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 dPC (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 dPC, suggests that the base stacking interaction between the substituted propynyl group in dC and the nearest-neighbor base grew stronger. Thus, substituting dPC for dC in the catalytic loop would be one of the best ways to increase the catalytic activity of the deoxyribozyme.