title: Characterization of deoxyribozymes that synthesize branched RNA.
abstract: 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 Mn(2+) than with Mg(2+). The K(d,app)(Mg(2+)) > 400 mM but K(d,app)(Mn(2+)) approximately 20-50 mM, and the ligation rates k(obs) are orders of magnitude faster with Mn(2+) than with Mg(2+) (e.g., 9F7 approximately 0.3 min(-1) with 20 mM Mn(2+) versus 0.4 h(-1) with 100 mM Mg(2+), both at pH 7.5 and 37 degrees C). Of the other tested transition metal ions Zn(2+), Ni(2+), Co(2+), and Cd(2+), only Co(2+) 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.
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
authors.forename: Yangming
authors.initials: Y
authors.lastname: Wang
authors.forename: Scott K
authors.initials: SK
authors.lastname: Silverman
mesh_terms: D001665:Binding Sites; D002413:Cations, Divalent; D021881:DNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000737:chemistry; D036103:Nanotechnology; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000737:chemistry; D012313:RNA / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D011130:RNA Ligase (ATP) / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity; D013698:Templates, Genetic
chemical_list: D002413:Cations, Divalent; D021881:DNA, Catalytic; D008670:Metals; D009711:Nucleotides; D012313:RNA; D011130:RNA Ligase (ATP)