In-vivo miRNA Detection Using Structurally Interacting RNA (sxRNA)

描述（由申请人提供）：我们建议开发一种跨分子rna开关，供科学家在体内测量miRNA活性。虽然有许多方法可以在体外量化mirna，但我们不知道有任何方法可以进行体内分析。在不破坏细胞的情况下观察活单细胞中特定miRNA生成的能力将是一个重要的工具。我们正在开拓一种基于rna的开关技术，称为结构相互作用rna (sxRNA)，它利用转录后基因调控作为miRNA检测的报告基因。通常情况下，rna结合蛋白（RBP）与一个3'茎环结构相关联，以促进上游编码区翻译多达一个数量级。有可能通过调控RBP的结合来修饰mRNA来调节报告基因的翻译。这是通过改变自然的茎环结构来实现的，这样它就不会形成，除非有一个额外的RNA，如miRNA，通过与定制设计的茎环的侧翼区域进行碱基配对来结合和稳定功能结构。我们的目标是首先评估体外开关机制，并测量相互作用的特异性和可能的信号强度。其次，我们将测量对翻译的影响，并确定这种转换机制在细胞中是否有效。如果在产品层面成功地将sxRNA用作诊断工具，该技术就有可能被用于其他用途。在miRNA表达水平是疾病、细胞或组织类型或状况的指标的任何地方，它都可能有效。sxRNA可能是一种基于rna的替代基因疗法，用于治疗蛋白质缺乏性疾病，如囊性纤维化，或癌症治疗。

DESCRIPTION (provided by applicant): We propose developing a trans-molecular RNA-switch for scientists to measure miRNA activity in-vivo. While there are numerous methods to quantify miRNAs in vitro, we are not aware of any method that allows for in vivo analysis. The ability to observe specific miRNA generation in living single cells without requiring the destruction of the cell would be a significant tool. We are pioneering an RNA-based switch technology called structurally interacting RNAs (sxRNA) which utilizes post-transcriptional gene regulation as a reporter for miRNA detection. Normally, RNA-binding proteins (RBP) associate with a 3' stem-loop structure to facilitate translation of an upstream coding region by as much as an order of magnitude. It is possible to modify the mRNA to modulate translation of the reporter gene by controlling the binding of RBP. This is accomplished by altering the natural stem-loop structure so that it does not form unless an additional RNA, such as a miRNA, binds and stabilizes the functional structure by base- pairing with the flanking regions of the custom designed stem-loop. Our goal with this proposal is first to evaluate the switch mechanism in-vitro and measure the specificity of interaction and signal strength possible. Secondly, we will measure the effect on translation and determine whether this switching mechanism is effective in a cell. Success at the product level using sxRNA as a diagnostic tool should ideally position the technology for additional uses. It could be effective anywhere that miRNA expression levels are an indicator of disease, cell or tissue type, or condition. sxRNA could be an RNA-based alternative to gene therapy for protein deficiency diseases, such as cystic fibrosis, or a cancer therapy. PUBLIC HEALTH RELEVANCE: While there are numerous methods to quantify miRNAs in vitro, we are not aware of any method that allows for in vivo analysis. The ability to observe specific miRNA generation in living single cells without requiring the destruction of the cell would be a significant tool. For example, stem cell researchers could ensure stem cells had not differentiated by in-vivo detection of miRNAs that are preferentially expressed during differentiation.