Claims
- 1. An RNA molecule containing an adenosine derivative covlently attached at the 5′ end of the RNA molecule.
- 2. The RNA molecule of claim 1, wherein the adenosine derivative is a 5′ derivative of adenosine or an N6 derivative of adenosine.
- 3. The RNA molecule of claim 1, wherein the adenosine derivative is coenzyme A, FAD, NAD, S-(5′-adenosyl)-cysteine, S-(5′-adenosyl)-homocysteine, or S-(5-adenosyl)-methionine.
- 4. The RNA molecule of claim 1, wherein the adenosine derivative is a fluorescein derivative of adenosine or a biotin derivative of adenosine.
- 5. The RNA molecule of claim 1, wherein the adenosine derivative is an adenosine 5′-amino-linker phosphoramidate.
- 6. The RNA molecule of claim 5, wherein the linker is an alkyl group, an alkoxy group, an alkoxy alkyl group, an alklamine group, or a polyalkylamine group.
- 7. The RNA molecule of claim 1, wherein the adenosine derivative is adenosine 5′-aminoethyl phosphoramidate (5′-EDA-AMP), adenosine 5′-(4-aminobutyl) phosphoramidate (5′-BDA-AMP), adenosine 5′-(6-aminohexyl) phosphoramidate (5′-HDA-AMP), adenbsine 5′-(2-aminoethoxyethyl) phosphoramidate (5′-ODEA-AMP), adenosine 5′-(3,6-dioxa-8-aminooctyl) phosphoramidate (5′-EDODEA-AMP), or adenosine 5′-(4,7,10-trioxa-13-aminotridecyl) phosphoramidate (5′-TOTDDA-AMP).
- 8. The RNA molecule of claim 1, wherein the adenosine derivative is adenosine 5′-(5)carboxyfluorescein aminoethyl phosphoramidate (5′-(5)FAM-EDA-AMP), adenosine 5′-(6) carboxyfluorescein aminoethyl phosphoramidate (5′-(6)FAM-EDA-AMP), adenosine 5′-(5)carboxyfluorescein (4-aminobutyl) phosphoramidate (5′-(5)FAM-BDA-AMP), adenosine 5′-(6) carboxyfluorescein (4-aminobutyl) phosphoramidate (5′-(6) FAM-BDA-AMP), adenosine 5′-(5)carboxyfluorescein (6-aminohexyl) phosphoramidate (5′-(5)FAM-HDA-AMP), adenosine 5′-(6)carboxyfluorescein (6-aminohexyl) phosphoramidate (5′-(6)FAM-HDA-AMP), adenosine 5′-(5) carboxyfluorescein (2-aminoethoxyethyl) phosphoramidate (5′-(5)FAM-ODEA-AMP), adenosine 5′-(6)carboxyfluorescein (2-aminoethoxyethyl) phosphoramidate (5′-(6)FAM-ODEA-AMP), adenosine 5′-(5)carboxyfluorescein (3,6-dioxa-8-aminooctyl) phosphoramidate (5′-(5)FAM-EDOEA-AMP), adenosine 5′-(6)carboxyfluorescein (3,6-dioxa-8-aminooctyl) phosphoramidate (5′-(6)FAM-EDODEA-AMP), adenosine 5′-(5)carboxyfluorescein (4,7,10-trioxa-13-aminotridecyl) phosphoramidate (5′-(5)FAM-TOTDDA-AMP), or adenosine 5′-(6)carboxyfluorescein (4,7,10-trioxa-13-aminotridecyl) phosphoramidate (5′-(6)FAM-TOTDDA-AMP).
- 9. The RNA molecule of claim 1, wherein the adenosine derivative is adenosine 5′-biotin (6-aminohexyl) phosphoramidate (5′-biotin-HDA-AMP), adenosine 5′-biotin (3,6-dioxa-8-aminooctyl) phosphoramidate (5′-biotin-EDODEA-AMP), or adenosine 5′-biotin (4,7,10-trioxa-13-aminotridecyl) phosphoramidate (5′-biotin-TOTDDA-AMP).
- 10. The RNA molecule of claim 1, wherein the adenosine derivative is an N6-amino-linker adenosine 5′-monophosphate.
- 11. The RNA molecule of claim 10, wherein the linker is an alkyl group, an alkoxy group, an alkoxy alkyl group, an alkylamine group, or a polyalkylamine group.
