Factors that Contribute to Efficient Catalytic Activity of a Small Ca²⁺-Dependent Deoxyribozyme in Relation to Its RNA Cleavage Function^†

Recently, we found a small Ca²⁺-dependent deoxyribozyme (unmodified), d(GCCTGGCAG₁G₂C₃T₄A₅C₆A₇A₈C₉G₁₀A₁₁GTCCCT), with cleavage activity for its RNA substrate, r(AGGGACA↓UGCCAGGC) (↓ denotes the RNA cleavage site), in the presence of Ca²⁺ and developed a functional SPR sensor chip with this deoxyribozyme [Okumoto, Y., Ohmichi, T., and Sugimoto, N. (2002) Biochemistry 41, 2769−2773]. In the study presented here, to clarify the factors contributing to the efficient catalytic activity of the unmodified deoxyribozyme, RNA cleavage reactions were carried out using 24 mutant deoxyribozymes containing one unnatural DNA nucleotide, such as dI (2‘-deoxyinosine), 7-deaza-dG, 2-aminopurine, 7-deaza-dA, 2-amino-dA, dm⁵C (5-methyl-2‘-deoxycytosine), or d^PC (5-propynyl-2‘-deoxycytosine). The K_m values (Michaelis constants) with the mutants that lacked N7 and O6 of G₁ and O6 of G₂ were 4.5 and 6.6 times that of the unmodified one, respectively. The k_cat value (cleavage rate constant) with the mutants that lacked O6 of G₁₀ was 0.025 times that of the unmodified one. The results of UV melting curves, SPR kinetics, and CD spectra supported the quantitative idea that the catalytic activity of the unmodified form was achieved using Ca²⁺. On the basis of these results, a preliminary model for two G₁·A₈ and G₂·A₇ mismatched base pairs such as G(anti)·A(anti) formed in the catalytic loop is proposed. The factor of 10 increase in the k_cat/K_m value of the mutant deoxyribozyme, which has C₉ substituted with d^PC, suggests that the base stacking interaction between the substituted propynyl group in dC and the nearest-neighbor base grew stronger. Thus, substituting d^PC for dC in the catalytic loop would be one of the best ways to increase the catalytic activity of the deoxyribozyme.