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

项目总结 转录后，RNA分子经历了一系列的处理步骤来调节剪接的 序列，RNA表达水平，亚细胞定位，以及翻译的起始和速度，并且每个 受RNA结合蛋白(RBPs)活性控制。RNA加工在正常情况下起着关键作用 发展和RNA加工的改变已被证明会导致家族性脊髓肌萎缩， 肌萎缩侧索硬化症和多种癌症类型。确定突变的RBP的RNA靶标 这些疾病是朝着对这种疾病的机械性理解迈出的关键的第一步，最终， 靶向疗法的发展。然而，识别RBP靶标的标准化方法 整个转录组都缺乏，因为他们需要大量的样本输入量，实验失败率很高 而且需要高扩增，导致大量的测序浪费和非定量结果。 最近我们开发了增强的Clip-Seq(ECLIP)方法，它显示了数千倍的 提高了从RBP图谱实验生成高通量测序文库的效率 通过结合成对的大小匹配的RBP靶标分析，实现了高度健壮和可重复性的靶标分析 投入。在这里，我们将开发eCLIP方法，供学术界和商界广泛使用 生物技术产业的以下目标： 1.开发eCLIP套件，并进行优化和稳定性测试，确保数据健壮 从标准化的套件格式生成。 2.执行标准化eCLIP试剂盒和协议的阿尔法测试，以验证实验成功 并找出需要进一步优化的领域。 3.开发和验证各种细胞类型、组织和样本类型的标准条件 (包括快速冷冻与固定组织、少量样本和非人类样本)。 4.开发用于eCLIP数据标准数据处理和初级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者 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人选。以上四个目标 将把eCLIP从一种学术研究方法转变为一种可以由生物医学标准化执行的方法 学术界和商业实体的研究人员，而不需要在RNA生物学方面的广泛专业知识。 芯片序列快速分析DNA结合蛋白靶标商品化试剂盒的研制 导致这项技术的大规模采用，并为DNA结合的研究提供了显著的好处 参与各种疾病的蛋白质；eCLIP试剂盒的开发将类似地使RNA结合的研究成为可能 蛋白质。此外，我们在圣地亚哥的位置靠近许多学术研究机构(包括 UCSD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。 [[当前超过30行限制的4行]]

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]]