- 12. The RNA molecule of claim 1, wherein the adenosine derivative is N6-aminoethyl adenosine 5′-monophosphate (N6- EDA-AMP), N6-(4-aminobutyl) adenosine 5′-monophosphate (N6-BDA-AMP), N6-(6-aminohexyl) adenosine 5′-monophosphate (N6-HDA-AMP), N6-(2-aminoethoxyethyl) adenosine 5′-monophosphate (N6-ODEA-AMP), N6-(3,6-dioxa-8-aminooctyl) adenosine 5′-monophosphate (N6-EDODEA-AMP), or N6-(4, 7, 10-trioxa-13-aminotridecyl) adenosine 5′-monophosphate (N6-TOTDDA-AMP).
- 13. The RNA molecule of claim 1, wherein the adenosine derivative is N6-(5)carboxyfluorescein aminoethyl adenosine 5′-monophosphate (N6-(5)FAM-EDA-AMP), N6-(6) carboxyfluorescein aminoethyl adenosine 5′-monophosphate (N6-(6)FAM-EDA-AMP), N6-(5)carboxyfluorescein (4-aminobutyl) adenosine 5′-monophosphate (N6-(5)FAM-BDA-AMP), N6-(6)carboxyfluorescein (4-aminobutyl) adenosine 5′-monophosphate (N6-(6)FAM-BDA-AMP), N6-(5) carboxyfluorescein (6-aminohexyl) adenosine 5′-monophosphate (N6-(5)FAM-HDA-AMP), N6-(6) carboxyfluorescein (6-aminohexyl) adenosine 5′-monophosphate (N6-(6)FAM-HDA-AMP), N6-(5) carboxyfluorescein (2-aminoethyl) adenosine 5′-monophosphate (N6-(5)FAM-ODEA-AMP), N6-(6) carboxyfluorescein (2-aminoethyl) adenosine 5′-monophosphate (N6-(6)FAM-ODEA-AMP), N6-(5) carboxyfluorescein (3,6-dioxa-8-aminooctyl) adenosine 5′-monophosphate (N6-(5)FAM-EDODEA-AMP), N6-(6) carboxyfluorescein (3,6-dioxa-8-aminooctyl) adenosine 5′-monophosphate (N6-(6)FAM-EDODEA-AMP), N6-(5) carboxyfluorescein (4, 7, 10-trioxa-13-aminotridecyl) adenosine 5′-monophosphate (N6-(5)FAM-TOTDDA-AMP), or N6-(6)carboxyfluorescein (4, 7, 10-trioxa-13-aminotridecyl) adenosine 5′-monophosphate (N6-(6)FAM-TOTDDA-AMP).
- 14. The RNA molecule of claim 1, wherein the adenosine derivative is an adenosine 5′-sulfhydryl-linker phosphoramidate.
- 15. The RNA molecule of claim 14, wherein the linker is an alkyl group, an alkoxy group, an alkoxy alkyl group, or an alkylamine group.
- 16. The RNA molecule of claim 1, wherein the adenosine derivative is adenosine 5′-mercaptoethyl phosphoramidate.
- 17. The RNA molecule of claim 1, wherein the adenosine derivative is adenosine 5′-(5)carboxyfluorescein thioethyl phosphoramidate or adenosine 5′-(6)carboxyfluorescein thioethyl phosphoramidate.
- 18. The RNA molecule of claim 1, wherein the adenosine derivative is adenosine 5′-biotin thioethyl phosphoramidate.
- 19. The RNA molecule of claim 1, wherein the adenosine derivative is an N6-sulfhydryl-linker adenosine 5′-monophosphate.
- 20. The RNA molecule of claim 19, wherein the linker is an alkyl group, an alkoxy group, an alkoxy alkyl group, or an alkylamine group.
- 21. The RNA molecule of claim 1, wherein the adenosine derivative is N6-mercaptoethyl adenosine 5′-monophosphate.
- 22. The RNA molecule of claim 1, wherein the adenosine derivative is N6-(5)carboxyfluorescein thioethyl adenosine 5′-monophosphate or N6-(6)carboxyfluorescein thioethyl adenosine 5′-monophosphate.
- 23. The RNA molecule of claim 1, wherein the adenosine derivative is N6-biotin thioethyl adenosine 5′-monophosphate.
- 24. The RNA molecule of claim 1, wherein the adenosine derivative is adenosine 5′-O-thiomonophosphate, adenosine 5′-[Î2-thio]diphosphate, or adenosine 5′-[Î3-thio]triphosphate.
