Abstract:

The 8–17 deoxyribozyme is a small RNA-cleaving DNA enzyme of significant applicative interest. We measured the kinetics of over 60 variants of 8–17, mutated within the "core" region. The data were analyzed according to a conceptual framework in which deleterious substitutions can either decrease the stability of the reaction's transition state, or favor unreactive ground-state conformations. In agreement with earlier in vitro evolution studies, the most severe functional effects were observed upon mutating four conserved residues, whose role was further explored by replacing them with non-standard nucleotides. Removal or modification of individual functional groups on the A6 and G7 bases suggested that these residues are involved in a close-contact interaction and form a network of functionally important hydrogen bonds. Mutagenesis of residues C13 and G14 was less revealing, but argued strongly against a role of C13 as a general acid/base catalyst. The use of non-standard nucleotides also led to the identification of one deoxyribozyme variant that, under some ionic conditions, is substantially more active than the wild-type construct. Finally, the effects of mutations in the intramolecular "core stem" correlated only in part with changes in helical stability, suggesting that a stable stem is required but not sufficient for optimal activity.