Method for base sequencing and biologically active nucleic acids

• US 20040115665A1
• Filed: 08/14/2003
• Published: 06/17/2004
• Est. Priority Date: 11/28/2000
• Status: Active Grant

• Current Assignee: Riken
• Original Assignee: Riken
• Inventors: Hayashizaki, Yoshihide
• Granted Patent: US 7,374,882 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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: C12N 15/115 Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
• CPC Class Codes: C12N 2310/33 of the base
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures

Abstract

Aptamers are nucleic acids and similar molecules, such as peptide-nucleic acids, that specifically bind to a ligand such as a protein or peptide. The present invention provides aptamers comprising at least one base capable of base pairing and different from the standard Watson-Crick bases. The present invention also relates to a method for preparation of such aptamers and to methods for sequencing nucleic acids that comprise at least one base capable of base pairing and different from the standard Watson-Crick bases.

Claims

1. A method for preparing nucleic acid aptamers comprising at least one base capable of base pairing and different from the standard Watson-Crick (W-C) base comprising:

a) providing a specific ligand;

b) synthesizing a pool of nucleic acid aptamers comprising at least one base capable of base pairing and different from the standard W-C bases;

c) mixing the pool of aptamers with the specific ligand;

d) selecting and amplifying a specific aptamer that binds to the specific ligand.

2. The method of claim 1, wherein the aptamer comprises at least one standard W-C base A, C, G, T or U and at least one base capable of base pairing and different from the standard W-C bases.

3. The method of claim 1, wherein the base capable of base pairing and different from the W-C standard bases is selected from the group consisting of:

iso-C;

iso-G;

2,6-diaminopyrimidine;

xanthine;

6-amino-5-substituted pyrazin-2(1H)-one;

1-methyl-pyrazolo[4,3-d]pyrimidine-5,7)(4H,6H)-dione;

5-aza-7-deazaguanine;

6-amino-3-substituted pyrazin-2(1H)-one;

3-amino-1-methylpurin-2-one;

2,4-diamino-5,6-dihydropyryimidine;

2-amino-6-(N,N-dimethylamino)purine;

pyridin-2-one;

3-methyl isocarbostyril;

5-methyl isocarbostyril;

7-propynyl isocarbostyril;

m-xylene;

1,3,4-trimethylbenzene;

2-methylanaphthalene;

1,4-dimethylnaphthalene;

1-methylanaphthalene;

naphthalene;

7-azaindole;

isocarbostyril;

6-methyl-7-asaindole;

3-propynyl-7-azaindole;

imidazopyridine;

pyrrolopyridine; and

a heterocyclic base having the structural formula

 

wherein R designates the point of attachment of the base to position 1 of ribose or deoxyribose ring, X is either a nitrogen atom or a carbon atom bearing a substitutent Z. Z is either a hydorgen, an unfunctionalized lower alkyl chain, or a lower alkyl chain bearing an amino, carboxyl, hydroxyl, thiol aryl, indole, or imidazolyl group, Y is either N or CH, and each ring contains no more than three nitrogens consecutively bonded.

4. The method of claim 1, wherein the at least one base capable of base pairing and different from the standard W-C base is no isoC and/or isoG.

5. The method of any one of claims 1-4, wherein the amplification of step d) is performed by polymerase chain reaction and wherein the base triphosphates different from W-C bases have a different concentration from the concentration of the standard W-C base triphosphates.

6. The method of any one of claims 1-4, wherein the amplification of step d) is performed by polymerase chain reaction and wherein a base different from the standard W-C bases is isoC and/or isoG and the concentration of isoC and isoG triphosphate is lower than the concentration of the standard W-C bases triphosphates.

7. The method of claim 1, wherein the ligand is selected from the group consisting of amino acids, peptides, proteins, lipids, oligosaccharides, alkaloids, terpenes, co-enzymes, antibiotics, and their derivatives and their complexes.

8. The method of claim 1, wherein the ligand is a protein or a modified protein.

9. The method of claim 1, wherein the molecular weight of the ligand is less than 5,000 daltons.

10. An isolated aptamer comprising at least one base capable of base pairing and different from the standard Watson-Crick (W-C) bases.

11. The aptamer of claim 10, wherein the aptamer comprises at least one standard W-C base A, C, G, T or U and at least one base capable of base pairing and different from the standard W-C bases.