- 25. The RNA molecule of claim 24, wherein the adenosine derivative is a fluorescein derivative of adenosine or a biotin derivative of adenosine.
- 26. The RNA molecule of claim 1, wherein the adenosine derivative is an adenosine 5′-(α,Î2-methylene)diphosphate, adenosine 5′-(Î2,Î3-imido)triphosphate, adenosine 5′-triphosphate Î3-(1-(2-nitrophenyl)ethyl) ester, or 5′-p-fluorosulfonylbenzoyl adenosine.
- 27. The RNA molecule of claim 1, wherein the adenosine derivative is N6-biotin (4-aminobutyl) adenosine 5′-monophosphate (N6-biotin-BDA-AMP), N6-biotin (6-aminohexyl) adenosine 5′-monophosphate (N6-biotin-HDA-AMP), N6-biotin (3,6-dioxa-8-aminooctyl) adenosine 5′-monophosphate (N6-biotin-EDODEA-AMP), or N6-biotin (4, 7, 10-trioxa-13-aminotridecyl) adenosine 5′-monophosphate (N6-biotin-TOTDDA-AMP).
- 28. The RNA molecule of claim 1, further comprising an amino acid, a peptide, a neurotransmitter, an amino acid-sugar, or a protein covalently attached to the adenosine derivative.
- 29. A method for the preparation of a RNA molecule containing an adenosine derivative covalently attached at the 5′ end of the RNA molecule, the method comprising:
providing a DNA template comprising a RNA polymerase promoter sequence; contacting the DNA template, an RNA polymerase enzyme, an adenosine derivative, ATP, UTP, GTP, and CTP to prepare a reaction mixture; and incubating the reaction mixture under conditions suitable for RNA transcription to prepare the RNA molecule.
- 30. The method of claim 29, wherein the promoter sequence is SEQ ID NO:7.
- 31. The method of claim 29, wherein the adenosine derivative is a 5′ derivative of adenosine or an N6 derivative of adenosine.
- 32. The method of claim 29, wherein the adenosine derivative is coenzyme A, FAD, NAD, S-(5′-adenosyl)-cysteine, S-(5′-adenosyl)-homocysteine, or S-(5′-adenosyl)-methionine.
- 33. The method of claim 29, wherein the adenosine derivative is adenosine 5′-O-thiomonophosphate, adenosine 5′-[Î2-thio]diphosphate, adenosine 5′-[Î3-thio]triphosphate, adenosine 5′-(α,Î2-methylene)diphosphate, adenosine 5′-(Î2,Î3-imido) triphosphate, adenosine 5′-triphosphate Î3-(1-(2-nitrophenyl) ethyl) ester, or 5′-p-fluorosulfonylbenzoyladenosine.
- 34. The-method of claim 29, OLE_LINK1 wherein the adenosine derivative is a fluorescein derivative of adenosine, a biotin derivative of adenosine, an Alexa derivative of adenosine, a BODIPY derivative of adenosine, a Cy3 derivative of adenosine, or a Cy5 derivative of adenosine.
- 35. The method of claim 29, wherein the adenosine derivative is covalently attached to an amino acid, a peptide, a neurotransmitter, an amino acid-sugar, or a protein.
- 36. The method of claim 29, wherein the RNA polymerase enzyme is the T7 RNA polymerase enzyme.
- 37. The method of claim 29, further comprising purifying the RNA molecule after the incubating step.
- 38. The method of claim 29, wherein the reaction mixture further comprises at least one radiolabel selected from the group consisting of [α-32P]ATP, [α-32P]GTP, [α-32P]CTP, [α-32P]UTP, [α-35S]ATP, [Î-35S]GTP, [α-35S]CTP, and [α-35S]UTP.
- 39. A kit comprising a RNA polymerase enzyme, an adenosine derivative, ATP, UTP, GTP, and CTP.
- 40. The kit of claim 39, wherein the adenosine derivative is a 5′ derivative of adenosine or an N6 derivative of adenosine.
- 41. The kit of claim 39, wherein the adenosine derivative is coenzyme A, FAD, NAD, S-(5′-adenosyl)-cysteine, S-(5′-adenosyl)-homocysteine, or S-(5′-adenosyl)-methionine.
