Substrate specificity and kinetic framework of a DNAzyme with an expanded chemical repertoire: a putative RNaseA mimic that catalyzes RNA hydrolysis independent of a divalent metal cation.
This work addresses the binding, cleavage and dissociation rates for the substrate and products of a synthetic RNaseA mimic that was combinatorially selected using chemically modified nucleoside triphosphates. This trans-cleaving DNAzyme, 9(25)-11t, catalyzes sequence-specific ribophosphodiester hydrolysis in the total absence of a divalent metal cation, and in low ionic strength at pH 7.5 and in the presence of EDTA. It is the first such sequence capable of multiple turnover. 9(25)-11t consists of 31 bases, 18 of which form a catalytic domain containing 4 imidazole and 6 allylamino modified nucleotides. This sequence cleaves the 15 nt long substrate, S1, at one embedded ribocytosine at the eighth position to give a 5'-product terminating in a 2',3'-phosphodiester and a 3'-product terminating in a 5'-OH. Under single turnover conditions at 24 degrees C, 9(25)-11t displays a maximum first-order rate constant, k(cat), of 0.037 min(-1) and a catalytic efficiency, k(cat)/K(m), of 5.3 x 10(5) M(-1) min(-1). The measured value of k(cat) under catalyst excess conditions agrees with the value of k(cat) observed for steady-state multiple turnover, implying that slow product release is not rate limiting with respect to multiple turnover. The substrate specificity of 9(25)-11t was gauged in terms of k(cat) values for substrate sequence variants. Base substitutions on the scissile ribose and at the two bases immediately downstream decrease k(cat) values by a factor of 4 to 250, indicating that 9(25)-11t displays significant sequence specificity despite the lack of an apparent Watson-Crick base-pairing scheme for recognition.
Ting
Richard
R
Chemistry Department, 2036 Main Mall, University of British Columbia, Vancouver, BC V6T 1Z1, Canada.
Thomas
Jason M
JM
Lermer
Leonard
L
Perrin
David M
DM
D001483:Base Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D021881:DNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D004722:Endoribonucleases / Q000737:chemistry* / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D002413:Cations, Divalent; D021881:DNA, Catalytic; C495395:DNAzyme, 9(25)-11t; D009838:Oligodeoxyribonucleotides; D012313:RNA; D004492:Edetic Acid; D004722:Endoribonucleases; D012259:Ribonuclease, Pancreatic; D008274:Magnesium