Characterization of Deoxyribozymes That Synthesize Branched RNA

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 K_d,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.