- 42. The kit of claim 39, wherein the adenosine derivative is adenosine 5′-O-thiomonophosphate, adenosine 5′-[Î2-thio]diphosphate, adenosine 5′-[Î3-thio]triphosphate, adenosine 5′-(α,Î2-methylene)diphosphate, adenosine 5′-(Î2,Î3-imido) triphosphate, adenosine 5′-triphosphate Î3-(1-(2-nitrophenyl) ethyl) ester, or 5′-p-fluorosulfonylbenzoyladenosine.
- 43. The kit of claim 39, wherein the adenosine derivative is a fluorescein derivative of adenosine, a biotin derivative of adenosine, an Alexa derivative of adenosine, a BODIPY derivative of adenosine, a Cy3 derivative of adenosine, or a Cy5 derivative of adenosine.
- 44. The kit of claim 39, wherein the RNA polymerase enzyme is the T7 RNA polymerase enzyme.
- 45. The kit of claim 39, further comprising at least one radiolabel selected from the group consisting of [α-32P]ATP, [α-32P]GTP, [α-32P]CTP, [α-32P]UTP, [α-35S]ATP, [α-32S]GTP, [α-35S]CTP, and [α-35S]UTP.
- 46. An adenosine derivative defined as an adenosine 5′-amino-linker phosphoramidate or a 5′-sulfhydryl-linker phosphoramidate.
- 47. The adenosine derivative of claim 46, wherein the linker is an alkyl group, an alkoxy group, an alkoxy alkyl group, an alkylamine group, or a polyalkylamine group.
- 48. The adenosine derivative of claim 46, wherein the adenosine derivative is adenosine 5′-aminoethyl phosphoramidate (5′-EDA-AMP), adenosine 5′-(4-aminobutyl) phosphoramidate (5′-BDA-AMP), adenosine 5′-(6-aminohexyl) phosphoramidate (5′-HDA-AMP), adenosine 5′-(2-aminoethoxyethyl) phosphoramidate (5′-ODEA-AMP), adenosine 5′-(3,6-dioxa-8-aminooctyl) phosphoramidate (5′-EDODEA-AMP), adenosine 5′-(4,7,10-trioxa-13-aminotridecyl) phosphoramidate (5′-TOTDDA-AMP), or adenosine 5′-mercaptoethyl phosphoramidate.
- 49. The adenosine derivative of claim 46, wherein the adenosine derivative is adenosine 5′-(5)carboxyfluorescein aminoethyl phosphoramidate (5′-(5)FAM-EDA-AMP), adenosine 5′-(6)carboxyfluorescein aminoethyl phosphoramidate (5′-(6)FAM-EDA-AMP), adenosine 5′-(5) carboxyfluorescein (4-aminobutyl) phosphoramidate (5′-(5) FAM-BDA-AMP), adenosine 5′-(6)carboxyfluorescein (4-aminobutyl) phosphoramidate (5′-(6)FAM-BDA-AMP), adenosine 5′-(5)carboxyfluorescein (6-aminohexyl) phosphoramidate (5′-(5)FAM-HDA-AMP), adenosine 5′-(6) carboxyfluorescein (6-aminohexyl) phosphoramidate (5′-(6) FAM-HDA-AMP), adenosine 5′-(5)carboxyfluorescein (2-aminoethoxyethyl) phosphoramidate (5′-(5)FAM-ODEA-AMP), adenosine 5′-(6)carboxyfluorescein (2-aminoethoxyethyl) phosphoramidate (5′-(6)FAM-ODEA-AMP), adenosine 5′-(5) carboxyfluorescein (3,6-dioxa-8-aminooctyl) phosphoramidate (5′-(5)FAM-EDODEA-AMP), adenosine 5′-(6) carboxyfluorescein (3,6-dioxa-8-aminooctyl) phosphoramidate (5′-(6)FAM-EDODEA-AMP), adenosine 5′-(5) carboxyfluorescein (4,7,10-trioxa-13-aminotridecyl) phosphoramidate (5′-(5)FAM-TOTDDA-AMP), adenosine 5′-(6) carboxyfluorescein (4,7,10-trioxa-13-aminotridecyl) phosphoramidate (5′-(6)FAM-TOTDDA-AMP), adenosine 5′-(5) carboxyfluorescein thioethyl phosphoramidate, or adenosine 5′-(6)carboxyfluorescein thioethyl phosphoramidate.
