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Evolution of a T7 RNA polymerase variant that transcribes 2′-O-methyl RNA

Nature Biotechnology volume 22, pages 1155–1160 (2004)Cite this article

Abstract

Modified RNA and DNA molecules have novel properties that their natural counterparts do not possess, such as better resistance to degradation in cells and improved pharmacokinetic behavior1,2. In particular, modifications at the 2′-OH of ribose are important for enhancing the stability of RNA3,4. Unfortunately, it is difficult to enzymatically synthesize modified nucleic acids of any substantial length because natural polymerases incorporate modified nucleotides inefficiently. Previously, we reported an activity-based method for selecting functional T7 RNA polymerase variants based on the ability of a T7 RNA polymerase to reproduce itself5. Here, we have modified the original procedure to identify polymerases that can efficiently incorporate multiple modified nucleotides at the 2′ position of the ribose. Most important, our method allows the selection of polymerases that have good processivities and can be combined to simultaneously incorporate several different modified nucleotides in a transcript.

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Figure 1: Autogene selection for active T7 RNA polymerase variants.
Figure 2: Polymerase activities with multiple 2′-O-methyl modifications and synthesis of long and complex modified transcripts using the selected polymerases.
Figure 3: Assays demonstrating the fidelity of the mutant enzyme, 'RGVG', E593G, V685A and the resistance of the 2′-O-methyl-modified RNA to cleavage by nucleases.

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References

  1. Opalinska, J.B. & Gewirtz, A.M. Nucleic-acid therapeutics: basic principles and recent applications. Nat. Rev. Drug Discov. 1, 503–514 (2002).

    Article  CAS  Google Scholar 

  2. Sullenger, B.A. & Gilboa, E. Emerging clinical applications of RNA. Nature 418, 252–258 (2002).

    Article  CAS  Google Scholar 

  3. Pieken, W.A., Olsen, D.B., Benseler, F., Aurup, H. & Eckstein, F. Kinetic characterization of ribonuclease-resistant 2′-modified hammerhead ribozymes. Science 253, 314–317 (1991).

    Article  CAS  Google Scholar 

  4. Kurreck, J. Antisense technologies Improvement through novel chemical modifications. Eur. J. Biochem. 270, 1628–1644 (2003).

    Article  CAS  Google Scholar 

  5. Chelliserrykattil, J., Cai, G. & Ellington, A.D. A combined in vitro/in vivo selection for polymerases with novel promoter specificities. BMC Biotechnol. 1, 13 (2001).

    Article  CAS  Google Scholar 

  6. Patel, P.H. & Loeb, L.A. DNA polymerase active site is highly mutable: evolutionary consequences. Proc. Natl. Acad. Sci. USA 97, 5095–5100 (2000).

    Article  CAS  Google Scholar 

  7. Ghadessy, F.J., Ong, J.L. & Holliger, P. Directed evolution of polymerase function by compartmentalized self-replication. Proc. Natl. Acad. Sci. USA 98, 4552–4557 (2001).

    Article  CAS  Google Scholar 

  8. Patel, P.H. & Loeb, L.A. Multiple amino acid substitutions allow DNA polymerases to synthesize RNA. J. Biol. Chem. 275, 40266–40272 (2000).

    Article  CAS  Google Scholar 

  9. Xia, G. et al. Directed evolution of novel polymerase activities: mutation of a DNA polymerase into an efficient RNA polymerase. Proc. Natl. Acad. Sci. USA 99, 6597–6602 (2002).

    Article  CAS  Google Scholar 

  10. Fa, M., Radeghieri, A., Henry, A.A. & Romesberg, F.E. Expanding the substrate repertoire of a DNA polymerase by directed evolution. J. Am. Chem. Soc. 126, 1748–1754 (2004).

    Article  CAS  Google Scholar 

  11. Roychowdhury, A., Illangkoon, H., Hendrickson, C.L. & Benner, S.A. 2′-deoxycytidines carrying amino and thiol functionality: synthesis and incorporation by vent (exo(-)) polymerase. Org. Lett. 6, 489–492 (2004).

    Article  CAS  Google Scholar 

  12. Hutter, D. & Benner, S.A. Expanding the genetic alphabet: non-epimerizing nucleoside with the pyDDA hydrogen-bonding pattern. J. Org. Chem. 68, 9839–9842 (2003).

    Article  CAS  Google Scholar 

  13. Mitsui, T., Kimoto, M., Sato, A., Yokoyama, S. & Hirao, I. An unnatural hydrophobic base, 4-propynylpyrrole-2-carbaldehyde, as an efficient pairing partner of 9-methylimidazo[(4,5)-b]pyridine. Bioorg. Med. Chem. Lett. 13, 4515–4518 (2003).

