Claims
- 1. A composition comprising an orthogonal aminoacyl-tRNA synthetase (O-RS), wherein the O-RS preferentially aminoacylates an orthogonal tRNA (O-tRNA) with an unnatural amino acid.
- 2. The composition of claim 1, wherein the O-RS comprises a nucleic acid comprising a polynucleotide sequence selected from the group consisting of: SEQ ID NO: 4-34 and a complementary polynucleotide sequence thereof.
- 3. The composition of claim 1, wherein the O-RS aminoacylates the O-tRNA with the unnatural amino acid in vivo.
- 4. The composition of claim 1, wherein the unnatural amino acid is selected from the group consisting of: an O-methyl-L-tyrosine, an L-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl-GlcNAcβ-serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, a p-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, a p-bromophenylalanine, a p-amino-L-phenylalanine, and an isopropyl-L-phenylalanine.
- 5. The composition of claim 1, wherein the unnatural amino acid is selected from the group consisting of: an unnatural analogue of a tyrosine amino acid; an unnatural analogue of a glutamine amino acid; an unnatural analogue of a phenylalanine amino acid; an unnatural analogue of a serine amino acid; an unnatural analogue of a threonine amino acid; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or any combination thereof; an amino acid with a photoactivatable cross-linker; a spin-labeled amino acid; a fluorescent amino acid; an amino acid with a novel functional group; an amino acid that covalently or noncovalently interacts with another molecule; a metal binding amino acid; a metal-containing amino acid; a radioactive amino acid; a photocaged amino acid, a photoisomerizable amino acid; a biotin or biotin-analogue containing amino acid; a glycosylated or carbohydrate modified amino acid; a keto containing amino acid; an amino acid comprising polyethylene glycol; an amino acid comprising polyether; a heavy atom substituted amino acid; a chemically cleavable or photocleavable amino acid; an amino acid with an elongated side chain; an amino acid containing a toxic group; a sugar substituted amino acid; a sugar substituted serine; a carbon-linked sugar-containing amino acid; a redox-active amino acid; an α-hydroxy containing acid; an amino thio acid containing amino acid; an α,α disubstituted amino acid; a β-amino acid; and a cyclic amino acid other than proline.
- 6. The composition of claim 1, wherein the O-RS has one or more improved or enhanced enzymatic properties, selected from the groups consisting of: Km and Kcat, for the unnatural amino acid as compared to a natural amino acid.
- 7. A polypeptide comprising an amino acid sequence encoded by a coding polynucleotide sequence, the coding polynucleotide sequence selected from the group consisting of:
a) a coding polynucleotide sequence selected from SEQ ID NO:4-34; b) a coding polynucleotide sequence that encodes a polypeptide selected from SEQ ID NO:35-66; c) a polynucleotide sequence which hybridizes under highly stringent conditions over substantially an entire length of a polynucleotide sequence of (a) or (b); and, d) a complementary sequence of (a), (b), or (c).
- 8. The polypeptide of claim 7, wherein the encoded polypeptide encodes an orthogonal aminoacyl tRNA sythetase.
- 9. A polypeptide comprising an amino acid sequence selected from SEQ ID NO:35-66.
- 10. A nucleic acid comprising: a polynucleotide sequence selected from the group consisting of:
a) a polynucleotide sequence selected from SEQ ID NO:1 to SEQ ID NO:3, or a complementary polynucleotide sequence thereof; and, b) a polynucleotide sequence which hybridizes under highly stringent conditions over substantially the entire length of polynucleotide sequence (a).
- 11. The nucleic acid of claim 10, wherein the polynucleotide sequence comprises an orthogonal tRNA.
- 12. The nucleic acid of claim 10, wherein the polynucleotide sequence forms a complementary pair with an orthogonal aminoacyl-tRNA synthetase.
- 13. The nucleic acid of claim 12, wherein the polypeptide sequence of the orthogonal aminoacyl-tRNA synthetase is selected from the group consisting of SEQ ID:35 to SEQ ID NO:66.
- 14. A composition comprising an orthogonal tRNA (O-tRNA), wherein the O-tRNA recognizes a selector codon and wherein the O-tRNA is preferentially aminoacylated with an unnatural amino acid by an orthogonal aminoacyl-tRNA synthetase.
- 15. The composition of claim 14, wherein the O-tRNA comprises a nucleic acid comprising a polynucleotide sequence selected from the group consisting of: SEQ ID NO: 1-3 and a complementary polynucleotide sequence thereof.
