, wherein conversion is partial.

9. The method of claim 6, wherein conversion is complete.

10. The method of claim 1, wherein said conversion occurs at a temperature sufficient to dissociate the 3′

-substitution group to form an open 3′

-hydroxyl group.

11. The method of claim 1, wherein said 3′

-substitution group converts to an open 3′

-hydroxyl group at a temperature between about 37°

C.-100°

C.

12. The method of claim 1, wherein said 3′

-substitution group converts to an open 3′

-hydroxyl group at a temperature between about 70°

C.-100°

C. or at about 50°

C.

13. The method of claim 1, wherein t^1/2 of conversion of said 3′

-substitution group to an open 3′

-hydroxyl group is between about 1-120 minutes at about 95°

C. or at about 50°

C.

14. The method of claim 1, wherein the amplification reaction comprises 3′

-substituted NTPs with two or more different 3′

-substitution groups.

15. The method of claim 14, wherein the different 3′

-substitution groups convert to open 3′

-hydroxyl groups at different temperatures.

16. The method of claim 15, wherein a first 3′

-substitution group converts to an open 3′

-hydroxyl group at about 50°

C. and a second 3′

-substitution group converts at about 95°

C.

17. The method of claim 1, wherein nucleic acid replication is polymerase chain reaction (PCR), Allele-specific PCR, Assembly PCR or Polymerase Cycling Assembly (PCA), Asymmetric PCR, Colony PCR, Emulsion PCR, Fast PCR, Gap Extension Ligation PCR (GEXL-PCR), Gap Ligation Chain Reaction (Gap LCR), Helicase-dependent amplification, Hot-start PCR, Intersequence-specific (ISSR) PCR, Inverse PCR, Ligation-mediated PCR, Linear-After-The-Exponential-PCR (LATE-PCR), Methylation-specific PCR (MSP), Multiplex Ligation-dependent Probe Amplification, (MLPA), Multiplex-PCR, Nested PCR, Overlap-extension PCR, PAN-AC, Quantitative PCR (Q-PCR), Quantitative real-time PCR (QRT-PCR), Real-Time PCR, Reverse Transcription (RT), Rapid Amplification of cDNA Ends (RACE PCR), Single molecule amplification PCR (SMA PCR), Thermal asymmetric interlaced PCR (TAIL-PCR), Touchdown PCR, long PCR, nucleic acid ligation, DNA sequencing or reverse transcription-PCR (RT-PCR).

18. The method of claim 1, wherein nucleic acid replication reaction comprises one or more enzymes selected from the group consisting of DNA dependent DNA polymerases, RNA dependent DNA polymerases, DNA dependent RNA polymerases, RNA dependent RNA polymerases, DNA ligases, RNA ligases, synthetases, nucleases, and restrictases.

19. The method of claim 1, wherein said nucleic acid is DNA, RNA, LNA, PNA or a combination thereof.

20. The method of claim 1, wherein said NTPs are one or more 3′

-substituted NTPs selected from the group consisting of 3′

-substituted dATP, 3′

-substituted dTTP, 3′

-substituted dGTP, 3′

-substituted dCTP, and 3′

-substituted dUTP.

21. The method of claim 1, wherein said 3-substituted NTPs comprise 3′

-substituted dATP, 3′

-substituted dTTP, 3′

-substituted dGTP, and 3′

-substituted dCTP.

22. The method of claim 1, wherein said 3′

-substituted NTPs comprise 3′

-substituted dATP, 3′

-substituted dUTP, 3′

-substituted dGTP, and 3′

-substituted dCTP.

23. The method of claim 1, wherein said 3′

-substituted NTPs comprise 25% or less of total NTPs in the replication reaction.

24. The method of claim 1 wherein said 3′

-substituted NTPs comprise 25% to 100% of total NTPs in the replication reaction.

25. The method of claim 1, further comprising one or more modification groups on a sugar or phosphate group of said 3′

-substituted NTPs.

26. The method of claim 1, wherein said 3′

-substituted NTP further comprises a detectable label.

27. The method of claim 1, wherein the 3′

-substituted NTPs are a chirally pure, racemic or diastereomeric mixture.

28. The method of claim 1, wherein the presence of the 3′

-substituted NTPs in a replication reaction reduces formation and subsequent replication of nonspecific products as compared with replication using a corresponding unsubstituted NTP.

29. The method of claim 1, wherein the 3′

-substitution group is selected from the group consisting of O-phenoxyacetyl;

O-methoxyacetyl;

O-acetyl;

O-(p-toluene)sulfonate;

O-phosphate;

O-nitrate;

O-[4-methoxy]-tetrahydrothiopyranyl;

O-tetrahydrothiopyranyl;

O-[5-methyl]-tetrahydrofuranyl;

O-[2-methyl,4-methoxy]-tetrahydropyranyl;

O-[5-methyl]-tetrahydropyranyl; and

O-tetrahydrothiofuranyl.

30. The method of claim 1, wherein the 3′

-substituted NTP and derivatives thereof has the structure of

31. The method of claim 30, wherein X¹, X³, and X⁴ are H;

W is CH₂;

Z⁰, Z¹, Z⁶ and Z⁷ are O; and

Z⁵ and Z⁸ are OH.

32. The method of claim 30, wherein A is NH, O, CH₂, or S;

X¹, X², X³, X⁴ and X⁵ are H;

W is CH₂;

Z⁰, Z¹, Z³, Z⁴, Z⁶ and Z⁰ are O; and

Z², Z⁵, Z⁸ and Z⁹ are OH.

