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RNA-catalysed carbon–carbon bond formation

Nature volume 389, pages 54–57 (1997)Cite this article

The ‘RNA world’ hypothesis1,2,3, which assumes that the chemical processes that led to the appearance of life were carried out by RNA molecules, has stimulated interest in catalytic reactions involving oligonucleotides such as catalytic RNA (ribozymes)4. Naturally occurring ribozymes have, for example, been shown to efficiently catalyse the formation and cleavage of nucleic-acid phosphodiester bonds4,5,6,7,8, and this narrow range of RNA-catalysed reactions has been subsequently expanded by in vitro selection methods to include ester9 and amide10,24 bond formation SN2 reactions and porphyrin metallations11,12. Carbon–carbon bond formation and the creation of asymmetric centres are both of great importance biochemically, but have not yet been accomplished by RNA catalysis. A widely used reaction that creates two new carbon–carbon bonds and up to four stereo-centres is the Diels–Alder cycloaddition, which occurs between a 1,3-butadiene and an alkene. Here we report the successful application of in vitro selection to isolate pyridine-modified RNA molecules that catalyse a Diels–Alder cycloaddition. We find that the RNA molecules accelerate the reaction rate by a factor of up to 800 relative to the uncatalysed reaction.

Rounds of in vitro selection for DAase activity were conducted by incubating the RNA–diene construct with the maleimide dienophile (compound 1 in Fig. 1; BMCC) in the presence of transition metals that could form pyridyl–Lewis acid complexes and/or coordinate to the RNA to enhance tertiary structure. The maleimide was attached to biotin so that RNA DAases could be partitioned away from unreacted RNA using streptavidin binding and denaturing polyacrylamide electrophoresis.

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Figure 1: The Diels–Alder reaction between the acyclic diene conjugated to the RNA through a long PEG linker and the maleimide dienophile 1 (BMCC).
Figure 2: a, Random region (∼100N) sequences of 11 isolates obtained from the DAase in vitro selection.
Figure 3: The observed rate constant (kobs) increases as a function of BMCC concentration for isolate 22.
Figure 4: Inhibition of the DAase activity of isolate 22 by the free cycloaddition product 3 with an apparent Ki shown.

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Acknowledgements

We thank L. Gold for support, guidance and vision; the scientific community at NeXstar, especially members of the Medicinal Chemistry group, for helpful discussions and ideas; S. Wayland for the synthesis of compound 2; and T. Wiegand and D. Nieuwlandt for inspiring dialogue and technical assistance. We also thank S. C. Pomerantz, P. F. Crain and J. A. McCloskey for ESI–MS/MS analysis.

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  1. NeXstar Pharmaceuticals, Inc., 2860 Wilderness Place, Boulder, 80301, Colorado, USA

    Theodore M. Tarasow, Sandra L. Tarasow & Bruce E. Eaton

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Tarasow, T., Tarasow, S. & Eaton, B. RNA-catalysed carbon–carbon bond formation. Nature 389, 54–57 (1997). https://doi.org/10.1038/37950

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