Claims
- 1. An isolated nucleic acid comprising a region encoding a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:15.
- 2. The nucleic acid of claim 1, wherein said nucleic acid comprises the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:14.
- 3. The nucleic acid of claim 1, wherein said nucleic acid is operatively linked to a promoter.
- 4. The nucleic acid of claim 3, wherein said promoter is an N4 vRNAP promoter set forth in SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29.
- 5. The nucleic acid of claim 3, wherein said promoter is a P2 sequence set forth in SEQ ID NO:16 or SEQ ID NO:28.
- 6. A recombinant host cell comprising a DNA segment encoding a N4 virion RNA polymerase.
- 7. The recombinant host cell of claim 6, wherein said DNA segment is a single-stranded DNA segment.
- 8. The recombinant host cell of claim 6, wherein said DNA segment is a double-stranded DNA segment.
- 9. The recombinant host cell of claim 6, wherein said DNA segment encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:4.
- 10. The recombinant host cell of claim 6, wherein said DNA segment encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:6.
- 11. The recombinant host cell of claim 6, wherein said cell is an E. coli cell.
- 12. A recombinant vector comprising a DNA segment encoding a N4 virion RNA polymerase polypeptide under the control of a promoter.
- 13. An isolated polynucleotide comprising a sequence identical or complementary to SEQ ID NO:1.
- 14. An isolated polynucleotide comprising a sequence identical or complementary to SEQ ID NO:3.
- 15. A purified N4 virion RNA polymerase comprising the polypeptide sequence of SEQ ID NO:2.
- 16. An isolated nucleic acid comprising a region encoding a polypeptide comprising at least 6 contiguous amino acids of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8, wherein said polypeptide has RNA polymerase activity under appropriate reaction conditions.
- 17. The nucleic acid of claim 16, wherein said polypeptide comprises at least 20 contiguous amino acids of said amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8.
- 18. The nucleic acid of claim 17, wherein said polypeptide comprises at least 40 contiguous amino acids of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8.
- 19. The nucleic acid of claim 18, wherein said polypeptide comprises at least 100 contiguous amino acids of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8.
- 20. The nucleic acid of claim 16, wherein said polypeptide comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8.
- 21. The nucleic acid of claim 16, wherein said polypeptide has at least one histidine tag.
- 22. The nucleic acid of claim 16, wherein said polypeptide has a mutation at position Y678.
- 23. A method of making RNA comprising:
(a) obtaining a N4 virion RNA polymerase; (b) obtaining DNA; (c) admixing said RNA polymerase and said DNA; and (d) culturing said RNA polymerase and said DNA under conditions effective to allow RNA synthesis.
- 24. The method of claim 23, further comprising synthesizing polynucleotides containing modified ribonucleotides or deoxyribonucleotides.
- 25. The method of claim 23, wherein said DNA is single-stranded DNA.
- 26. The method of claim 23, wherein said DNA is double-stranded DNA.
- 27. The method of claim 23, wherein said admixing occurs in a host cell.
- 28. The method of claim 27, wherein said host cell is an E. coli host cell.
- 29. The method of claim 23, wherein said RNA polymerase has the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:15.
- 30. The method of claim 29, wherein said RNA polymerase has the amino acid sequence set forth in SEQ ID NO:4.
- 31. The method of claim 23, wherein said RNA polymerase is a mutant of an RNA polymerase having the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:6.
- 32. The method of claim 31, wherein said mutant has a mutation at position number Y678.
- 33. The method of claim 32, wherein said mutant is histidine tagged.
- 34. The method of claim 23, wherein said RNA contains derivatized nucleotides.
- 35. The method of claim 23, further comprising using a promoter.
- 36. The method of claim 35, wherein said promoter is an N4 vRNAP promoter set forth in SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29.
- 37. The method of claim 36, wherein said promoter is a P2 sequence set forth in SEQ ID NO:16 or SEQ ID NO:28.
- 38. The method of claim 35, wherein the promoter comprises a set of inverted repeats forming a hairpin with a 2-7 base pair long stem and 3-5 base loop having purines in the central and/or next to the central position of the loop.
- 39. The method of claim 35, wherein said promoter sequence is upstream of the transcription initiation site.