33. The method of claim 30, wherein X² is H, OH, F, CH₃, OCH₃, N₃, NH₂, or NHCH₃;

A is O;

X¹, X³, X⁴ and X⁵ are H;

W is CH₂;

Z⁰, Z¹, Z³, Z⁴, Z⁶ and Z⁷ are O;

Z², Z⁵, Z⁸ and Z⁹ are OH.

34. The method of claim 30, wherein X⁵ is H, SH, CH₃, F, OCH₃, NH₂, or NHCH₃;

A is O;

X¹, X², X³ and X⁴ are H;

W is CH₂;

Z⁰, Z¹, Z³, Z⁴, Z⁶, and Z⁷ are O; and

Z², Z⁵, Z⁸ and Z⁹ are OH.

35. The method of claim 30, wherein Z² is OH, SH, BH₃, CH₃, OCH₃, or OCH₂CH₃;

A is O;

X¹, X², X³ X⁴ and X⁵ are H;

W is CH₂;

Z⁰, Z¹, Z³, Z⁴, Z⁶ and Z⁷ are O; and

Z⁵, Z⁸ and Z⁹ are OH.

36. The method of claim 30, wherein Z⁴ is O or S;

n is 1;

A is O;

X¹, X², X³ X⁴ and X⁵ are H, W is CH₂;

Z⁰, Z¹, Z³, Z⁶ and Z⁷ are O; and

Z², Z⁵, Z⁸ and Z⁹ are OH.

37. The method of claim 30, wherein Z⁹ is SH, SCH₂CH₂CN, OH, F, OCH₃, OCH₂CH₃, OCH , NHCH₃, NH₂, NHCH₂CH₂NH₂, NHCH₂CH₂CH₂CH₂CH₂CH₂CH₂ or a phosphate group;

n is 1;

A is O;

X¹, X², X³ X⁴ and X⁵ are H;

W is CH₂;

Z⁰, Z¹, Z³, Z⁴, Z⁶ and Z⁷ are O; and

Z², Z⁵ and Z⁸ are OH.

38. The method of claim 30, wherein n is 1;

A is O;

X¹, X², X³ X⁴ and X⁵ are H;

W is CH₂;

Z⁰, Z¹, Z³, Z⁴, Z⁶ and Z⁷ are O; and

Z², Z⁵, Z⁸ and Z⁹ are OH.

39. The method of claim 30, wherein B is thymine, cytosine, adenine, guanine, uracil, 5-aminoallyl-uracil, 7-deazaguanine, 7-deaza-7-methylguanine, 7-deaza-7-iodoguanine, 7-deaza-7-aminoallyl-guanine, 7-deaza-8-azaguanine, 7-deazadenine, 2,6-diaminopurine, 5-nitro-cytosine, 5-aminoallyl-cytosine, 5-(Biotin-16)-cytosine, 5-(Fluorescein-11)-cytosine, 4-methylamino-cytosine, 2-thio-5-methyluracil, or 4-thio-5-methyluracil.

40. The method of claim 30, wherein B is recognizable by a nucleic acid polymerase.

41. A kit for nucleic acid amplification comprising one or more 3′

-substituted NTPs of claim 1.

42. The kit of claim 41 further comprising one or more selected from the group consisting of instructions for performing a method using said one or more 3′

-substituted NTPs to perform said method, container marked for nucleic acid amplification, unmodified dNTPs, modified NTPs, nucleic acid polymerase, magnesium chloride or other divalent cation, and reaction buffer.

43. The kit of claim 42, comprising nucleic acid polymerase and one or more additional enzymes.

44. The method of claim 30, wherein R is:

45. The method of claim 44, wherein Z¹⁰ is O.

46. The method of claim 45, wherein R⁷ is an optionally substituted alkyl group having 1-6 carbon atoms.

47. The method of claim 46, wherein the alkyl includes an alkoxy substituent.

48. The method of claim 46, wherein the alkyl includes a cycloalkyl substituent.

51. The method of claim 1, wherein the 3′

-substitution group is O-tetrahydrofuranyl, O-tetrahydropyranyl or O-[4-methoxy]-tetrahydropyranyl.

52. The method of claim 1, wherein the 3′

-substitution group is O-(1-cyclohexoxy)ethyl, O-(tert-butoxy)ethyl or 2-(iso-butoxy)ethyl.

53. The method of claim 30, wherein R is

54. The method of claim 30, wherein R is

55. The method of claim 30, wherein R is

56. The method of claim 30, wherein R is

57. The method of claim 30, wherein R is

58. The method of claim 30, wherein R is

59. The method of claim 30, wherein R is

60. The method of claim 30, wherein R is

61. The method of claim 30, wherein R is

62. The method of claim 30, wherein R is

63. The method of claim 30, wherein R is

49. A method for improving specificity of replicating nucleic acids, said method comprising:

replicating nucleic acid, wherein at least one NTP added to a replication reaction mixture comprises a thermally labile 3′

-substitution group and wherein said replication reaction mixture does not contain chemical reagents for removal of said thermally labile 3′

-substitution group.

50. A method for improving efficiency of replicating nucleic acids, said method comprising:

replicating nucleic acid, wherein at least one NTP added to a replication reaction mixture comprises a thermally labile 3′

-substitution group and wherein said replication reaction mixture does not contain chemical reagents for removal of said thermally labile 3′

-substitution group.