| Ribozyme (RNAzyme) is a kind of single-stranded RNA molecules with catalytic activity. It can specifically cleave RNA molecules. By the beginning of the 1980s, the American scientists, Cech and Altman respectively found that RNA has biocatalysis function. Their discovery has striked traditional idea of lasted-semicentennial that all enzymes are protein, arosing a revolution of enzyme. Because of this discovery, Cech and Altman gained the Nobel Prize. In all discovered ribozymes, the hammerhead ribozyme is the best representative, because it has many excellences including simple structure, small molecule, being apt to design, synthesize and transfer, further more it doesn`t like hairpin ribozyme that has specifical to its substrate. The ideal ribozyme should satisfy with requires of highly catalytic efficiency, well specificity and stabilization. By the 1990s, with the development of molecular biology and molecular evolution in vitro, Breaker and his colleagues firstly obtained DNA molecule with cleaving RNA activity depending on Pb2+ ion, and denominated DNAzyme. In nature, DNA molecules almost exist in double-stranded mode, but in especial condition, single-stranded DNA maybe appear similar structure like synthesized DNAzyme in theory. Up to now,the natural DNAzyme hasn`t been found. The reported DNAzymes at present are obtained by an in vitro selection strategy. Because DNAzyme has many predominances including relatively small molecule, structural stabilization, highly catalytic efficiency, being apt to synthesize and modify etc. It has represented a potentially useful way in gene therapy, disease diagnose and molecular biology research etc. It is possible that we design a RNA-DNAzyme gene with double-activities of deoxyribozyme and ribozyme. Then in the process of replication and transcription , RNA-DNAzyme would show the activity of deoxyribozyme and ribozyme respectively. We designed and constructed a RNA-DNAzyme gene (Rz-Dz), which cleaves mRNA coding β-lactamase. Rz-Dz gene was ligated into the pBluescript II KS(+) phagemid at the multiple clone sites BamH I and Hind III. The ligation products were then transformed into bacterial host XL1-Blue MRF'by using electrotransformation. Then a single strand DNA molecule (dRz-Dz)with the deoxyribozyme sequence was secreted when Rz-Dz recombinant was replicated in the presence of VCSM 13 helper phage. The cleavage activity of dRz-Dz was detected by mixing dRz-Dz with 32P-labeled RNA substrate. The result was separated by 16% denaturing polyacrylamide gel, visualized by autoradiograph and quantified. In vitro experiment approved that dRz-Dz has obvious activity of cleaving RNA substrate. The catalytic activity of dRz-Dz is less than the activity of 10-23 deoxyribozyme. Kinetics measured in 10mmol/L MgCl2, at 37℃, with a RNA substrate, yielded a KM value of ~35.8nmol/L, kcat value of ~0.42min-1 and Kobs value of ~1.18×107 mol-1·L·min-1 respectively. The activity depended on the present of divalent metal ions which was the same as other deoxyriboszymes. dRz-Dz showed highest activity in Mn2+. Pb2+, Ca2+ and Mg2+ take second place, Zn2+,Co2+and Ba2+ almost have no support to the activity. The activity of deoxyribozyme can be regulated by concentrary of metal ions. We compared the cleavage efficience of dRz-Dz in different concentrate of magnesium at 37℃for 30 minutes. The result showed that enzyme activity was obviously influenced by Mg2+ concentrate. The linearized recombinant Rz-Dz phagemid was used as templates for in vitro transcription with MAXIscript T3 Kit. The transcript of 175 nt (rRz-Dz)was the sequence with the designed ribozyme . We mixed the transcript with the 32P-labeled RNA substrate to measured the cleavage activity. The denaturing 16% PAGE was used to isolate the substrates and the products. The result confirmed that rRz-Dz can cleave the RNA substrate. Kinetics were measured in 100mmol/L MgCl2, 50mmol/L (pH7.5) Tris-HCl, at 37℃.The KM value was ~2801nmol/L, kcat value was ~0.12min-1 and Kobs value was ~0.43×... |
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