The Preliminary Study Of A New Nucleic Acid Interventional Molecule In Targeting T Cell CTLA-4 Receptor

Abstract: Ribozyme and DNAzyme possess the function of hydrolyzing mRNA molecular, and they are the important tool of blocking gene expression and anti-virus. There are two key techniques in the Ribozyme and DNAzyme study. One problem is the effect of Ribozyme and DNAzyme, their effection are concerned with the senior structure of mRNA, ie be concerning with the availability of the target site. The other is the delivery efficiency of Ribozyme and DNAzyme, high-efficient delivery is the precondition of playing their roles. Gene therapy requires safety, efficient and targeted molecular carrier. pRNA isolated from Bacillus subtilis is a small RNA molecules of about 30nm size(Fig. 1), it easily passes through the cell membranes. Then it possesses both the structural characteristics of nucleic acids and similar proteins functions, so it can be designed as an ideal non-viral vectors. pRNA molecule carrier have the safe and efficient features. It can act on the effector cells for a longer time, at the same time it cann't cause immune response and exclusion phenomenon, so it is the very promising ideal molecular carrier in the gene therapy[1]. In this study, we restart the T cell activation and proliferation through CTLA4 intervention pRNA-Ribozyme molecular; in order to seek a new immunosuppressive program for radioactive immune cells. Methods: Firstly amplification and cloning of the CTLA4 gene, transcription of RNA in vitro, we screened the availability targets of CTLA4-mRNA by using RASS method and to verify its effectiveness then confirm Ribozyme'targets. So we design and construct the DNA plasmid of CTLA4-pRNA-Ribozyme according to the targets, then construct and prepare the CTLA4-pRNA-Ribozyme molecules. We analyzed their cleavage activity in vitro, then compared and analysed their advantages and disadvantages. pRNA carries the effect molecule Ribozyme to make Ribozyme to possess a specific target activity, binding and cutting initiatively pRNA-Ribozyme of genes RNA. The RNA molecule which base sequences are more than the 100nt is synthesized by chemical methods difficultly, so we recombine and prepare 170nt pRNA-Ribozyme through of the molecular biology methods. We prepare DNA plasmid in accordance with the pRNA-Ribozyme linking to the cis-connected open architecture and Ribozyme embedded into pRNA to the closed inner structure through using PCR amplification and cloning of reorganization, according to pRNA sequence and Ribozyme molecular sequences; then transcribe and prepare pRNA- Ribozyme molecules in vitro. Results: We prepared the pRNA-Ribozyme molecules and the formation of polymer structure automatically in vitro through two ways. pRNA-Ribozyme restructured and prepared can be transported accurately to the targets through the pRNA vector, that is it can entrance initiatively into the cell, recognize actively, cut mRNA actively and block CTLA-4 gene's expression, consequently the Ribozyme function is bringed into play. We prepared the specific and efficient CTLA4-pRNA-Ribozyme in vitro. Conclusions: The pRNA-Ribozyme(cis-connected open architecture and Ribozyme embedded into pRNA to a closed inner structure of pRNA-Ribozyme) is prepared through the biosynthesis in vitro. And the inhibition effect is obvious that interfere CTLA-4 gene expression in vitro. Preparation and biosynthesis of DNA plasmid of CTLA4-pRNA-Ribozyme in vitro provides a intracellular expression basis.