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 Ca(2+)-dependent deoxyribozyme (unmodified), d(GCCTGGCAG(1)G(2)C(3)T(4)A(5)C(6)A(7)A(8)C(9)G(10)A(11)GTCCCT), with cleavage activity for its RNA substrate, r(AGGGACA downward arrow UGCCAGGC) ( downward arrow denotes the RNA cleavage site), in the presence of Ca(2+) 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, dm(5)C (5-methyl-2'-deoxycytosine), or d(P)C (5-propynyl-2'-deoxycytosine). The K(m) values (Michaelis constants) with the mutants that lacked N7 and O6 of G(1) and O6 of G(2) 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 G(10) 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 Ca(2+). On the basis of these results, a preliminary model for two G(1) x A(8) and G(2) x A(7) mismatched base pairs such as G(anti) x 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 C(9) substituted with d(P)C, suggests that the base stacking interaction between the substituted propynyl group in dC and the nearest-neighbor base grew stronger. Thus, substituting d(P)C for dC in the catalytic loop would be one of the best ways to increase the catalytic activity of the deoxyribozyme.
Okumoto
Yasuhide
Y
Department of Chemistry, Faculty of Science and Engineering, Konan University, 8-9-1 Okamoto, Higashinada-ku, Kobe 658-8501, Japan.
Tanabe
Yoshiatsu
Y
Sugimoto
Naoki
N
D020137:Base Pair Mismatch / Q000235:genetics; D002118:Calcium / Q000737:chemistry*; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D002413:Cations, Divalent; D021881:DNA, Catalytic / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics; D003839:Deoxyadenosines / Q000138:chemical synthesis / Q000235:genetics; D003841:Deoxycytidine / Q000138:chemical synthesis / Q000235:genetics; D003849:Deoxyguanosine / Q000138:chemical synthesis / Q000235:genetics; D006151:Guanosine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007288:Inosine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000235:genetics; D009154:Mutation; D009692:Nucleic Acid Heteroduplexes / Q000138:chemical synthesis / Q000235:genetics; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000235:genetics; D016337:RNA, Catalytic / Q000138:chemical synthesis / Q000737:chemistry*
D002413:Cations, Divalent; D021881:DNA, Catalytic; D003839:Deoxyadenosines; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides; D016337:RNA, Catalytic; D003841:Deoxycytidine; D006151:Guanosine; D007288:Inosine; C068218:7-deazaguanosine; D003849:Deoxyguanosine; C012271:deoxyinosine; D002118:Calcium