Unambiguous transcriptome-wide identification of microRNA:mRNA interactions through enhanced chimeric read amplification

项目摘要 哺乳动物细胞中的蛋白质合成不仅依赖于从DNA转录RNA， 基因组，但也对RNA的稳定性及其翻译速率。一类约21个核苷酸的RNA 被称为“微小RNA”的蛋白质合成调控中起着至关重要的作用， 蛋白质编码RNA分子中的序列，并引起RNA切割或抑制翻译。 microRNA调控在几乎所有研究的人类生理系统中起着关键作用，而错误的基因调控则是一个很大的问题。 个体microRNA或整体microRNA加工的调节与癌症、心脏病和 肾脏疾病、病毒感染反应和许多其他疾病。确定个人目标 microRNA提供了对每个microRNA的功能生物学作用的基本见解。此外，委员会认为， 作为功能性调节分子，microRNA现在被高度追求用于治疗 试剂，其中需要直接映射microRNA靶标的能力来测定适当的靶向 结合以及低脱靶相互作用。然而，目前的方法是缺乏的，因为它们 高假阳性率，缺乏将靶点分配给单个microRNA的能力，或者不能扩展到 以定量准确性分析所有（特别是低丰度）microRNA。 最近，我们开发了增强型CLIP-seq（eCLIP）方法，其具有1000倍的改进。 从RBP分析实验产生高通量测序文库的效率， 通过掺入成对的大小匹配的输入，高度稳健和可重现的RBP靶点分析。 Eclipse已经成功地将eCLIP开发为一个高利润的合同服务产品，现在已经有了一个 eCLIP套件正在进行beta测试。在这里，我们将开发eCLIP方法的一个专用变体， microRNA的定位靶向转录组范围内的以下三个目标： 1.使用嵌合eCLIP（chim-eCLIP）明确直接分析miRNA靶点。 2.将简化的chim-eCLIP方法商业化。 3.将嵌合eCLIP转化为结构化和记录良好的试剂盒形式。 Eclipse Bio是执行上述目标的理想候选人，因为我们在基因组学方面的专业知识， 计算生物学，特别是在RNA加工和分析RNA结合蛋白靶点方面。三 上述目标将使所有生物医学领域都能以标准方法进行microRNA靶向定位。 研究人员在学术界和工业界，并建立一个严格的标准，验证特异性的治疗 microRNAs。正确评估治疗性miRNA样分子的能力将提供显著的益处 在各种生物学背景下研究microRNA调控的研究人员和开发 RNA疗法在临床上的应用此外，在圣地亚哥，我们靠近许多研究机构和生物技术公司 做RNA研究，可以提供科学和商业化的专业知识和援助。

PROJECT SUMMARY Protein synthesis in mammalian cells depends not only on the transcription of RNA from the DNA genome, but also on the stability of that RNA and its rate of translation. A class of ~21 nucleotide RNAs known as ‘microRNAs’ play critical roles in regulating protein synthesis by recognizing complementary sequences in protein-coding RNA molecules and causing either RNA cleavage or inhibition of translation. MicroRNA regulation plays key roles in nearly every studied human physiological system, and mis- regulation of individual microRNAs or global microRNA processing has been linked to cancer, cardiac and kidney disease, viral infection response, and many other diseases. Identifying the targets of individual microRNAs provides an essential insight into the functional biological role of each microRNA. Furthermore, as functional regulatory molecules microRNAs are now being highly pursued for their use as therapeutic agents, where the ability to directly map microRNA targets is required to assay both proper on-target binding as well as low off-target interactions. However, current methods are lacking, as they have either high false-positive rates, lack the ability to assign targets to individual microRNAs, or do not scale to profiling all (particularly low-abundance) microRNAs with quantitative accuracy. Recently we developed the enhanced CLIP-seq (eCLIP) methodology, with 1000-fold improved efficiency of generating high-throughput sequencing libraries from RBP profiling experiments, enabling highly robust and reproducible RBP target profiling through the incorporation of paired size-matched inputs. Eclipse has successfully developed eCLIP as a highly profitable contract service product, and now has an eCLIP kit in beta testing. Here we will develop a specialized variant of the eCLIP method for unambiguous mapping of microRNA targets transcriptome-wide in the following three aims: 1. Validate unambiguous direct profiling of miRNA targets with chimeric eCLIP (chim-eCLIP). 2. Validate simplified chim-eCLIP method for commercialization. 3. Conversion of chimeric eCLIP into a structured and well-documented kit format. Eclipse Bio is an ideal candidate to perform the aims described above due to our expertise in genomics and computational biology, particularly in RNA processing and profiling RNA binding protein targets. The three aims above will enable microRNA target mapping to be performed in a standard method by all biomedical researchers in academia and industry, and create a rigorous standard for validating specificity of therapeutic microRNAs. The ability to properly assess therapeutic miRNA-like molecules will provide significant benefits to researchers studying microRNA regulation in various biological contexts and drug companies developing RNA therapies in the clinic. Additionally, in San Diego we are close to many research institutes and biotechs doing RNA research that can provide scientific and commercialization expertise and assistance.