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An alcohol dehydrogenase ribozyme

Abstract

We report an RNA molecule that exhibits activity analogous to that of alcohol dehydrogenase (ADH). Directed in vitro evolution was used to enrich nicotinamide adenine dinucleotide (NAD+)–dependent redox-active RNAs from a combinatorial pool. The most active ribozyme in the population forms a compact pseudoknotted structure and oxidizes an alcohol seven orders of magnitude faster than the estimated spontaneous rate. Moreover, this ADH RNA was coupled with a redox relay between NADH and flavin adenine dinucleotide to give a NAD+-regeneration system. Our demonstration of the redox ability of RNA adds support to an RNA-based metabolic system in ancient life.

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Figure 1: In vitro selection of ADH ribozymes.
Figure 2: Product analyses of the benzaldehyde generated by ribox02.
Figure 3: The proposed secondary structure of 1-ribox02.
Figure 4: A multicomponent redox system involving 1-ribox02, NAD+/NADH and FAD/FADH2.

References

  1. Berg, J.M., Tymoczko, J.L. & Stryer, L. Biochemistry (Freeman, New York, 2002).

    Google Scholar 

  2. Gray, H.B. & Ellis, J.W.R. Electron transfer. In Bioinorganic Chemistry (eds. Bertini, I., Gray, H.B., Lippard, S.J. & Valentine, J.S.) 315–363 (University Science Books, Sausalito, California, USA, 1994).

    Google Scholar 

  3. Gilbert, W. The RNA world. Nature 319, 618 (1986).

    Article  Google Scholar 

  4. White, H.B., III. Coenzymes as fossils of an earlier metabolic state. J. Mol. Evol. 7, 101–104 (1976).

    Article  CAS  Google Scholar 

  5. Joyce, G.F. & Orgel, L.E. Prospects for understanding the origin of the RNA world. In The RNA World (eds. Gesteland, R.F., Cech, T.R. & Atkins, J.F.) 49–77 (Cold Spring Harbor Laboratory Press, New York, 1999).

    Google Scholar 

  6. Benner, S.A., Ellington, A.D. & Tauer, A. Modern metabolism as a palimpsest of the RNA world. Proc. Natl. Acad. Sci. USA 86, 7054–7058 (1989).

    Article  CAS  Google Scholar 

  7. Bertini, I. & Luchinat, C. The reaction pathways of zinc enzymes and related biological catalysts. In Bioinorganic Chemistry (eds. Bertini, I., Gray, H.B., Lippard, S.J. & Valentine, J.S.) 37–106 (University Science Books, Sausalito, California, USA, 1994).

    Google Scholar 

  8. Konarska, M.M., Grabowski, P.J., Padgett, R.A. & Sharp, P.A. Characterization of the branch site in lariat RNAs produced by splicing of mRNA precursors. Nature 313, 552–557 (1985).

    Article  CAS  Google Scholar 

  9. Burgin, A.B. & Pace, N.R. Mapping the active site of ribonuclease P RNA using a substrate containing a photoaffinity agent. EMBO J. 9, 4111–4118 (1990).

    Article  CAS  Google Scholar 

  10. Nakamura, M., Toda, M., Saito, H. & Ohkura, Y. Fluorimetric determination of aromatic aldehydes with DDB. Anal. Chim. Acta. 134, 39–45 (1982).

    Article  CAS  Google Scholar 

  11. Blankenhorn, G. Intermolecular complexes between N-methyl-1,4-dihydronicotinamide and flavines. The influence of steric and electronic factors on complex formation and the rate of flavine-dependent dihydronicotinamide dehydrogenation. Biochemistry 14, 3172–3176 (1975).

    Article  CAS  Google Scholar 

  12. Kimura, E., Shionoya, M., Hoshino, A., Ikeda, T. & Yamada, Y. A model for catalytically active zinc(II) ion in liver alcohol dehydrogenase: a novel “hydride transfer” reaction catalyzed by zinc(II)-macrocyclic polyamine complexes. J. Am. Chem. Soc. 114, 10134–10137 (1992).

    Article  CAS  Google Scholar 

  13. Shinkai, S., Era, H., Tsuno, T. & Manabe, O. Oxidation of alcohols via alkoxymagnesium halides by an NAD+ model compound. Bull. Chem. Soc. Jpn. 57, 1435–1436 (1984).