- 16. The composition of claim 14, wherein the selector codon comprises a unique three base codon, a nonsense codon, a rare codon, an unnatural codon, or at least a four base codon.
- 17. The composition of claim 14, further comprising the orthogonal aminoacyl-tRNA synthetase (O-RS).
- 18. The composition of claim 17, wherein the O-tRNA and the O-RS are complementary.
- 19. The composition of claim 17, wherein the composition comprises a mutRNATyr-mutTyrRS pair.
- 20. The composition of claim 19, wherein the composition comprises a mutRNATyr-SS12TyrRS pair.
- 21. The composition of claim 17, wherein the composition comprises a mutRNALeu-mutLeuRS pair.
- 22. The composition of claim 17, wherein the composition comprises a mutRNAThr-mutThrRS pair.
- 23. The composition of claim 17, wherein the composition comprises a mutRNAGlu-mutGluRS pair.
- 24. The composition of claim 17, wherein the O-tRNA and the O-RS are derived by mutation of a naturally occurring tRNA and an RS from at least one organism, wherein the at least one organism is a prokaryotic organism.
- 25. The composition of claim 24, wherein the at least one organism is selected from the group consisting of: Methanococcus jannaschii, Methanobacterium thennoautotrophicum, and a Halobacterium.
- 26. The composition of claim 17, wherein the O-tRNA and the O-RS are derived by mutation of a naturally occurring tRNA and RS from at least one organism, wherein the at least one organism is a eukaryotic organism.
- 27. The composition of claim 26, wherein the at least one organism is selected from the group consisting of: yeasts, mammals, fungi, insects, plants and protists.
- 28. The composition of claim 17, wherein the O-tRNA is derived by mutation of a naturally occurring tRNA from a first organism and the O-RS is derived by mutation of a naturally occurring RS from a second organism.
- 29. The composition of claim 17, wherein the O-tRNA and the O-RS are isolated from at least one organism, wherein the at least one organism is a prokaryotic organism.
- 30. The composition of claim 29, wherein the at least one organism is selected from the group consisting of: Methanococcus jannaschii, Methanobacterium thermoautotrophicum, and a Halobacterium.
- 31. The composition of claim 17, wherein the O-tRNA and the O-RS are isolated from at least one organism, wherein the at least one organism is a eukaryotic organism.
- 32. The composition of claim 31, wherein the at least one organism is selected from the group consisting of: yeasts, mammals, fungi, insects, plants and protists.
- 33. The composition of claim 17, wherein the O-tRNA is isolated from a first organism and the O-RS is isolated from a second organism.
- 34. The composition of claim 17, wherein one or more of the O-tRNA and the O-RS is isolated from one or more library, which one or more library comprises an O-tRNA or an O-RS from one or more organism.
- 35. The composition of claim 34, wherein the one or more organism comprises a prokaryote or a eukaryote.
- 36. The composition of claim 17, wherein the composition is in a cell.
- 37. The composition of claim 17, wherein the composition comprises an in vitro translation system.
- 38. A method for producing at least one recombinant orthogonal aminoacyl-tRNA synthetase (O-RS), the method comprising:
(a) generating a library of RSs derived from at least one aminoacyl-tRNA synthetase (RS) from a first organism; (b) selecting or screening the library of RSs for members that aminoacylate an orthogonal tRNA (O-tRNA) in the presence of an unnatural amino acid and a natural amino acid, thereby providing a pool of active RSs; and, (c) selecting or screening the pool for active RSs that preferentially aminoacylate the O-tRNA in the absence of the unnatural amino acid, thereby providing the at least one recombinant O-RS; wherein the at least one recombinant O-RS preferentially aminoacylates the O-tRNA with the unnatural amino acid.
- 39. The method of claim 38, wherein the first organism is a prokaryotic organism.
- 40. The method of claim 38, wherein the first organism is selected from the group consisting of: Methanococcus jannaschii, Methanobacterium thermoautotrophicum, and a Halobacterium.
- 41. The method of claim 38, wherein the at least one RS is an inactive RS.
- 42. The method of claim 41, where the inactive RS is generated by mutating an active RS.
- 43. The method of claim 42, wherein the inactive RS comprises at least about 1, at least about 2, at least about 3, at least about 4, about 5, at least about 6, or at least about 10 or more amino acids mutated to alanine amino acids.
- 44. The method of claim 38, wherein the library of RSs comprises a library of mutant RS.
- 45. The method of claim 44, wherein step (a) comprises random mutation.