12. The aptamer of claim 10, wherein said base different from the standard W-C bases is selected from the group consisting of:

iso-C;

iso-G;

2,6-diaminopyrimidine;

xanthine;

6-amino-5-substituted pyrazin-2(1H)-one;

1-methyl-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione;

5-aza-7-deazaguanine;

6-amino-3-substituted pyrazin-2(1H)-one;

3-amino-1-methylpurin-2-one;

2,4-diamino-5,6-dihydropyryimidine;

2-amino-6-(N,N-dimethylamino)purine;

pyridin-2-one;

3-methyl isocarbostyril;

5-methyl isocarbostyril;

7-propynyl isocarbostyril;

m-xylene;

1,3,4-trim ethylbenzene;

2-methyl anaphthalene;

1,4-dimethylnaphthalene;

1-methylanaphthalene;

naphthalene;

7-azaindole;

isocarbostyril;

6-methyl-7-asaindole;

3-propynyl-7-azaindole;

imidazopyridine;

pyrrolopyridine and a heterocyclic base having a structural formula

 

wherein R designates the point of attachment of the base to position 1 of a ribose or deoxyribose ring, X is either a nitrogen atom or a carbon atom bearing a substituent Z. Z is either a hydrogen, an unfunctionalized lower alkyl chain, or a lower alkyl chain bearing an amino, carboxy, hydroxyl, thiol, aryl, indole, or imidazolyl group, Y is either N or CH, and each ring contains no more than three nitrogens consecutively bonded.

13. The aptamer of claim 10, wherein said base capable of base pairing and different from the standard W-C bases is isoC and/or isoG.

14. The aptamer of claim 10, which is a singe, double or triple stranded nucleic acid.

15. The aptamer of any one of claims 10-14, which is DNA, RNA or PNA.

16. The aptamer of any one of claims 10-15, wherein the number of bases of the aptamer is 1-300.

17. A method for isolating a specific ligand from a pool of ligands comprising:

i) providing at least one specific aptamer according to one of claims 10-16;

ii) mixing the aptamer with a pool of ligands; and

iii) recovering the specific ligand bound to the specific aptamer.

18. The method of claim 17, wherein the ligand is selected from the group consisting of amino acids, peptides, proteins, lipids, oligosaccharides, alkaloids, terpenes, co-enzymes, antibiotics, and their derivatives and their complexes.

19. The method of claim 17, wherein the ligand is a protein or a modified protein.

20. The method of claim 19, wherein the protein is phosporylated.

21. The method of claim 17, wherein the aptamer discriminates between phosphorylated and non-phosphorylated ligand proteins.

22. The method of claim 19, wherein the protein is glycosylated.

23. The method of claim 17, wherein the aptamer discriminates between glycosylated and non-glycosylated ligand proteins.

24. The method of claim 17, wherein the aptamer is fixed on an insoluble substrate.

25. The method of claim 24, wherein the aptamer is fixed on an insoluble substrate by means of a spacer sequence.

26. The method of claim 24 or 25, wherein the insoluble substrate is a chip substrate.

27. The method of claim 17, wherein the pool of ligand is labeled.

28. An insoluble substrate having at least one nucleic acid aptamer according to any one of claims 10-16 fixed thereto.

29. The insoluble substrate of claim 28, wherein the aptamer is fixed to the substrate by means of a spacer sequence.

30. The insoluble substrate of claim 28, comprising a plurality of aptamers that specifically bind to particular known ligands.

31. The insoluble substrate of any one of claims 28-30 that is a chip substrate.

32. A method for the detection of specific ligand from a biological sample, comprising:

I) selecting at least one specific aptamer, according to any one of claims 10-16, capable of binding to a specific ligand from a biological sample;

II) mixing the at least one the specific aptamer with a biological sample to allow binding of the ligand to the at least one aptamer;

III) detecting the presence and/or quantity of the specific ligand from the biological sample bound to the at least one aptamer.

33. The method of claim 32, wherein the aptamer is in solution or fixed to a substrate.

34. The method of claim 32, wherein the ligand in the biological sample is labeled, and the presence and/or quantity of the specific ligand is determined by detecting the label.

35. The method of claim 32, wherein the method is a diagnostic method and a diagnosis of a disease is based upon the amount or presence or absence of the ligand bound to the aptamer.