    Article  CAS  Google Scholar 

  14. Mathews, D.H., Sabina, J., Zuker, M. & Turner, D.H. Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J. Mol. Biol. 288, 911–940 (1999).

    Article  CAS  Google Scholar 

  15. Sassanfar, M. & Szostak, J.W. An RNA motif that binds ATP. Nature 364, 550–553 (1993).

    Article  CAS  Google Scholar 

  16. Jiang, F., Kumar, R.A., Jones, R.A. & Patel, D.J. Structural basis of RNA folding and recognition in an AMP-RNA aptamer complex. Nature 382, 183–186 (1996).

    Article  CAS  Google Scholar 

  17. Feigon, J., Dieckmann, T. & Smith, F.W. Aptamer structures from A to zeta. Chem. Biol. 3, 611–617 (1996).

    Article  CAS  Google Scholar 

  18. Shinkai, S., Yamada, S. & Kunitake, T. Coenzyme models. 10. Rapid oxidation of NADH by a flavin immobilized in cationic polyelectrolytes. Macromolecules 11, 65–68 (1978).

    Article  CAS  Google Scholar 

  19. Hamachi, I. & Kobuke, Y. Facilitated electron transfer from NADH to flavolipid bound in ammonium bilayer. J. Chem. Soc., Chem. Commun., 130–131 (1989).

  20. Kokubo, T., Sassa, S. & Kaiser, E.T. Flavohemoglobin: a semisynthetic hydroxylase acting in the absence of reductase. J. Am. Chem. Soc. 109, 606–607 (1987).

    Article  CAS  Google Scholar 

  21. Ferre-D'Amare, A.R., Zhou, K. & Doudna, J.A. Crystal structure of a hepatitis delta virus ribozyme. Nature 395, 567–574 (1998).

    Article  CAS  Google Scholar 

  22. Hilbers, C.W., Michiels, P.J. & Heus, H.A. New developments in structure determination of pseudoknots. Biopolymers 48, 137–153 (1998).

    Article  CAS  Google Scholar 

  23. Yarus, M. Boundaries for an RNA world. Curr. Opin. Chem. Biol. 3, 260–267 (1999).

    Article  CAS  Google Scholar 

  24. Bartel, D.P. & Unrau, P.J. Constructing an RNA world. Trends Cell Biol. 9, M9–M13 (1999).

    Article  CAS  Google Scholar 

  25. Huang, F., Bugg, C.W. & Yarus, M. RNA-catalyzed CoA, NAD, and FAD synthesis from phosphopantetheine, NMN, and FMN. Biochemistry 39, 15548–15555 (2000).

    Article  CAS  Google Scholar 

  26. Breaker, R.R. & Joyce, G.F. Self-incorporation of coenzymes by ribozymes. J. Mol. Evol. 40, 551–558 (1995).

    Article  CAS  Google Scholar 

  27. Burgstaller, P. & Famulok, M. Isolation of RNA aptamers for biological cofactors by in vitro selection. Angew. Chem. Int. Ed. Engl. 33, 1084–1087 (1994).

    Article  Google Scholar 

  28. Lauhon, C.T. & Szostak, J.W. RNA aptamers that bind flavin and nicotinamide redox cofactors. J. Am. Chem. Soc. 117, 1246–1257 (1995).

    Article  CAS  Google Scholar 

  29. Roychowdhury-Saha, M., Lato, S.M., Shank, E.D. & Burke, D.H. Flavin recognition by an RNA aptamer targeted toward FAD. Biochemistry 41, 2492–2499 (2002).

    Article  CAS  Google Scholar 

  30. Cadwell, R.C. & Joyce, G.F. Mutagenic PCR. PCR Methods Appl. 3, S136–S140 (1994).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by a grant from the US National Science Foundation (H.S). S.T. acknowledges the JSPS Research Fellowships for Research Abroad. We thank all members of Suga and Bright groups, in particular D.R.W. Hodgson and H. Murakami for invaluable discussions and C.A. Munson for assistance with fluorescent measurements.

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Correspondence to Hiroaki Suga.

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Tsukiji, S., Pattnaik, S. & Suga, H. An alcohol dehydrogenase ribozyme. Nat Struct Mol Biol 10, 713–717 (2003). https://doi.org/10.1038/nsb964

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