Exploration Of Cis-cleaving Natural DNAzymes

Early1980s, Cech et al. discovered RNA with catalytic activity in Tetrahymena,namely ribozymes (catalytic RNA, RNAzyme, ribozyme), ribozymes found to breakthe traditional concept enrich "nature of the enzyme is protein" and develop theconcepts and types of enzymes.1994, Breaker and Joyce obtained RNA cleavageactivity of DNA molecules in the presence of Pb2+by in vitro evolution, calledDeoxyribozyme (catalytic DNA, DNAzyme, deoxyribozyme). This proves that notonly RNA and DNA molecules carry genetic information, but may have abiocatalytic function. Up to now variety of ribozymes and DNAzymes with differenttypes of catalysis has been found. Although only four kinds of bases constitute,Breaker, have already proven "mechanism strategy if applied properly, the catalyticability of the ribozyme and DNAzyme with protease match."Since1994, for the first time Deoxyribozyme obtained in vitro evolution,DNAzyme has been identified dozen. Relative to the ribozyme, the DNAzyme has"small molecular weight, low cost synthesis, high stability, selectivity and widelycatalyze chemical reactions, the role of the program and the time easy to control,"and so on, these advantages decisions Deoxyribozyme irreplaceable and necessity ofresearch in the life sciences. However, all this Deoxyribozyme are obtained by invitro evolution. Although many scientists actively trying to search naturalDeoxyribozyme, but has not been reported.Our research group dedicated to the research and exploration of naturalDeoxyribozyme,in early research work we have confirmed trans-DNAzyme, thereis a single chain of natural DNAzyme sequence, and the species is widely distributed,from lower to higher prokaryotes plants, animals and even humans. This study isbased on previous work and further expand.Classical pistol Deoxyribozyme is one of the classic DNAzyme need factor-mediated of Cu2+and ascorbic acid(Vc) to supplemented self-oxidation. Inthis paper, we use classic type pistol Deoxyribozyme motif as a reference sequence,using NCBI BLAST method to compare the GenBank database analysis, search cis,single-stranded natural Deoxyribozyme, and the resulting cis single-stranded naturalDeoxyribozyme13enzymatic properties characterized. The results showed that cis,single-stranded natural Deoxyribozyme13self-cleaving depends on the Cu2+andascorbic acid(Vc), optimal catalytic conditions: Cu2+,50μM/L, ascorbic acid(Vc),1mM/L. In the pH range6.5to8.5, the catalytic activity of natural DNAzymes13is increased as the pH increased, the optimum catalytic pH is8.0. In the reactiontemperature range of4-42℃, the catalytic activity of natural Deoxyribozyme13decreased with increasing temperature. Under optimal catalytic conditions, weobtained cis, single-stranded natural Deoxyribozyme13apparent first-order rateconstant kobsof0.029. Further, we resarch the cleavage site of cis, single-strandednatural DNAzyme13, the results show that the class of their cleavage sites aresimilar prototype pistol Deoxyribozyme pistol Deoxyribozyme prototype based onthe class analysis. In addition, we also reseach double-stranded natural DNAzymesequence, single-stranded natural cis-trans Deoxyribozyme complementarysequence and reverse strands cofactor activity and other cis-impact single-strandednatural DNAzymes were studied at this stage did not get a clear positive findings.The cis-claeving natural Deoxyribozyme found abundant the development ofnucleic acid enzyme theory, opening up a new DNAzyme enzyme source, laid thefoundation for further study of the biological effects of natural Deoxyribozyme andevolutionary significance.