Mimicking the first step of RNA splicing: An artificial DNA enzyme can synthesize branched RNA using an oligonucleotide leaving group as a 5′-exon analogue

Rebecca L. Coppins, Scott K. Silverman

Research output: Contribution to journalArticle

Abstract

The 7S11 deoxyribozyme synthesizes 2′,5′-branched RNA by mediating the nucleophilic attack of an internal 2′-hydroxyl group of one RNA substrate into the 5′-triphosphate of a second RNA substrate, with pyrophosphate as the leaving group. Here we comprehensively examined the role of the leaving group in the 7S11-catalyzed reaction by altering the 5′-phosphorylation state and the length of the second RNA substrate. When the leaving group is the less stabilized phosphate or hydroxide anion as provided by a 5′-diphosphate or 5′-monophosphate, the same 2′,5′-branched product is formed as when pyrophosphate is the leaving group, but with an ∼50- or ∼1000-fold lower rate (Brønsted βLG = -0.40). When the 5′-end of the RNA substrate that bears the leaving group is longer by one or more nucleotides, either the new 5′-terminal α-phosphate or the original α-phosphate can be attacked by the branch-site 2′-hydroxyl group; in the latter case, the leaving group is an oligonucleotide. The choice between these α-phosphate reaction sites is determined by the subtle balance between the length of the single-stranded 5′-extension and the stability of the leaving group. Because the branch-site adenosine is a bulged nucleotide flanked by Watson-Crick duplex regions, we earlier concluded that 7S11 structurally mimics the first step of natural RNA splicing. The observation of 7S11-catalyzed branch formation with an oligonucleotide leaving group strengthens this resemblance to natural RNA splicing, with the oligonucleotide playing the role of the 5′-exon in the first step. These findings reinforce the notion that splicing-related catalysis can be achieved by artificial nucleic acid enzymes that are much smaller than the spliceosome and group II introns.

Original languageEnglish (US)
Pages (from-to)13439-13446
Number of pages8
JournalBiochemistry
Volume44
Issue number41
DOIs
StatePublished - Oct 18 2005

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ASJC Scopus subject areas

  • Biochemistry

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