Abstract：
Synthetic riboswitches are promising regulatory devices due to their small size, lack of immunogenicity, and ability to fine-tune gene expression in the absence of exogenous trans-acting factors. Based on a gene inhibitory system developed at our lab, termed U1snRNP interference (U1i), we developed tetracycline (TC)-inducible riboswitches that modulate mRNA polyadenylation through selective U1 snRNP recruitment. First, we engineered different TC-U1i riboswitches, which repress gene expression unless TC is added, leading to inductions of gene expression of 3-to-4-fold. Second, we developed a technique called Systematic Evolution of Riboswitches by Exponential Enrichment (SEREX), to isolate riboswitches with enhanced U1 snRNP binding capacity and activity, achieving inducibilities of up to 8-fold. Interestingly, by multiplexing riboswitches we increased inductions up to 37-fold. Finally, we demonstrated that U1i-based riboswitches are dose-dependent and reversible and can regulate the expression of reporter and endogenous genes in culture cells and mouse models, resulting in attractive systems for gene therapy applications. Our work probes SEREX as a much-needed technology for the in vitro identification of riboswitches capable of regulating gene expression in vivo.
摘要：
合成核糖开关由于其体积小，是有前途的调控设备，缺乏免疫原性，以及在不存在外源反式作用因子的情况下微调基因表达的能力。基于我们实验室开发的基因抑制系统，称为U1snRNP干扰(U1i)，我们开发了四环素（TC）诱导型核糖开关，通过选择性U1snRNP募集调节mRNA多聚腺苷酸化。首先,我们设计了不同的TC-U1i核糖开关，除非添加TC，否则会抑制基因表达，导致3至4倍的基因表达诱导。第二,我们开发了一种称为指数富集的核糖开关系统演化(SEREX)的技术，为了隔离具有增强的U1snRNP结合能力和活性的核糖开关，实现高达8倍的诱惑力。有趣的是,通过多路复用核糖开关，我们将诱导增加了37倍。最后,我们证明了基于U1i的核糖开关是剂量依赖性和可逆的，可以调节培养细胞和小鼠模型中报告基因和内源基因的表达，导致基因治疗应用的有吸引力的系统。我们的工作探讨了SEREX作为一种急需的技术，用于体外鉴定能够在体内调节基因表达的核糖开关。
