The Mammalian Gene Regulation Through Ligand-induced Artificial Ribozyme Switches

Numerous no-coding RNA-based control devices that integrate sensing and gene-regulatory function have been developed in synthetic biology.Riboswitches are natural RNA-based genetic switches that sense small-molecule metabolites and can regulate genes expression at the level of transcription and translation without any protein factors.It can be widely found in various organisms.Based on the principles of riboswitch regulation,a versatile set of synthetic riboswitches named aptazymes,have been engineered by combing aptamers domain with natural ribozymes.These ligand-dependent self-cleaving riboswitches can be customized for induction by various small molecules and have been applied for the triggering of mRNA self-cleavage in variety of organisms including mammalian cells,and can be applied in conditional gene regulation system.In recent years,riboswitches were also used for new applications of therapeutic approaches to address human diseases,but further study is needed to translate these RNA-based regulatory systems to functional gene control for clinical applications.HSV-TK/GCV system and RNA interference are two effective ways for the gene therapy.In this study,we described a theophylline-responsive ribozyme-based device provided a powerful platform for HSV-TK gene cis-regulation in tumor cells.Results indicated that ribozyme switch enables a dynamic and reversible control over gene expression and lower toxic regulatory system retains functionality over long time period when being stably integrated.We further developed a system that activates RNA interference in response to small molecule by integrating a theophylline aptamer-fused HDV ribozyme with a pri-miRNA analogue,which can be induced into self-cleaving and release siRNA of Bcl-2 gene in dose dependent fashion.The system allowed us to have a temporal or effective modulation of RNA interference,which provided the theory basis and method for the practical applications of gene therapy.Additionally,the thiamin pyrophosphate(TPP)responsive fluorescence sensor ribozyme switches were developed in the mammallian cells this research.By tuning TPP concentration changes into the visual EGFP expression in vivo,the ribozyme switches enables an efficient development of label-free,noninvasive and high-specific biosensors in living mammalian cells.