Development of chimeric read amplification into a robust method for profiling targets of specific microRNAs and global amplification of all microRNA:mRNA pairings transcriptome-wide

项目摘要 microRNA的转录后调节是细胞稳态的基础。微小rna是 小的非编码RNA，其通过与靶细胞中的互补序列进行碱基配对来微调基因表达， 信使RNA（mRNA）分子。一个microRNA可以有多个靶点， mRNA网络的调控。microRNA调控的重要性不仅体现在它们的 进化保守，而且几乎每一个生物过程中的microRNA的参与 包括增殖、炎症、发育和代谢。个别microRNA的失调或 全球microRNA加工对发育、生理和疾病具有重要影响。 microRNA表达异常与从癌症到心力衰竭等许多疾病有关， microRNA已成为治疗方法的有吸引力的靶标和工具。精确目标识别 microRNA的研究对于理解它们的功能作用至关重要。识别microRNA靶标的现有方法 由于假阳性率高，无法明确区分直接和间接 microRNA-靶标相互作用或不能分析低丰度microRNA。为此，我们已申请 我们先前开发的增强交联和免疫沉淀（eCLIP）技术， 专门的替代方案，嵌合eCLIP，以明确识别转录组范围内的microRNA靶点， 方式嵌合eCLIP基于AGO 2 eCLIP，包括连接步骤，其中microRNA和mRNA 片段彼此连接形成嵌合RNA分子，然后可以对其进行测序。直接 使用microRNA：mRNA，从嵌合eCLIP数据中可以很容易地识别单个microRNA的靶标 嵌合读段。本文提出的工作将扩展先前开发的嵌合eCLIP技术， 包括微RNA和基因特异性读数富集方法，以及用户友好分析包 从而产生了一个强大的和多功能的嵌合eCLIP试剂盒，供学术和生物技术领域广泛使用， 行业，如以下目标所述： 1.使用基于探针的嵌合eCLIP扩展microRNA靶标的直接分析 2.完善用于分析嵌合eCLIP数据的软件工具，以加强面向客户的关键功能 3.进行扩大的beta测试，并最终完成全方位嵌合eCLIP试剂盒的商业化 Eclipse Bio是执行上述目标的理想候选人，因为我们在基因组学，RNA， 处理和计算生物学。上述目标将使强大的microRNA靶点定位成为可能。 由所有生物医学研究人员使用标准方法进行，并将允许严格验证 microRNA特异性正确评估治疗性microRNA样分子的能力将提供 这对研究microRNA调控的研究人员和开发RNA疗法的公司都有重大益处。

PROJECT SUMMARY Post-transcriptional regulation by microRNAs is fundamental for cellular homeostasis. MicroRNAs are small non-coding RNAs that fine tune gene expression by base pairing to complementary sequences in target messenger RNA (mRNA) molecules. A single microRNA can have multiple targets allowing for coordinated regulation of mRNA networks. The importance of microRNA regulation is evidenced not only by their evolutionary conservation but also by the involvement of microRNAs in nearly every biological process including proliferation, inflammation, development, and metabolism. Misregulation of individual microRNAs or global microRNA processing has important consequences for development, physiology, and disease. Aberrant microRNA expression has been linked to many diseases ranging from cancer to cardiac failure, and microRNAs have become attractive targets and tools for therapeutic approaches. Precise target identification of microRNAs is essential to understand their functional role. Current methods to identify microRNA targets are limited, as they have high false-positive rates, are unable to definitively distinguish direct from indirect microRNA-target interactions or are unable to profile low-abundance microRNAs. To this end we have applied our previously developed enhanced crosslinking and immunoprecipitation (eCLIP) technology to develop a specialized alternative, chimeric-eCLIP, to unambiguously identify microRNA targets in a transcriptome-wide manner. Chimeric-eCLIP is based on AGO2 eCLIP and includes a ligation step where microRNA and mRNA fragments are ligated to each other forming chimeric RNA molecules that can then be sequenced. Direct targets of individual microRNAs are easily identifiable from chimeric-eCLIP data using microRNA:mRNA chimeric reads. The work proposed here will expand the previously developed chimeric-eCLIP technology to include microRNA- and gene-specific read enrichment methods, and a user-friendly analysis package resulting in a robust and versatile chimeric-eCLIP kit for wide-spread use by academic and biotechnology industries, as outlined in the following aims: 1. Expand direct profiling of microRNA targets with probe-based chimeric-eCLIP 2. Refine software tools for analysis of chimeric-eCLIP data to strengthen key customer-facing features 3. Conduct expanded beta testing and finalize commercialization of the all-inclusive chimeric-eCLIP kit Eclipse Bio is an ideal candidate to perform the aims described above due to our expertise in genomics, RNA processing, and computational biology. The above aims will enable robust microRNA target mapping to be performed using a standard method by all biomedical researchers and will allow for rigorous validation of microRNA specificity. The ability to properly assess therapeutic microRNA-like molecules will provide significant benefit to researchers studying microRNA regulation and companies developing RNA therapies.