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Kinetic characterization of a cis- and trans-acting M2+-independent DNAzyme that depends on synthetic RNaseA-like functionality - Burst-phase kinetics from the coalescence of two active DNAzyme folds

TitleKinetic characterization of a cis- and trans-acting M2+-independent DNAzyme that depends on synthetic RNaseA-like functionality - Burst-phase kinetics from the coalescence of two active DNAzyme folds
Publication TypeJournal Article
Year of Publication2007
AuthorsTing, R, Thomas, JM, Perrin, DM
JournalCanadian Journal of Chemistry-Revue Canadienne De Chimie
Volume85
Pagination313-329
Date PublishedApr
ISBN Number0008-4042
Abstract

This work describes the kinetics of the DNAzyme 9(25)-11, a combinatorially selected, M(2+-)independent ribophosphodiesterase that is covalently modified with both cationic amines and imidazoles. At 13 degrees C, cis- and trans-cleaving constructs of 9(25)-11 demonstrate the highest rate constants reported to date for any M(2+-)independent nucleic acid catalyst, investigated at physiological ionic strength and pH 7.5 (0.3 min(-1) for self cleavage and 0.2 min(-1) for intermolecular cleavage). In contrast to the cis-cleaving species, single-turnover experiments with the trans-cleaving species exhibit biphasic cleavage data, suggesting the presence of two conformations of the catalyst-substrate complex. Pulse-chase experiments demonstrate that both complexes lead to substrate cleavage. Under multiple-turnover conditions, the higher rate constant appears in a burst phase that decays to a slower steady state exhibiting a rate constant of 0.0077 min(-1), a value approximating that of the slow-cleaving phase seen in single-turnover experiments. Slow product release is excluded as the source of the burst phase. An integrated rate equation is derived to describe burst-phase kinetics based on the funneling of the initial population of fast-cleaving conformation into a steady-state population composed largely of the slow-cleaving conformation.

URL<Go to ISI>://000246576200010