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Nucleic Acids Research 2004 32(22):6660-6672; doi:10.1093/nar/gkh1007
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Published online 29 December 2004

Nucleic Acids Research, Vol. 32 No. 22 © Oxford University Press 2004; all rights reserved

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

Richard Ting, Jason M. Thomas, Leonard Lermer and David M. Perrin*

Chemistry Department, 2036 Main Mall, University of British Columbia, Vancouver, BC V6T 1Z1, Canada

* To whom correspondence should be addressed. Tel: +1 604 822 0567; Email: dperrin{at}chem.ubc.ca

Received October 9, 2004; Revised and Accepted November 24, 2004

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 x 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.


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