Orthogonal suppressor tRNAs and aminoacyl-tRNA synthetases and uses thereof

• US 20060134748A1
• Filed: 11/21/2005
• Published: 06/22/2006
• Est. Priority Date: 11/20/2004
• Status: Abandoned Application

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: RajBhandary, Uttam, Koehrer, Caroline
• Application Number:
  US11/284,259
• Publication Number:
  US 20060134748A1
• Time in Patent Office: Days
• US Class Current: 435/69.100
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression

Abstract

The present invention provides novel orthogonal suppressor tRNAs, aminoacyl-tRNA synthetases, and suppressor tRNA/aminoacyl-tRNA synthetase pairs. In preferred embodiments the suppressor tRNAs function in mammalian cells and in certain embodiments they are not aminoacylated by any of the mammalian cytoplasmic aminoacyl-tRNA synthetases. The invention provides a novel ochre suppressor tRNA that fulfills these criteria. The invention further provides novel amber and opal suppressor tRNAs having a range of different translation efficiencies. The invention also provides mammalian cells containing the suppressor tRNAs, aminoacyl-tRNA synthetases, or both. The suppressor tRNAs may be introduced into the cell from the exterior or may be expressed by the cell. They may be aminoacylated with either a natural or an unnatural amino acid. The suppressor tRNAs, aminoacyl-tRNA synthetases, or both, may be expressed by a mammalian cell in a regulated manner. The invention further provides methods for synthesizing proteins using the inventive suppressor tRNAs and aminoacyl-tRNA synthetases and proteins produced by the methods.

Claims

1. An ochre suppressor tRNA that is orthogonal to a mammalian cell.

2. The ochre suppressor of claim 1, wherein the ochre suppressor tRNA has a translation efficiency of between approximately 0.03 and approximately 4.5% when present in a mammalian cell that contains an aminoacyl-tRNA synthetase that aminoacylates the ochre suppressor tRNA.

3. The ochre suppressor of claim 1, wherein the ochre suppressor tRNA has a translation efficiency of approximately 4.5% when present in a mammalian cell that contains an aminoacyl-tRNA synthetase that aminoacylates the ochre suppressor tRNA.

4. The ochre suppressor of claim 1, wherein the ochre suppressor tRNA has a translation efficiency of at least 4.5% when present in a mammalian cell that contains an aminoacyl-tRNA synthetase that aminoacylates the ochre suppressor tRNA.

5. The ochre suppressor of claim 1, wherein the ochre suppressor is not a substrate for any native aminoacyl-tRNA synthetase in the cell.

6. The ochre suppressor tRNA of claim 1, wherein the ochre suppressor tRNA is aminoacylated with a natural or unnatural amino acid.

7. An isolated mammalian cell containing the ochre suppressor tRNA of claim 1.

8. The mammalian cell of claim 7, wherein the suppressor tRNA was not synthesized by the cell.

9. The mammalian cell of claim 7, wherein the suppressor tRNA is expressed by the cell.

10. The mammalian cell of claim 7, wherein the activity or expression of the suppressor tRNA is regulatable.

11. The mammalian cell of claim 7, wherein the ochre suppressor tRNA is not a substrate for any native aminoacyl-tRNA synthetase in the cell.

12. The mammalian cell of claim 7, further comprising an aminoacyl-tRNA synthetase that aminoacylates the ochre suppressor tRNA.

13. The mammalian cell of claim 12, wherein the aminoacyl-tRNA synthetase is orthogonal to the cell.

14. The mammalian cell of claim 12, wherein the cell expresses the aminoacyl-tRNA synthetase.

15. The mammalian cell of claim 12, wherein the cell expresses the aminoacyl-tRNA synthetase in a regulatable manner.

16. The mammalian cell of claim 12, wherein the cell expresses both the ochre suppressor tRNA and the aminoacyl-tRNA synthetase.

17. The mammalian cell of claim 12, wherein the cell further comprises a heterologous polynucleotide that comprises an open reading frame containing an ochre codon.

18. The mammalian cell of claim 17, wherein the open reading frame contains an amber codon, an opal codon, or both, and the cell further comprises an orthogonal amber suppressor tRNA-aaRS pair, an orthogonal opal suppressor tRNA-aaRS pair, or both.

19. The mammalian cell of claim 7, further comprising an amber suppressor tRNA that is orthogonal to the cell.

20. The mammalian cell of claim 19, wherein the amber suppressor tRNA has a translation efficiency of between approximately 2.8% and approximately 34% when present in a mammalian cell that contains an aminoacyl-tRNA synthetase that aminoacylates the amber suppressor tRNA.

21. The mammalian cell of claim 19, wherein the amber suppressor tRNA is not a substrate for any native aminoacyl-tRNA synthetase in the cell.

22. The mammalian cell of claim 19, further comprising an aminoacyl-tRNA synthetase that aminoacylates the ochre suppressor tRNA, the amber suppressor tRNA, or both.

