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A DNA enzyme that mimics the first step of RNA splicing

Abstract

We have discovered an artificial DNA enzyme that mimics the first step of RNA splicing. In vitro selection was used to identify DNA enzymes that ligate RNA. One of the new DNA enzymes carries out splicing-related catalysis by specifically recognizing an unpaired internal adenosine and facilitating attack of its 2′-hydroxyl onto a 5′-triphosphate. This reaction forms 2′,5′-branched RNA and is analogous to the first step of in vivo RNA splicing, in which a ribozyme cleaves itself with formation of a branched intermediate. Unlike a natural ribozyme, the new DNA enzyme has no 2′-hydroxyl groups to aid in the catalytic mechanism. Our finding has two important implications. First, branch-site adenosine reactivity seems to be mechanistically favored by nucleic acid enzymes. Second, hydroxyl groups are not obligatory components of nucleic acid enzymes that carry out biologically related catalysis.

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Figure 1: Selection and sequence of a DNA enzyme that ligates RNA.
Figure 2: Analysis of the 2′,5′-branched RNA product formed by the 7S11 DNA enzyme.
Figure 3: Analysis of the role of the left-hand DNA binding arm.
Figure 4: Interactions between the 7S11 DNA enzyme and the RNA substrates.
Figure 5: The leaving group plays little if any role in the 7S11-mediated RNA ligation reaction.
Figure 6: Close relationship between the branch-forming reaction catalyzed by 7S11 and the first step of in vivo RNA splicing.

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Acknowledgements

This research was supported by the Burroughs Wellcome Fund (New Investigator Award in the Basic Pharmacological Sciences), the March of Dimes Birth Defects Foundation (Basil O'Connor Starter Scholar Research Award), the US National Institutes of Health, the Petroleum Research Fund administered by the American Chemical Society, and the University of Illinois at Urbana-Champaign Department of Chemistry (all to S.K.S.). S.K.S. is the recipient of a fellowship from The David and Lucile Packard Foundation. We thank members of the Silverman lab for discussions, H. Imker for technical assistance with the background reaction rate experiments, Y. Wang for testing lariat RNA formation with 7S11 and P. Hergenrother and J. Cottell for comments on the manuscript.

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Correspondence to Scott K Silverman.

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Coppins, R., Silverman, S. A DNA enzyme that mimics the first step of RNA splicing. Nat Struct Mol Biol 11, 270–274 (2004). https://doi.org/10.1038/nsmb727

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