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

Abstract

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, 925-11t, 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. 925-11t 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°C, 925-11t displays a maximum first-order rate constant, kcat, of 0.037 min-1 and a catalytic efficiency, kcat/Km, of 5.3 × 105 M-1 min-1. The measured value of kcat under catalyst excess conditions agrees with the value of kcat observed for steady-state multiple turnover, implying that slow product release is not rate limiting with respect to multiple turnover. The substrate specificity Of 925-11t was gauged in terms of kcat values for substrate sequence variants. Base substitutions on the scissile ribose and at the two bases immediately downstream decrease kcat values by a factor of 4 to 250, indicating that 925-11t displays significant sequence specificity despite the lack of an apparent Watson-Crick base-pairing scheme for recognition. © Oxford University Press 2004; all rights reserved.