- 50. The adenosine derivative of claim 46, wherein the adenosine derivative is adenosine 5′-biotin (6-aminohexyl) phosphoramidate (5′-biotin-HDA-AMP), adenosine 5′-biotin (3,6-dioxa-8-aminooctyl) phosphoramidate (5′-biotin-EDODEA-AMP), adenosine 5′-biotin (4,7,10-trioxa-13-aminotridecyl) phosphoramidate (5′-biotin-TOTDDA-AMP), or adenosine 5′ biotin thioethyl phosphoramidate.
- 51. An adenosine derivative defined as an N6-amino-linker adenosine 5′-monophosphate or a 5′-sulfhydryl-linker phosphoramidate.
- 52. The adenosine derivative of claim 51, wherein the linker is an alkyl group, an alkoxy group, an alkoxy alkyl group, an alkylamine group, or a polyalkylamine group.
- 53. The adenosine derivative of claim 51, wherein the adenosine derivative is N6-aminoethyl adenosine 5′-monophosphate (N6-EDA-AMP), N6-(4-aminobutyl) adenosine 5′-monophosphate (N6-BDA-AMP), N6-(6-aminohexyl) adenosine 5′-monophosphate (N6-HDA-AMP), N6-(2-aminoethoxyethyl) adenosine 5′-monophosphate (N6-ODEA-AMP), N6-(3,6-dioxa-8-aminooctyl) adenosine 5′-monophosphate (N6-EDODEA-AMP), N6-(4, 7, 10-trioxa-13-aminotridecyl) adenosine 5′-monophosphate (N6-TOTDDA-AMP), or N6-mercaptoethyl adenosine 5′-monophosphate.
- 54. The adenosine derivative of claim 51, wherein the adenosine derivative is N6-(5)carboxyfluorescein aminoethyl adenosine 5′-monophosphate (N6-(5)FAM-EDA-AMP), N6-(6) carboxyfluorescein aminoethyl adenosine 5′-monophosphate (N6-(6)FAM-EDA-AMP), N6-(5)carboxyfluorescein (4-aminobutyl) adenosine 5′-monophosphate (N6-(5)FAM-BDA-AMP), N6-(6)carboxyfluorescein (4-aminobutyl) adenosine 5′-monophosphate (N6-(6)FAM-BDA-AMP), N6-(5) carboxyfluorescein (6-aminohexyl) adenosine 5′-monophosphate (N6-(5)FAM-HDA-AMP), N6-(6) carboxyfluorescein (6-aminohexyl) adenosine 5′-monophosphate (N6-(6)FAM-HDA-AMP), N6-(5) carboxyfluorescein (2-aminoethyl) adenosine 5′-monophosphate (N6-(5)FAM-ODEA-AMP), N6-(6) carboxyfluorescein (2-aminoethyl) adenosine 5′-monophosphate (N6-(6)FAM-ODEA-AMP), N6-(5) carboxyfluorescein (3,6-dioxa-8-aminooctyl) adenosine 5′-monophosphate (N6-(5)FAM-EDODEA-AMP), N6-(6) carboxyfluorescein (3,6-dioxa-8-aminooctyl) adenosine 5′-monophosphate (N6-(6)FAM-EDODEA-AMP), N6-(5) carboxyfluorescein (4, 7, 10-trioxa-13-aminotridecyl) adenosine 5′-monophosphate (N6-(5)FAM-TOTDDA-AMP), N6-(6)carboxyfluorescein (4, 7, 10-trioxa-13-aminotridecyl) adenosine 5′-monophosphate (N6-(6)FAM-TOTDDA-AMP), N6-(5)carboxyfluorescein thioethyl adenosine 5′-monophosphate, or N6-(6)carboxyfluorescein thioethyl adenosine 5′-monophosphate.
- 55. The adenosine derivative of claim 51, wherein the adenosine derivative is N6-biotin (4-aminobutyl) adenosine 5′-monophosphate (N6-biotin-BDA-AMP), N6-biotin (6-aminohexyl) adenosine 5′-monophosphate (N6-biotin-HDA-AMP), N6-biotin (3,6-dioxa-8-aminooctyl) adenosine 5′-monophosphate (N6-biotin-EDODEA-AMP), N6-biotin (4, 7, 10-trioxa-13-aminotridecyl) adenosine 5′-monophosphate (N6-biotin-TOTDDA-AMP), or N6-biotin thioethyl adenosine 5′-monophosphate.
FEDERAL RESEARCH STATEMENT
[0001] The government may own rights in the present invention pursuant to grant number MCB9974487 from the National Science Foundation and grant number NAG5-10668 from the National Aeronautics and Space Administration.