23. The mammalian cell of claim 7, further comprising an opal suppressor tRNA that is orthogonal to the cell.

24. The mammalian cell of claim 23, wherein the opal suppressor is not a substrate for any native aminoacyl-tRNA synthetase in the cell.

25. The mammalian cell of claim 23, wherein the opal suppressor has a translation efficiency of between approximately 0.05% and 10% when present in a mammalian cell that contains an aminoacyl-tRNA synthetase that aminoacylates the opal suppressor tRNA.

26. The mammalian cell of claim 23, further comprising an aminoacyl-tRNA synthetase that aminoacylates the ochre suppressor tRNA, the opal suppressor tRNA, or both.

27. The mammalian cell of claim 7, further comprising an amber suppressor tRNA that is orthogonal to the cell and an opal suppressor tRNA that is orthogonal to the cell.

28. The mammalian cell of claim 27, wherein the amber suppressor tRNA, the opal suppressor tRNA, the ochre suppressor tRNA, or any combination thereof, is not a substrate for any native aminoacyl-tRNA synthetase in the cell.

29. The mammalian cell of claim 27, further comprising an aminoacyl-tRNA synthetase that aminoacylates the ochre suppressor tRNA, the amber suppressor tRNA, the opal suppressor tRNA, or any combination thereof.

30. The mammalian cell of claim 27, further comprising at least two different aminoacyl-tRNA synthetases, each of which aminoacylates at least one of the suppressor tRNAs.

31. The mammalian cell of claim 7, wherein the cell comprises (i) a heterologous polynucleotide that comprises an open reading frame containing an ochre codon and (ii) an aaRS that aminoacylates the ochre suppressor tRNA.

32. A polynucleotide that comprises a template for transcription of the ochre suppressor tRNA of claim 1, operably linked to a promoter sequence.

33. A mammalian cell comprising the polynucleotide of claim 32.

34. The mammalian cell of claim 33, further comprising a polynucleotide that comprises a template for transcription of an aaRS that aminoacylates the ochre suppressor tRNA.

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88. A method for synthesizing a protein in a mammalian cell by translation of genes containing at least one stop codon within the open reading frame, the method comprising steps of:

(a) providing an isolated mammalian cell containing;

(i) at least one gene that includes at least one ochre codon within the open reading frame;

(ii) an ochre suppressor tRNA that is orthogonal to the cell; and

(iii) an aminoacyl-tRNA synthetase (aaRS) that aminoacylates the ochre suppressor tRNA; and

(b) maintaining the cell for a period of time under conditions in which protein synthesis can occur.

89. The method of claim 88, wherein the step of providing the mammalian cell comprises the step of:

contacting a mammalian cell with an ochre suppressor tRNA that was not synthesized within the cell, so that the tRNA is taken up into the cell at a level sufficient to allow readthrough of the ochre codon in the cell.

90. The method of claim 88, wherein the ochre suppressor tRNA is aminoacylated with an unnatural amino acid.

91. The method of claim 88, wherein the step of providing the mammalian cell comprises the step of:

introducing a polynucleotide that contains a template for transcription of the ochre suppressor tRNA into the cell, so that the cell expresses the tRNA.

92. The method of claim 88, wherein the step of providing the mammalian cell comprises the step of:

introducing a polynucleotide that contains a template for transcription of the aminoacyl-tRNA synthetase into the cell, so that the cell expresses the aminoacyl-tRNA synthetase.

93. The method of claim 88, wherein the ochre suppressor tRNA is not a substrate for any native aminoacyl-tRNA synthetase in the cell.

94. The method of claim 88, wherein the gene further contains an amber or opal stop codon, or both, in the open reading frame, and wherein the cell further comprises an orthogonal amber suppressor tRNA, an orthogonal opal suppressor tRNA, or both.

95. The method of claim 88, wherein the cell is contacted with an unnatural amino acid that is taken up by the cell and used by the aaRS to aminoacylate the ochre suppressor tRNA, so that the unnatural amino acid is inserted into a growing amino acid chain at a position defined by the ochre codon in the open reading frame.

96. The method of claim 88, wherein (i) the at least one gene includes three different stop codons within the open reading frame;

(ii) the cell comprises three suppressor tRNAs, wherein the suppressor tRNAs read through three different stop codons; and

(iii) the cell comprises a set of one or more aminoacyl-tRNA synthetases, wherein aminoacyl-tRNA synthetases in the set of aminoacyl-tRNA synthetases aminoacylate the suppressor tRNAs.