    Article  CAS  Google Scholar 

  14. Switzer, C.Y., Moroney, S.E. & Benner, S.A. Enzymatic recognition of the base pair between isocytidine and isoguanosine. Biochemistry 32, 10489–10496 (1993).

    Article  CAS  Google Scholar 

  15. Hirao, I., Mitsui, T., Kimoto, M., Harada, Y. & Yokoyama, S. An unnatural base pair for efficient incorporation of nucleotide analogs into RNAs. Nucleic Acids Res. Suppl. 3, 215–216 (2003).

    Article  CAS  Google Scholar 

  16. Sismour, A.M. et al. PCR amplification of DNA containing non-standard base pairs by variants of reverse transcriptase from Human Immunodeficiency Virus-1. Nucleic Acids Res. 32, 728–735 (2004).

    Article  CAS  Google Scholar 

  17. Padilla, R. & Sousa, R. A Y639F/H784A T7 RNA polymerase double mutant displays superior properties for synthesizing RNAs with non-canonical NTPs. Nucleic Acids Res. 30, e138 (2002).

    Article  Google Scholar 

  18. Padilla, R. & Sousa, R. Efficient synthesis of nucleic acids heavily modified with non-canonical ribose 2′-groups using a mutant T7 RNA polymerase (RNAP). Nucleic Acids Res. 27, 1561–1563 (1999).

    Article  CAS  Google Scholar 

  19. Huang, Y., Eckstein, F., Padilla, R. & Sousa, R. Mechanism of ribose 2′-group discrimination by an RNA polymerase. Biochemistry 36, 8231–8242 (1997).

    Article  CAS  Google Scholar 

  20. Brieba, L.G. & Sousa, R. Roles of histidine 784 and tyrosine 639 in ribose discrimination by T7 RNA polymerase. Biochemistry 39, 919–923 (2000).

    Article  CAS  Google Scholar 

  21. Yin, Y.W. & Steitz, T.A. Structural basis for the transition from initiation to elongation transcription in T7 RNA polymerase. Science 298, 1387–1395 (2002).

    Article  CAS  Google Scholar 

  22. Temiakov, D. et al. Structural basis for substrate selection by T7 RNA polymerase. Cell 116, 381–391 (2004).

    Article  CAS  Google Scholar 

  23. Sousa, R. & Padilla, R. A mutant T7 RNA polymerase as a DNA polymerase. EMBO J. 14, 4609–4621 (1995).

    Article  CAS  Google Scholar 

  24. Braasch, D.A. et al. RNA interference in mammalian cells by chemically-modified RNA. Biochemistry 42, 7967–7975 (2003).

    Article  CAS  Google Scholar 

  25. Rong, M., Durbin, R.K. & McAllister, W.T. Template strand switching by T7 RNA polymerase. J. Biol. Chem. 273, 10253–10260 (1998).

    Article  CAS  Google Scholar 

  26. delCardayre, S.B. & Raines, R.T. Structural determinants of enzymatic processivity. Biochemistry 33, 6031–6037 (1994).

    Article  CAS  Google Scholar 

  27. Jansen, B. & Zangemeister-Wittke, U. Antisense therapy for cancer–the time of truth. Lancet Oncol. 3, 672–683 (2002).

    Article  CAS  Google Scholar 

  28. Majlessi, M., Nelson, N.C. & Becker, M.M. Advantages of 2′-O-methyl oligoribonucleotide probes for detecting RNA targets. Nucleic Acids Res. 26, 2224–2229 (1998).

    Article  CAS  Google Scholar 

  29. Lesnik, E.A. & Freier, S.M. What affects the effect of 2′-alkoxy modifications? 1. Stabilization effect of 2′-methoxy substitutions in uniformly modified DNA oligonucleotides. Biochemistry 37, 6991–6997 (1998).

    Article  CAS  Google Scholar 

  30. Chelliserrykattil, J. & Ellington, A.D. in Methods Mol Biol, Directed enzyme evolution vol. 230 (eds. Arnold, F.H. & Georgiou, G.) 27–43, (Humana Press, Totowa, New Jersey, 2003).

    Book  Google Scholar 

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Acknowledgements

This work was supported by a grant from the Office of Naval Research and by the Robert A. Welch Foundation.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, 1 University Station A4800, University of Texas at Austin, Austin, 78712-0159, Texas, USA

    Jijumon Chelliserrykattil & Andrew D Ellington

  2. Institute for Cellular and Molecular Biology, 1 University Station A4800, University of Texas at Austin, Austin, 78712-0159, Texas, USA

    Andrew D Ellington

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  1. Jijumon Chelliserrykattil
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Correspondence to Andrew D Ellington.

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Chelliserrykattil, J., Ellington, A. Evolution of a T7 RNA polymerase variant that transcribes 2′-O-methyl RNA. Nat Biotechnol 22, 1155–1160 (2004). https://doi.org/10.1038/nbt1001

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