- 40. The method of claim 23, wherein step (c) is carried out at a pH of between 6 and 9.
- 41. The method of claim 40, wherein step (c) is carried out at a pH of between 7.5 and 8.5.
- 42. The method of claim 23, further comprising admixing Mg+2 or Mn+2.
- 43. The method of claim 42, comprising admixing Mg+2.
- 44. The method of claim 23, further defined as carried out at a temperature of 25° C. to 50° C.
- 45. The method of claim 44, further defined as carried out at a temperature of 30° C. to 45° C.
- 46. The method of claim 45, further defined as carried out at a temperature of 32° C. to 42° C.
- 47. The method of claim 23, further comprising the step of translation.
- 48. The method of claim 23, further comprising using a reporter gene.
- 49. The method of claim 48, wherein said reporter gene is an α-peptide of β-galactosidase.
- 50. The method of claim 23, wherein said admixing occurs in vivo.
- 51. The method of claim 23, wherein said admixing occurs in vitro.
- 52. The method of claim 23, further comprising admixing an E. coli single-stranded binding protein (EcoSSB), a SSB protein homologous to EcoSSB or another naturally occurring or chimeric SSB protein homologous to EcoSSB with said DNA and said polymerase
- 53. The method of claim 52, further comprising translation of said RNA.
- 54. The method of claim 23, wherein said DNA is single-stranded linear DNA.
- 55. The method of claim 23, wherein said DNA is single-stranded circular DNA.
- 56. The method of claim 55, wherein said circular DNA is bacteriophage M13 DNA.
- 57. The method of claim 23, wherein said DNA is denatured DNA.
- 58. The method of claim 57, wherein said denatured DNA is single-stranded DNA.
- 59. The method of claim 57, wherein said denatured DNA is double-stranded linear DNA.
- 60. The method of claim 57, wherein said denatured DNA is double-stranded circular DNA.
- 61. The method of claim 23, wherein said RNA is purified RNA.
- 62. The method of claim 23, wherein said RNA comprises modified nucleotides.
- 63. The method of claim 23, wherein mixed RNA-DNA oligonucleotides are made.
- 64. The method of claim 23, wherein no EcoSSB is admixed with said RNA polymerase and said DNA and wherein said RNA is in the form of a DNA/RNA hybrid.
- 65. The method of claim 23, wherein said RNA comprises a detectable label.
- 66. The method of claim 65, wherein said detectable label is a fluorescent tag.
- 67. The method of claim 65, wherein said detectable label is biotin.
- 68. The method of claim 65, wherein said detectable label is digoxigenin.
- 69. The method of claim 65, wherein said detectable label is 2′-fluoro nucleoside triphosphate.
- 70. The method of claim 65, wherein said detectable label is a radiolabel.
- 71. The method of claim 70, wherein said radiolabel is a 35S- or 32P-label.
- 72. The method of claim 65, wherein said RNA is adapted for use as a probe for blotting experiments or in-situ hybridization.
- 73. The method of claim 23, wherein nucleoside triphosphates (NTPs) are incorporated into said RNA.
- 74. The method of claim 73, wherein said NTPs comprise a detectable label.
- 75. The method of claim 75, wherein said NTPs are derivatized NTPs.
- 76. The method of claim 23, wherein deoxynucleoside triphosphates are incorporated into said RNA.
- 77. The method of claim 23, wherein said RNA is adapted for NMR structural determination.
- 78. The method of claim 77, wherein said RNA has between 10 and 1000 bases.
- 79. The method of claim 78, wherein said RNA has between 10 and 300 bases.
- 80. The method of claim 23, wherein said RNA is adapted for spliceosome assembly.
- 81. The method of claim 23, wherein said RNA is adapted for splicing reactions.
- 82. The method of claim 23, wherein said RNA is adapted for use in antisense experiments.
- 83. The method of claim 23, wherein said RNA is adapted for use in probing for a complementary nucleotide sequence.
- 84. The method of claim 23, wherein said RNA is adapted for use as a probe in RNase protection studies.
- 85. The method of claim 23, further comprising the step of delivering said RNA into a cell.
- 86. The method of claim 85, wherein delivering is by microinjection.
