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，供科学家测量 miRNA 体内活性。虽然有多种体外定量 miRNA 的方法，但我们不知道有任何方法可以进行体内分析。无需破坏细胞即可观察活单细胞中特定 miRNA 生成的能力将是一个重要的工具。我们正在开创一种基于 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，RNA 结合蛋白 (RBP) 与 3' 茎环结构结合，以促进上游编码区的翻译达一个数量级。可以通过控制 RBP 的结合来修饰 mRNA 以调节报告基因的翻译。这是通过改变天然的茎环结构来实现的，除非额外的RNA（例如miRNA）通过与定制设计的茎环的侧翼区域碱基配对来结合并稳定功能结构，否则它不会形成。我们提出该提案的目标是首先评估体外开关机制并测量可能的相互作用和信号强度的特异性。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。使用 sxRNA 作为诊断工具在产品层面取得成功，应该可以理想地将该技术用于其他用途。它在 miRNA 表达水平作为疾病、细胞或组织类型或状况指标的任何地方都可能有效。 sxRNA 可能是一种基于 RNA 的替代基因疗法，用于治疗囊性纤维化等蛋白质缺乏疾病或癌症疗法。 公共健康相关性：虽然有多种体外定量 miRNA 的方法，但我们不知道有任何方法可以进行体内分析。无需破坏细胞即可观察活单细胞中特定 miRNA 生成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 miRNA 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.