- 46. The method of claim 38, wherein the step (b) comprises:
introducing a positive selection or screening marker and the library of RSs into a plurality of cells, wherein the positive selection or screening marker comprises at least one selector codon; growing the plurality of cells in the presence of a selection or screening agent; identifying cells that survive or show a specific screening response in the presence of the selection or screening agent by suppressing the at least one selector codon in the positive selection or screening marker, thereby providing a subset of positively selected or screened cells that contains the pool of active RSs.
- 47. The method of claim 46, wherein the selection or screening agent concentration is varied.
- 48. The method of claim 46, wherein the at least one selector codon comprises an amber codon, an ochre codon, or an opal stop codon.
- 49. The method of claim 46, wherein the positive selection marker is a chloramphenicol acetyltransferase (CAT) gene and wherein the at least one selector codon is an amber stop codon in the CAT gene.
- 50. The method of claim 46, wherein the positive selection marker is a β-lactamase gene and wherein the at least one selector codon is an amber stop codon in the β-lactamase gene.
- 51. The method of claim 46, wherein the positive screening marker comprises a fluorescent or luminescent screening marker.
- 52. The method of claim 46, wherein the positive screening marker comprises an affinity based screening marker.
- 53. The method of claim 38, wherein the step (c) comprises:
introducing a negative selection or screening marker with the pool of active RSs from step (b) into a plurality of cells of a second organism, wherein the negative selection or screening marker gene comprises at least one selector codon; identifying cells that survive or show a specific screening response in a 1st media supplemented with the unnatural amino acid and a selection or screening agent, but fail to survive or to show the specific screening response in a 2nd media not supplemented with the unnatural amino acid and the selection or screening agent, thereby providing surviving or screened cells with the at least one recombinant O-RS.
- 54. The method of claim 53, wherein concentration of the selection or screening agent is varied.
- 55. The method of claim 53, wherein the first and the second organism are different.
- 56. The method of claim 53, wherein the first and second organism comprise a prokaryote, a eukaryote, a mammal, an Escherichia coli, a fungi, a yeast, an archaebacterium, a eubacterium, a plant, an insect, or a protist.
- 57. The method of claim 53, wherein the negative selection marker comprises a chloramphenicol acetyltransferase (CAT) gene comprising at least one selector codon.
- 58. The method of claim 53, wherein the screening marker comprises a fluorescent or luminescent screening marker.
- 59. The method of claim 53, wherein the screening marker comprises an affinity based screening marker.
- 60. The method of claim 38, wherein step (c) comprises:
isolating the pool of active RSs from step (b); introducing a negative selection or screening marker, wherein the negative selection or screening marker gene comprises at least one selector codon, and the pool of active RSs into a plurality of cells of a second organism; identifying cells that survive or show a specific screening response in a 1st media not supplemented with the unnatural amino acid, but fail to survive or to show the specific screening response in a 2nd media supplemented with the unnatural amino acid, thereby providing surviving or screened cells with the at least one recombinant O-RS, wherein the at least one recombinant O-RS is specific for the unnatural amino acid.
- 61. The method of claim 60, wherein the at least one selector codon comprises two or more selector codons.
- 62. The method of claim 60, wherein the first and the second organism are different.
- 63. The method of claim 60, wherein the first and second organism comprise a prokaryote, a eukaryote, a mammal, an Escherichia coli, a fungi, a yeast, an archaebacteria, a eubacteria, a plant, an insect, or a protist.
- 64. The method of claim 60, wherein the negative selection marker comprises a ribonuclease barnase gene comprising at least one selector codon.
- 65. The method of claim 60, wherein the screening marker comprises a fluorescent or luminescent screening marker.
- 66. The method of claim 60, wherein the screening marker comprises an affinity based screening marker.
- 67. The method of claim 38, wherein step (b), (c) or both steps (b) and (c), comprise varying a selection or screening stringency.
- 68. The method of claim 38, further comprising:
(d) isolating the at least one recombinant O-RS; (e) generating a second set of O-RS derived from the at least one recombinant O-RS, wherein the second set of O-RS comprises a set of mutated O-RS; and, (f) repeating steps (b) and (c) until a mutated O-RS is obtained that comprises an ability to preferentially aminoacylate the O-tRNA.
- 69. The method of claim 68, further comprising repeating steps (d)-(f) at least about two times.
- 70. The method of claim 68, wherein step (e) comprises random mutagenesis, site-specific mutagenesis, recombination or any combination thereof.
- 71. The method of claim 38, wherein step (b), (c) or both steps (b) and (c) comprise using a reporter, wherein the reporter is detected by fluorescence-activated cell sorting (FACS) or wherein the reporter is detected by luminescence.
