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

描述(申请人提供)：我们建议为科学家开发一种跨分子RNA开关来测量体内miRNA的活性。虽然有许多方法可以在体外定量miRNAs，但我们还不知道有任何方法可以在体内进行分析。在活着的单个细胞中观察特定miRNA生成而不需要破坏细胞的能力将是一个重要的工具。我们正在开创一种基于RNA的开关技术，称为结构相互作用RNAs(SxRNA)，它利用转录后基因调控作为miRNA检测的报告。正常情况下，RNA结合蛋白(RBP)与3‘茎环结构结合，促进上游编码区的翻译多达一个数量级。通过控制RBP的结合，有可能通过修饰该基因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，以便除非额外的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并稳定功能结构，否则它不会形成。我们提出这一建议的目的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，我们将测量这种转换机制对翻译的影响，并确定这种转换机制在小区中是否有效。使用sxRNA作为诊断工具在产品层面上取得成功，应该会理想地将该技术定位于其他用途。只要miRNA表达水平是疾病、细胞或组织类型或状况的指标，它就可能有效。SxRNA可能是蛋白质缺乏性疾病(如囊性纤维化)基因治疗或癌症治疗的一种基于RNA的替代方法。 与公共卫生相关：虽然有许多方法可以在体外量化miRNAs，但我们还不知道有任何方法可以在体内进行分析。在活着的单个细胞中观察特定miRNA生成而不需要破坏细胞的能力将是一个重要的工具。例如，干细胞研究人员可以通过体内检测分化过程中优先表达的miRNAs来确保干细胞没有分化。

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.