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

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 similar to 50- or similar to 1000-fold lower rate (Bronsted beta(LG) = -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 alpha-phosphate or the original a-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 alpha-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.