- 87. The method of claim 23, further comprising the step of amplifying said RNA.
- 88. A method of making RNA comprising:
(a) obtaining a N4 virion RNA polymerase; (b) obtaining a single-stranded DNA oligonucleotide wherein said oligonucleotide contains a N4 virion RNA polymerase promoter sequence; (c) admixing said RNA polymerase and said oligonucleotide; and (d) culturing said RNA polymerase and said oligonucleotide under conditions effective to allow RNA synthesis.
- 89. The method of claim 88, wherein said RNA polymerase has the amino sequence set forth in SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8.
- 90. The method of claim 88, wherein said DNA has between 20 and 200 bases.
- 91. A method of making RNA comprising:
(a) obtaining a N4 virion RNA polymerase; (b) obtaining a single-stranded DNA wherein said DNA contains a N4 virion RNA polymerase promoter sequence; (c) obtaining a ribonucleoside triphosphate (XTP) or a derivatized ribonucleoside triphosphate; (d) admixing said RNA polymerase, said DNA and said XTP; and (e) culturing said RNA polymerase and said oligonucleotide under conditions effective to allow RNA synthesis wherein said RNA is a derivatized RNA.
- 92. The method of claim 91, wherein said RNA polymerase has the amino sequence set forth in SEQ ID NO:4.
- 93. The method of claim 91, wherein said RNA polymerase is a mutant of an RNA polymerase comprising the amino sequence essentially as set forth in SEQ ID NO:4 or SEQ ID NO:6.
- 94. The method of claim 93, wherein said mutant has a mutation at position number Y678.
- 95. The method of claim 91, wherein said RNA polymerase has the amino sequence set forth in SEQ ID NO:8.
- 96. A method for in vivo protein synthesis comprising:
(a) obtaining an RNA polymerase having the amino sequence set forth in SEQ ID NO:4 or a mutant thereof; (b) obtaining DNA wherein said DNA contains a N4 virion RNA polymerase promoter sequence; (c) admixing said RNA polymerase and said DNA; (d) culturing said RNA polymerase and said DNA under conditions effective to allow RNA synthesis; and (e) culturing said RNA in vivo under conditions effective to allow protein synthesis.
- 97. The method of claim 96, wherein step (e) comprises using a two plasmid system.
- 98. The method of claim 96, wherein step (e) comprises using a one plasmid system.
- 99. The method of claim 98, wherein a reporter gene and said RNA polymerase are on the same plasmid.
- 100. A method of making a full-length N4 vRNAP or mini-vRNAP comprising:
(a) expressing vRNAP, wherein said vRNAP has the amino sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:15 or a mutant thereof; and (b) purifying said vRNAP.
- 101. The method of claim 100, wherein said expressing occurs in a bacteria, yeast, CHO, Cos, HeLa, NIH3T3, Jurkat, 293, Saos, or PC12 host cell.
- 102. The method of claim 100, further comprising using a promoter appropriate for expression in the host cell line being used.
- 103. The method of claim 102, wherein said promoter is pBAD.
- 104. The method of claim 102, wherein said promoter is a promoter recognized by T7 RNA polymerase, T3 RNA polymerase or SP6 RNA polymerase.
- 105. The method of claim 102, wherein said promoter is a promoter recognized by T7-like RNA polymerase.
- 106. The method of claim 100, wherein said vRNAP has a specific recombinant sequence for use in purification.
- 107. The method of claim 106, wherein said vRNAP has at least one histidine, FLAG, hemaglutinin or c-myc tag.
- 108. The method of claim 106, wherein said vRNAP has at least one histidine tag.
- 109. The method of claim 107, wherein said purifying occurs in one step.
- 110. The method of claim 100, wherein said vRNAP does not have a tag.
Parent Case Info
[0001] This application claims the priority of U.S. Provisional Patent Application Serial No. 60/292,845, filed May 22, 2001, the entire disclosure of which is specifically incorporated herein by reference.
Government Interests
[0002] The government may own rights in the present invention pursuant to grant number R01 A1 12575 from the National Institute of Health.
Provisional Applications (1)
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Number |
Date |
Country |
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60292845 |
May 2001 |
US |