36. A detection or diagnostic kit for the determination of a specific ligand selected from a pool of ligands comprising at least one specific aptamer according to any one of claims 10-16, or at least one substrate having one or more aptamers according to any one of claims 25-31 fixed on it.

37. The kit of claim 36, further comprising one or more labels for labelling a pool of ligands.

38. A pharmaceutical composition comprising the aptamer according to any one of claims 10-16 and a pharmaceutically suitable diluent, excipient and/or carrier.

47. The method of claim 37, wherein the template is DNA, the synthesizing enzyme is DNA-dependent DNA polymerase, the synthesizing enzyme substrate is a plurality of dNTPs and substrate derivatives comprise ddNTPs.

48. The method of claim 47, wherein a dNTP or a ddNTP is labeled.

49. The method of claim 48, wherein the primer or initiator is labeled.

50. The method of claim 48 or 49, wherein the label is an isotope, a chromophore or a fluorophore.

39. A method for determining the nucleotide base sequence of a nucleic acid template comprising at least one base capable of base pairing and different from the standard Watson-Crick (W-C) bases comprising:

a) providing a nucleic acid template comprising at least one base capable of base pairing and different from the standard W-C bases;

b) elongating said template using a primer or a promoter or a promoter and an initiator in the presence of a nucleic acid synthesizing enzyme, nucleic acid synthesizing enzyme substrates and nucleic acid enzyme substrate derivatives;

c) determining the base sequence of the template as the reverse complement of the sequence of the elongation product obtained in b).

40. The method of claim 39, wherein the product of step b) comprises a plurality of polynucleotides and each of said polynucleotides is separated from the others differing in length by a single nucleotide.

41. The method of claim 39, wherein after step a) an annealing reaction between said template and a primer able to hybridize to said template is carried out.

42. The method of claim 41, wherein the nucleic acid template comprises at least one standard W-C base A, C, G, T or U and at least one base capable of base pairing and different from the standard W-C bases.

43. The method of claim 42, wherein the base triphosphates different from the standard W-C bases are present at a different concentration than the standard W-C base triphosphates are present at in the elongation step b).

44. The method of claim 39, wherein the base capable of base pairing and different from the standard W-C bases is selected from the group consisting of:

iso-C;

iso-G;

2,6-diaminopyrimidine;

xanthine;

6-amino-5-substituted pyrazin-2(1H)-one;

1-methyl-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione;

5-aza-7-deazaguanine;

6-amino-3-substituted pyrazin-2(1H)-one;

3-amino-1-methylpurin-2-one;

2,4-diamino-5,6-dihydropyryimidine;

2-amino-6-(N,N-dimethylamino)purine;

pyridin-2-one;

3-methyl isocarbostyril;

5-methyl isocarbostyril;

7-propynyl isocarbostyril;

m-xylene;

1,3,4-trimethylbenzene;

2-methylanaphthalene;

1,4-dimethylnaphthalene;

1-methylanaphthalene;

naphthalene;

7-azaindole;

isocarbostyril;

6-methyl-7-asaindole;

3-propynyl-7-azaindole;

imidazopyridine;

pyrrolopyridine and a heterocyclic base having a structural formula of

 

wherein R designates the point of attachment of the base to position 1 of a ribose or deoxyribose ring, X is either a nitrogen atom or a carbon atom bearing a substitutent Z. Z is either a hydrogen, an unfunctionalized lower alkyl chain, or a lower alkyl chain bearing an amino, carboxyl, hydroxyl, thiol, aryl, indole, or imidazolyl group, Y is either N or CH, and each ring contains no more than three nitrogens consecutively bonded.

45. The method of claim 39, wherein the base capable of base pairing and different from the standard W-C is isoC and/or isoG.

46. The method of claim 45, wherein the concentration of isoC and isoG triphosphate is lower than the concentration of the standard base triphosphates in the elongation step b).

51. The method of claim 39, wherein the template is DNA and the synthesizing enzyme is a DNA-dependent RNA polymerase, the synthesizing enzyme substrate is a plurality of NTPs and substrate derivatives comprise 3′

-dNTPs derivatives.

52. The method of claim 51, wherein a NTP or a 3′

-dNTP derivative is labeled.