- 72. The method of claim 38, wherein the step (b), (c) or both steps (b) and (c) comprise using a reporter, wherein the reporter is displayed on a cell surface or on a phage display.
- 73. The method of claim 38, wherein the unnatural amino acid is selected from the group consisting of: an O-methyl-L-tyrosine, an L-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl-GlcNAcβ-serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, a p-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, a p-bromophenylalanine, a p-amino-L-phenylalanine, and an isopropyl-L-phenylalanine.
- 74. The at least one recombinant O-RS produced by the method of claim 38.
- 75. A method for producing a recombinant orthogonal tRNA (O-tRNA), the method comprising:
(a) generating a library of tRNAs derived from at least one tRNA from a first organism; (b) selecting or screening the library for tRNAs that are aminoacylated by an aminoacyl-tRNA synthetase (RS) from a second organism in the absence of a RS from the first organism, thereby providing a pool of tRNAs; and, (c) selecting or screening the pool of tRNAs for members that are aminoacylated by an introduced orthogonal RS (O-RS), thereby providing at least one recombinant O-tRNA; wherein the at least one recombinant O-tRNA recognizes at least one selector codon and is not efficiency recognized by the RS from the second organism and is preferentially aminoacylated by the O-RS.
- 76. The method of claim 75, wherein the at least one tRNA is a suppressor tRNA.
- 77. The method of claim 75, wherein the selector codon comprises a unique three base codon of natural or unnatural bases, a nonsense codon, a rare codon, an unnatural codon or at least a four base codon.
- 78. The method of claim 75, wherein the selector codon comprises an amber codon, an ochre codon, or an opal stop codon.
- 79. The method of claim 75, wherein the at least one recombinant O-tRNA possesses an improvement of orthogonality.
- 80. The method of claim 75, wherein the first organism is a prokaryotic organism.
- 81. The method of claim 80, wherein the prokaryotic organism is selected from the group consisting of: Methanococcus jannaschii, Methanobacterium thermoautotrophicum, and a Halobacterium.
- 82. The method of claim 75, wherein the first and second organism comprise a prokaryote, a eukaryote, a mammal, an Escherichia coli, a fungi, a yeast, an archaebacteria, a eubacteria, a plant, an insect, or a protist.
- 83. The method of claim 75, wherein the first and the second organism are different.
- 84. The method of claim 75, wherein the recombinant tRNA is aminoacylated by an unnatural amino acid, wherein the unnatural amino acid is biosynthesized in vivo either naturally or through genetic manipulation.
- 85. The method of claim 75, wherein the unnatural amino acid is added to a growth media of one or more of the first or second organism.
- 86. The method of claim 75, wherein the step (b) comprises:
introducing a toxic marker gene or a gene that leads to the production of a toxic or static agent or a gene essential to the organism, wherein the marker gene comprises the at least one selector codon and the library of mutant tRNAs into a plurality of cells from the second organism; and, selecting surviving cells, wherein the surviving cells contain the pool of tRNAs comprising at least one orthogonal tRNA or nonfunctional tRNA.
- 87. The method of claim 86, wherein selecting surviving cells comprises a comparison ratio cell density assay.
- 88. The method of claim 86, wherein the selector codon comprises two or more selector codons.
- 89. The method of claim 86, wherein the marker gene is a ribonuclease barnase gene, wherein the ribonuclease barnase gene comprises at least one amber codon.
- 90. The method of claim 89, wherein the ribonuclease barnase gene comprises two or more amber codons.
- 91. The method of claim 75, wherein the step (c) comprises:
introducing a positive selection or screening marker gene, wherein the positive marker gene comprises a drug resistance gene or a gene essential to the organism, or a gene that leads to detoxification of a toxic agent, comprising at least one of the selector codons, the O-RS, and the pool of tRNAs into a plurality of cells from the second organism; and, identifying surviving or screened cells grown in the presence of a selection or screening agent, thereby providing a pool of cells possessing the at least one recombinant tRNA, wherein the at least recombinant tRNA is aminoacylated by the O-RS and inserts an amino acid into a translation product encoded by the positive marker gene, in response to the at least one selector codons.
- 92. The method of claim 91, wherein the selection agent is an antibiotic.
- 93. The method of claim 91, wherein concentration of the selection or screening agent is varied.
- 94. The method of claim 91, wherein the drug resistance gene is a β-lactamase gene.
- 95. The method of claim 94, wherein the β-lactamase gene comprises at least one amber stop codon.
- 96. The at least one recombinant O-tRNA produced by the method of claim 75.
