The Transgene Expression Regulation Of Artificial Ribozyme Riboswitch And Its Optimization

The regulation of transgene is quite significant in gene therapy. With the increasing study of RNA, more researchers chose noncoding RNA(nc RNA) as the tool to regulate transgenes. Especially, the artificial small nc RNAs with high efficiency and low immunogenicity, developing quickly as a ideal gene therapy tool. The discovery of riboswitch, an oriental type of RNA regulatory element, has turned a new page of the study of nc RNAs.Riboswitch includes two parts, aptamer and expression platform. The change of the second structure in aptamer caused by combining with the ligand will lead to the change in the platform, and then influence the expression of the target gene. The ON type riboswitch will lead to the increase of the transgene, and the OFF type one will lead to the dicrese. The artificial riboswitch is designed according to the natural fragement.Ribozyme riboswitch uses ribozyme as the expression platform. Ribozyme can catalyze the clipping of itself, the expression of the target gene can be regulated by this clipping or not. The mechanism is comparatively simply.The HTRS-1(Hammerhead Theophylline Riboswitch-1)ribozyme riboswitch, used in this paper, belongs to ON type riboswitches, constituted by theophylline aptamer and satellite tobacco ringspot virus hammerhead ribozyme, and constructed by rational design. This riboswitch can be transported by recombinated plasmid vector or recombinant adeno-associated virus(r AAV) into mammal cell.The results show that the HTRS-1 ribozyme riboswitch can regulate the expression of kinds of transgene in different mammal cells. The range of regulation is diverse, however. The regulation is dispose to the concentration of ligand, and the most fitable point is 1000μM. The regulation fold of one riboswitch is different from 1.7 to about 3.7 folds according to different target genes. And the different vector can causing various results.Some paper reported that the regulation of the ribozyme riboswitch has dynamic lacks, such as the high basic expression and the small regulation range(regulation fold). We reformed the structure of the riboswitch including the mutating of the start codon in the catalyzing centre hoop, inserting the riboswitches into two sites in the UTRs, or making two or more cascading riboswitches. Also, all of the reforms can change the regulation capability, no matter higher or lower, even turned to be OFF type. The riboswitch with the most vasted regulation is not the tandem one, but the type inserted in two sites. And the regulation fold can be 5. The results of this paper may offer some experimental basis for the optimization of artificial ribozyme riboswitch, and for the application of them in vivo or in clinic.