53. The method of claim 51, wherein the primer or initiator is labeled.

54. The method of claim 52 or 53, wherein the label is an isotope, a chromophore or a fluorophore.

55. The method of claim 39, wherein the template is RNA and the synthesizing enzyme is a RNA-dependent DNA polymerase, the synthesizing enzyme substrate is a plurality of dNTPs and the substrate derivatives comprise ddNTPs.

56. The method of claim 55, wherein a dNTP or a ddNTP is labeled.

57. The method of claim 56, wherein the primer or initiator is labeled.

58. The method of claim 56 or 57, wherein the label is an isotope, a chromophore or a fluorophore.

59. The method of claim 39, wherein the template is RNA and the synthesizing enzyme is a RNA-dependent RNA polymerase, the synthesizing enzyme substrate is a plurality of NTPs and the substrate derivatives comprise 3′

-dNTPs derivatives.

60. The method of claim 59, wherein a NTP or a 3′

-dNTP derivative is labeled.

61. The method of claim 59, wherein the primer or initiator is labeled.

62. The method of claim 60 or 61, wherein the label is an isotope, a chromophore or a fluorophore.

63. A method for determining the nucleotide base sequence of a nucleic acid template comprising at least one base capable of base pairing and different from the standard Watson-Crick (W-C) bases comprising:

a) providing a nucleic acid template comprising at least one base capable of base pairing and different from the standard W-C bases;

b) labeling one end of said template;

c) chemically degrading said labeled template;

d) determining the length of the products obtained in c, obtaining the sequence of the template as the sequence of the incremental lengths of the products.

64. The method of claim 63, wherein the nucleic acid template comprises at least one standard W-C base and at least one base capable of base pairing and different from the standard W-C bases.

65. The method of claim 63, wherein the base capable of base pairing and different from the standard W-C bases is selected from the group consisting of:

iso-C;

iso-G;

2,6-diaminopyrimidine;

xanthine;

6-amino-5-substituted pyrazin-2(1H)-one;

1-methyl-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione;

5-aza-7-deazaguanine;

6-amino-3-substituted pyrazin-2(1H)-one;

3-amino-1-methylpurin-2-one;

2,4-diamino-5,6-dihydropyryimidine;

2-amino-6-(N,N-dimethylamino)purine;

pyridin-2-one;

3-methyl isocarbostyril;

5-methyl isocarbostyril;

7-propynyl isocarbostyril;

m-xylene;

1,3,4-trimethylbenzene;

2-methylanaphthalene;

1,4-dimethylnaphthalene;

1-methylanaphthalene;

naphthalene;

7-azaindole;

isocarbostyril;

6-methyl-7-asaindole;

3-propynyl-7-azaindole;

imidazopyridine; and

pyrrolopyridine and having a hetrocyclic base having a structural formula of

 

wherein R designates the point of attachment of the base to position 1 of a ribose or deoxyribose ring, X is either a nitrogen atom or a carbon atom bearing, a substitutent Z. Z is either a hydrogen, an unfunctionalized lower alkyl chain, or a lower alkyl chain bearing an amino, carboxyl, hydroxyl, thiol, aryl, indole, or imidazolyl group, Y is either N or CH, and each ring contains no more than three nitrogens consecutively bonded.

66. The method of claim 63, wherein the base capable of base pairing and different from the standard W-C bases is isoC and/or isoG.

67. A method for the determination of the base sequence of a nucleic acid template comprising at least one base capable of base pairing and different from the standard Watson-Crick (W-C) bases,:

A) providing a nucleic acid template comprising at least one base capable of base pairing and different from the standard W-C bases;

B) elongating said template using a primer or a promoter or a promoter and an initiator in the presence of a nucleic acid synthesizing enzyme, nucleic acid synthesizing enzyme substrates and nucleic acid synthesizing enzyme substrate derivatives;

C) determining the base sequence of the elongation product obtained in B) using MALDI-TOF-MS analysis.

68. A method for the determining the base sequence of a nucleic acid template comprising at least one base capable of base pairing and different from the standard Watson-Crick (W-C) bases,:

a) providing a nucleic acid template comprising at least one base capable of base pairing and different from the standard W-C standard bases;

b) carrying out elongation by using a primer, a promoter, or a promoter and initiator in the presence of nucleoside triphosphates whose base comprises at least one base capable of base pairing and different from the standard W-C bases by adding a first nucleoside triphosphate and detecting PPi release and degrading the first nucleoside triphosphate, then adding a second nucleoside triphosphate and detecting PPi and degrading the second nucleoside triphosphate, and repeating the procedure according to the kind of base of the nucleoside triphosphates provided;

c) repeating step b) above according to the template bases number desired to be sequenced;

d) determining the sequence of the template as the sequence of the nucleoside triphosphates added.