97. A method for synthesizing a protein in a mammalian cell by translation of genes containing at least one stop codon within the open reading frame, the method comprising steps of:

(a) providing an isolated mammalian cell containing;

(i) at least one gene that includes at least one ochre, amber, or opal codon within the open reading frame;

(ii) one or more suppressor tRNAs selected from the group consisting of;

an ochre suppressor tRNA that is orthogonal to the cell, an amber suppressor tRNA having a translation efficiency of between approximately 2.8% and approximately 34% when present in a mammalian cell that contains an aminoacyl-tRNA synthetase that aminoacylates the amber suppressor tRNA, and an opal suppressor tRNA having a translation efficiency of between approximately 0.05% and approximately 10% when present in a mammalian cell that contains an aminoacyl-tRNA synthetase that aminoacylates the opal suppressor tRNA; and

(iii) one or more aminoacyl-tRNA synthetases (aaRSs) each of which aminoacylates at least one of the suppressor tRNAs; and

(b) maintaining the cell for a period of time under conditions in which protein synthesis can occur.

98. The method of claim 97, wherein the step of providing the mammalian cell comprises the step of:

contacting a mammalian cell with a suppressor tRNA that was not synthesized within the cell, so that the tRNA is taken up into the cell at a level sufficient to allow readthrough of a stop codon in the cell.

99. The method of claim 97, wherein the suppressor tRNA is aminoacylated with an unnatural amino acid.

100. The method of claim 97, wherein the step of providing the mammalian cell comprises the step of:

introducing a polynucleotide that contains a template for transcription of the suppressor tRNA into the cell, so that the cell expresses the tRNA.

101. The method of claim 97, wherein the step of providing the mammalian cell comprises the step of:

introducing a polynucleotide that contains a template for transcription of the aminoacyl-tRNA synthetase into the cell, so that the cell expresses the aminoacyl-tRNA synthetase.

102. The method of claim 97, wherein the suppressor tRNA is not a substrate for any native aminoacyl-tRNA synthetase in the cell.

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119. The kit of claim 117, wherein the kit comprises an ochre suppressor tRNA that is orthogonal to a mammalian cell, or a polynucleotide or expression vector that comprises a template for synthesis of the tRNA, or both.

120. The kit of claim 119, wherein the kit further comprises one or more items selected from the group consisting of:

(i) an amber suppressor tRNA that is orthogonal to a mammalian cell, or a polynucleotide or expression vector that comprises a template for synthesis of such an amber suppressor tRNA, or both;

(ii) an opal suppressor tRNA that is orthogonal to a mammalian cell, or a polynucleotide or expression vector that comprises a template for synthesis of such an opal suppressor tRNA, or both.

121. The kit of claim 117, wherein the kit comprises (i) an ochre suppressor tRNA that is orthogonal to a mammalian cell, or a polynucleotide or expression vector that comprises a template for synthesis of the tRNA, or both;

(ii) an amber suppressor tRNA that is orthogonal to a mammalian cell, or a polynucleotide or expression vector that comprises a template for synthesis of such an amber suppressor tRNA, or both; and

(iii) an opal suppressor tRNA that is orthogonal to a mammalian cell, or a polynucleotide or expression vector that comprises a template for synthesis of such an opal suppressor tRNA, or both;

122. The kit of claim 117, further comprising at least one item selected from the group consisting of:

(i) an aminoacyl-tRNA synthetases that aminoacylates a suppressor tRNA that is orthogonal to a mammalian cell, a polynucleotide or expression vector that comprises a template for synthesis of such an aminoacyl-tRNA synthetase, or both;

(ii) a mammalian cell;

(iii) one or more unnatural amino acids;

(iv) an agent that induces or represses transcription;

(v) a transfection reagent such as a lipid;

(vi) an in vitro translation system;

(vii) a reporter system;

(viii) a buffer;

(ix) tissue culture medium; and

(x) instructions for use.

123. The kit of claim 117, wherein one or more of the suppressor tRNAs is not a substrate for any native aminoacyl-tRNA in the cell.

124. The kit of claim 117, wherein the kit comprises at least one collection of suppressor tRNAs selected from the group consisting of:

a collection of ochre suppressor tRNAs that are orthogonal to a mammalian cell, wherein members of the collection suppress ochre codons in a mammalian cell with a range of different efficiencies;

a collection of amber suppressor tRNAs that are orthogonal to a mammalian cell, wherein members of the collection suppress amber codons in a mammalian cell with a range of different efficiencies; and

a collection of opal suppressor tRNAs that are orthogonal to a mammalian cell, wherein members of the collection suppress opal codons in a mammalian cell with a range of different efficiencies.

118. A kit comprising at least one suppressor tRNA that is orthogonal to a mammalian cell, or a polynucleotide or expression vector that comprises a template for synthesis of the suppressor tRNA, or both, wherein the suppressor tRNA is selected from the group consisting of:

an ochre suppressor tRNA, an amber suppressor tRNA having a translation efficiency of between approximately 2.8% and approximately 34% when present in a mammalian cell that contains an aminoacyl-tRNA synthetase that aminoacylates the amber suppressor tRNA, and an opal suppressor tRNA having a translation efficiency of between approximately 0.05% and approximately 10% when present in a mammalian cell that contains an aminoacyl-tRNA synthetase that aminoacylates the opal suppressor tRNA.

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