Quantitative analysis of a RNA-cleaving DNA catalyst obtained via in vitro selection.

Abstract

In vitro selections performed in the presence of Mg(2+) generated DNA sequences capable of cleaving an internal ribonucleoside linkage. Several of these, surprisingly, displayed intermolecular catalysis and catalysis independent of Mg(2+), features that the selection protocol was not explicitly designed to select. A detailed physical organic analysis was applied to one of these DNAzymes, termed 614. First, the progress curve for the reaction was dissected to identify factors that prevented the molecule from displaying clean first-order transformation kinetics and 100% conversion. Several factors were identified and quantitated, including (a) competitive intra- and intermolecular rate processes, (b) alternative reactive and unreactive conformations, and (c) mutations within the catalyst. Other factors were excluded, including "approach to equilibrium" kinetics and product inhibition. The possibility of complementary strand inhibition was demonstrated but was shown to not be a factor under the conditions of these experiments. The rates of the intra- and intermolecular processes were compared, and saturation models for the intermolecular process were built. The rate-limiting step for the intermolecular reaction was found to be the association/folding of the enzyme with the substrate and not the cleavage step. The DNAzyme 614 is more active in trans than in cis and more active at temperatures below the selection temperature than at the selection temperature. Many of these properties have not been reported in similar systems; these results therefore expand the phenomenology known for this class of DNA-based catalysts. A brief survey of other catalysts arising from this selection found other Mg(2+)-independent DNAzymes and provided a preliminary view of the ruggedness of the landscape, relating function to structure in sequence space. Hypotheses are suggested to account for the fact that a selection in the presence of Mg(2+) did not exploit this Mg(2+). This study of a specific catalytically active DNAzyme is an example of studies that will be necessary generally to permit in vitro selection to help us understand the distribution of function in sequence space.