69. A method for determining the base sequence of a nucleic acid template comprising at least one base capable of base pairing and different from the standard Watson-Crick (W-C) bases,:

a) providing a plurality of oligonucleotides fixed on a chip, the oligonucleotides comprising at least one base capable of base pairing and different from the standard W-C bases and having overlapping frames displaced by one or two bases;

b) hybridizing the oligonucleotides with a labeled template according to the invention comprising at least one base capable of base pairing and different from the standard W-C bases;

c) detecting the signal of the label;

d) determining the sequence of the template as the set of overlapping oligonucleotides that are labeled.

70. A compound 2′

,3′

-dideoxyisoguanosine 5′

-triphosphate (ddisoG) or 2′

,3′

-dideoxyisocytidine 5′

-triphosphate (ddisoC).

71. A method for the preparation of 2′

,3′

-dideoxyisoguanosine 5′

-triphosphate comprising;

preparing the N-oxide of ddATP by precipitation of a mixture of disodium salt of ddATP and a solution of monopermaleic acid;

purifying the prepared N-oxide of ddATP and irradiating the purified N-oxide of ddATP with light from a high pressure mercury arc lamp; and

recovering the final product 2′

,3′

-dideoxyisoguanosine 5′

-triphosphate.

72. A method for the preparation of 2′

,3′

-dideoxyisoguanosine 5′

-triphosphate comprising;

adding hydrogen peroxide to a solution of sodium carbonate in water;

adding maleic anhydride and stirring the mixture until all of the maleic acid is dissolved;

adding concentrated sulfuric acid in water at 0°

C.;

extracting the mixture with ether;

obtaining a solution of monopermaleic acid by evaporating the ether extract in the presence of water in a stream of air;

adjusting the pH of the solution 7.0 with hydroxide ion;

adding a solution of disodium salt of ddATP;

stirring the reaction mixture at room temperature;

adjusting the pH to 4.5;

adding absolute EtOH;

recovering the resulting precipitate by centrifugation;

dissolving the precipitate in water;

adjusting the pH to 4.5;

recovering the N-oxide of ddATP by precipitation with ether;

dissolving the N-oxide of ddATP in water;

placing the solution of the N-oxide of ddATP in a photochemical reaction tube and irradiating the solution;

adjusting the pH to 10;

stirring the solution at room temperature;

removing the water; and

purifying the product by HPLC.

73. A method for preparing 2′

,3′

-dideoxyisocytidine 5′

-triphosphate comprising;

preparing 2′

,3′

-deoxyisocytidine from a mixture of 2,5′

-anhydro-2′

,3′

-dideoxyuridine and methanol;

adding a solution of triethylammonium bicarbonate to 2′

,3′

-deoxyisocytidine and removing the solvent by evaporation;

purifying the product obtained above by HPLC; and

recovering 2′

,3′

-di deoxyisocytidine 5′

-triphosphate.

74. A method for preparing 2′

,3′

-dideoxyisocytidine 5′

-triphosphate comprising;

adding diethyl azodicarboxylate to a mixture of 2′

,3′

-dideoxyuridine and triphenylphosphine suspended in tetrahydrofuran;

stirring the mixture at room temperature overnight to obtain a pale yellow suspension containing 2,5′

-anhydro-2′

,3′

-dideoxyuridine;

adding the suspension of 2,5′

-anhydro-2′

,3′

-dideoxyuridine to methanol saturated with dry ammonia;

stirring the mixture at room temperature;

removing the solvent;

extracting the residue with water;

evaporating the water to obtain 2′

,3′

-deoxyisocytidine;

treating a mixture of 2′

,3′

-deoxyisocytidine in trimethyl phosphate with phosphoryl chloride at 0°

C.;

adding a solution of tris(tributylammonium) pyrophosphate in dimethylformamide to the mixture and agitating vigorously;

adding a solution of triethylammonium bicarbonate;

removing the solvents; and

purifying the product by HPLC.