- 97. A method for producing at least one specific O-tRNA/O-RS pair, the method comprising:
(a) generating a library of tRNAs derived from at least one tRNA from a first organism; (b) negatively selecting or screening the library for tRNAs that are aminoacylated by an aminoacyl-tRNA synthetase (RS) from a second organism in the absence of a RS from the first organism, thereby providing a pool of tRNAs; (c) selecting or screening the pool of tRNAs for members that are aminoacylated by an introduced orthogonal RS (O-RS), thereby providing at least one recombinant O-tRNA; wherein the at least one recombinant O-tRNA recognizes a selector codon and is not efficiency recognized by the RS from the second organism and is preferentially aminoacylated by the O-RS; (d) generating a library of RSs derived from at least one aminoacyl-tRNA synthetase (RS) from a third organism; (e) selecting or screening the library of RSs for members that preferentially aminoacylate the at least one recombinant O-tRNA in the presence of an unnatural amino acid and a natural amino acid, thereby providing a pool of active RSs; and, (f) negatively selecting or screening the pool for active RSs that preferentially aminoacylate the at least one recombinant O-tRNA in the absence of the unnatural amino acid, thereby providing the at least one specific O-tRNA/O-RS pair, wherein the at least one specific O-tRNA/O-RS pair comprises at least one recombinant O-RS that is specific for the unnatural amino acid and the at least one recombinant O-tRNA.
- 98. The specific O-tRNA/O-RS pair produced by the method of claim 97.
- 99. The method of claim 97, wherein the at least one specific O-tRNA/O-RS pair is a mutRNATyr-mutTyrRS pair.
- 100. The method of claim 97, wherein the first and the third organism are same.
- 101. The method of claim 97, wherein the first organism and the third organism are Methanococcus jannaschii.
- 102. A method for identifying an orthogonal tRNA-tRNA synthetase pair for use in an in vivo translation system of a second organism, the method comprising:
introducing a marker gene, a TRNA and an aminoacyl-tRNA synthetase (RS) isolated or derived from a first organism into a first set of cells from the second organism; introducing the marker gene and the tRNA into a duplicate cell set from the second organism; and selecting or screening for surviving cells or for cells showing a specific screening response in the first set that fail to survive or show said response in the duplicate cell set, wherein the first set and the duplicate cell set are grown in the presence of a selection or screening agent, wherein the surviving or screened cells comprise the orthogonal tRNA-tRNA synthetase pair for use in the in the in vivo translation system of the second organism.
- 103. The method of claim 102, wherein the comparing and selecting or screening comprises an in vivo complementation assay.
- 104. The method of claim 102, wherein concentration of the selection or screening agent is varied.
- 105. The method of claim 102, wherein the first organism is a prokaryotic organism.
- 106. The method of claim 102, wherein the second organism is a prokaryotic organism.
- 107. The method of claim 102, wherein the first and second organism are different.
- 108. The method of claim 102, wherein the first organism is selected from the group consisting of: Methanococcus jannaschii, Methanobacterium thermoautotrophicum, and a Halobacterium.
- 109. The method of claim 102, wherein the second organism is Escherichia coli.
- 110. The method of claim 38, 75, 97, or 102, wherein the selecting or screening comprises one or more positive or negative selection or screening chosen from the groups consisting of: a change in amino acid permeability, a change in translation efficiency, and a change in translational fidelity, and wherein the one or more change is based upon a mutation in one or more gene in an organism in which an orthogonal TRNA-tRNA synthetase pair are used to produce protein.
- 111. The method of claim 38, 75, 97, or 102, wherein the selecting or screening comprises selecting or screening at least 2 selector codons within one or more selection gene or within one or more screening gene.
- 112. The method of claim 111, wherein the at least 2 selector codons are in the same selection gene or the same screening gene.
- 113. The method of claim 111, wherein the at least 2 selector codons are in different selection or screening genes.
- 114. The method of claim 111, wherein the at least 2 selector codons comprise different selector codons.
- 115. The method of claim 111, wherein the at least 2 selector codons comprise the same selector codons.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional patent application Serial No. 60/285,030, filed Apr. 19, 2001, and U.S. patent application Ser. No. 60/355,514, filed Feb. 6, 2002, the specifications of which are incorporated herein in their entirety.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
[0002] The invention was made with United States Government support under Grant No. 6502573 from the Office of Naval Research and Grant No. GM2159 from the National Institutes. The United States Government has certain rights in the invention.
Provisional Applications (2)
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Number |
Date |
Country |
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60285030 |
Apr 2001 |
US |
|
60355514 |
Feb 2002 |
US |