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

This work describes the kinetics of the DNAzyme 925-11, a combinatorially selected, M2+-independent ribophosphodiesterase that is covalently modified with both cationic amines and imidazoles. At 13 °C, cis- and trans-cleaving constructs of 925-11 demonstrate the highest rate constants reported to date for any M2+-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.Key words: RNase mimics, DNAzymes, ribozymes, kinetics, RNA cleavage.