We recently reported deoxyribozymes (DNA enzymes) that synthesize 2′,5′-branched RNA. The in vitro-selected 9F7 and 9F21 deoxyribozymes mediate reaction of a branch-site adenosine 2′-hydroxyl on one RNA substrate with the 5′-triphosphate of another RNA substrate. Here we characterize these DNA enzymes with respect to their branch-forming activity. Both 9F7 and 9F21 are much more active with Mn2+ than with Mg 2+. The Kd,app(Mg2+) > 400 mM but K d,app(Mn2+) ≈ 20-50 mM, and the ligation rates k obs are orders of magnitude faster with Mn2+ than with Mg2+ (e.g., 9F7 ∼ 0.3 min-1 with 20 mM Mn 2+ versus 0.4 h-1 with 100 mM Mg2+, both at pH 7.5 and 37 °C). Of the other tested transition metal ions Zn2+, Ni2+, Co2+, and Cd2+, only Co2+ supports a trace amount of activity. 9F7 is more tolerant than 9F21 of varying the RNA substrate sequences. For the RNA substrate that donates the adenosine 2′-hydroxyl, 9F7 requires YUA, where Y = pyrimidine and A is the branch site. The 3′-tail emerging from the branch-site A may have indefinite length, but it must be at least one nucleotide long for high activity. The 5′-triphosphate RNA substrate requires several additional nucleotides with varying sequence requirements (5′-pppGRMWR). Outside of these regions that flank the ligation site, 9F7 and 9F21 tolerate any RNA substrate sequences via Watson-Crick covariation of the DNA binding arms that interact directly with the substrates. 9F7 provides a high yield of 2′,5′ -branched RNA on the preparative nanomole scale. The ligation reaction is effectively irreversible; the pyrophosphate leaving group in the ligation reaction does not induce 2′,5′-cleavage, and pyrophosphate does not significantly inhibit ligation except in 1000-fold excess. Deleting a specific nucleotide in one of the DNA binding arms near the ligation junction enhances ligation activity, suggesting an interesting structure near this region of the deoxyribozyme-substrate complex. These data support the utility of deoxyribozymes in creating synthetic 2′,5′-branched RNAs for investigations of group II intron splicing, debranching enzyme (Dbr) activity, and other biochemical reactions.
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