A robust kit, standards, and software for identifying RNA binding protein targets

项目摘要 转录后，RNA分子经历许多处理步骤，调节剪接的RNA分子。 序列、RNA表达水平、亚细胞定位以及翻译起始和翻译速率，并分别 由RNA结合蛋白（RBP）的活性控制。RNA加工在正常的 发育，改变的RNA加工已被证明会导致家族性脊髓性肌萎缩症， 肌萎缩侧索硬化症和多种癌症类型。鉴定RBP突变的RNA靶标， 这些疾病是对疾病的机械理解的关键的第一步，最终， 发展靶向治疗。然而，识别RBP目标的标准化方法 转录组范围内缺乏，因为它们需要大量的样本输入量，实验失败率高 并且需要高扩增，导致显著浪费的测序和非定量结果。 最近，我们开发了增强型CLIP-seq（eCLIP）方法，该方法显示了数千倍的 提高了从RBP谱分析实验产生高通量测序文库的效率， 通过掺入配对的大小匹配的RBP靶向分析， 输入。在这里，我们将开发eCLIP方法，供学术界和商业界广泛使用 生物技术产业，目标如下： 1.开发eCLIP试剂盒，并进行优化和稳定性测试，以确保可靠的数据 从标准化的试剂盒格式生成。 2.对标准化eCLIP试剂盒和方案进行alpha测试，以验证实验成功 并确定进一步优化的领域。 3.开发并验证各种细胞类型、组织和样本类型的标准条件 （包括快速冷冻与固定组织、小样本量和非人类样本）。 4.开发用于eCLIP数据的标准数据处理和原始数据分析的软件工具， 包括与公开可用的eCLIP数据资源的集成。 由于我们在基因组学方面的专业知识，Eclipse Bio是实现上述目标的理想候选人 和计算生物学（特别是在RNA处理领域），以及在早期阶段的开始 生物公司，使我们成为执行上述研究的理想候选人。上述四个目标 将把eCLIP从一种学术研究方法转变为一种可以通过生物医学标准执行的方法。 在学术界以及商业实体的研究人员，而不需要在RNA生物学的广泛的专业知识。 ChIP-seq快速分析DNA结合蛋白靶标的标准化商品化试剂盒的开发 导致了这种技术的大规模采用，并为DNA结合的研究提供了显着的好处 与各种疾病有关的蛋白质; eCLIP试剂盒的开发将同样能够研究RNA结合 proteins.此外，我们在圣地亚哥的位置靠近许多学术研究机构（包括 加州大学圣地亚哥分校和索尔克研究所）以及生物技术公司将提供科学和商业化 专业知识和援助。 [[当前4行超过30行限制]]

PROJECT SUMMARY After transcription, RNA molecules undergo a number of processing steps that regulate the spliced sequence, RNA expression level, subcellular localization, and initiation and rate of translation, and are each controlled by the activity of RNA binding proteins (RBPs). RNA processing plays critical roles in normal development, and altered RNA processing has been shown to cause familial Spinal Muscular Atrophy, Amyotrophic Lateral Sclerosis, and multiple cancer types. Identifying the RNA targets of the RBP mutated in these diseases is a critical first step towards a mechanistic understanding of the disease and, ultimately, development of targeted therapeutics. However, standardized methods for identification of RBP targets transcriptome-wide were lacking, as they required large sample input amounts, had high experimental failure rates, and required high amplification leading to significant wasted sequencing and non-quantitative results. Recently we developed the enhanced CLIP-seq (eCLIP) methodology, which showed thousand-fold improved efficiency of generating high-throughput sequencing libraries from RBP profiling experiments and enabled highly robust and reproducible RBP target profiling through the incorporation of paired size-matched inputs. Here we will develop the eCLIP method for wide-spread use by the academic and commercial biotechnology industry in the following aims: 1. Develop an eCLIP kit, and perform optimization and stability testing to ensure robust data generation from a standardized kit format. 2. Perform alpha testing of a standardized eCLIP kit and protocol to validate experimental success and identify areas for further optimization. 3. Develop and validate standard conditions for various cell-types, tissues, and sample types (including flash-frozen versus fixed tissues, small sample amounts, and non-human samples). 4. Develop software tools for standard data processing and primary data analysis of eCLIP data, including integration with publicly available eCLIP data resources. Eclipse Bio is an ideal candidate to perform the aims described above due to our expertise in genomics and computational biology (particularly in the area of RNA processing), as well as in the starting of early-phase biology companies, make us ideal candidates to perform the research proposed above. The four aims above will take eCLIP from an academic research method to one that can be standardly performed by biomedical researchers in academia as well as commercial entities, without requiring extensive expertise in RNA biology. The development of standardized commercial kits to profile DNA binding protein targets by ChIP-seq rapidly led to large-scale adoption of this technique, and has provided significant benefits to studies of DNA binding proteins involved in various diseases; development of an eCLIP kit will similarly enable studies of RNA binding proteins. Additionally, our location in San Diego proximal to numerous academic research institutes (including UCSD and the Salk Institute) as well as biotechnology companies will provide scientific and commercialization expertise and assistance. [[currently 4 lines over 30 line limit]]