| 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 stricked 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. We design and construct a circular DNAzyme randomized library which cleave mRNA coding β-lactamase , and simulate starting point of replication by inserting 40 randomized DNA sequence in which has four groups of GATC sequence. In vitro we make linear DNAzyme to link circular DNAzyme by using splint and T4 DNA ligase, and make use of denaturing 16% polyacrylamide gel to detect link result. And then transform namocircular DNAzyme into E.coli JM109 DE3 by using electrotransformation. We attempt to select a self-replicated nanocircular DNAzyme in vivo by using improved selection of colony hybridization. In selective ways we tried to use "isolated plamid way" and colony hybridization. Finally, we improve dot hybridization according as our experiment conditions to increase selective efficience. We selected about one hundred thousand clones, but hasn`t found positive clone by far. In the course of selection, we found some positive clones, but tesified negative clones at last.We will take experiments for the future from two aspects. One hand we might redesign nanocircular DNAzyme. The golden rule is also simple and applied. At the base of former design, we add a selective sign to increase selective efficiency. On other hand we also might continue to look for "High Throughput Screening". We can gain satisfied result if we joint the both.In addition we respectively design and synthesize hammerhead ribozyme and 10-23 DNAzyme, then we link linear ribozyme and linear DNAzyme respectively and turn them into circular "double catalytic domain" ribozyme and circular "double catalytic domain" DNAzyme. The key to the experiment is that linking linear ribozyme and linear DNAzyme into circular ribozyme and circular DNAzyme and isolating and purifying them. We tried to use some linking ways such as "one linking way", "two linking way" and "two linking and two isolating way". After comparing with them, we adopt "two linking and two isolating way". The advantage of this way is highly isolated purification, but its disadvantages are much losing and inconvenient operations. We quickly give up "one linking way" because of bad linking result. The "two linking way" is that processing the second linking with adding T4 DNA ligase and splint after the first linking. The linking result is between "one linking way" and "two linking and two isolating way". Finally <...
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