Protein Bound RNA Sequencing

描述(由申请人提供)：蛋白质-RNA的相互作用对基因表达的转录后控制的调控逻辑做出了深远的贡献。在全球范围内破译这一错综复杂的分子相互作用层的工具正走在分子生物学和基因组学的前沿。检测RNA结合蛋白(RBPs)与其靶标相互作用的最广泛的方法是IP微阵列，它受到几个实验陷阱的困扰。交联免疫沉淀(CLIP)方法克服了许多这些缺陷，同时有助于识别RNA靶标以及原位结合位点。开发一种新的工具来破译这一错综复杂的分子相互作用层是这一提议的一个重点。在开发过程中，我们将识别克隆读取产生的噪音，并提高程序的灵敏度。分析技术将被开发，以允许建立连接效率的模型并准确地估计克隆偏向。该项目的最终目标是创建最小偏差的基于Clip-Seq的文库，该文库易于和公平地进行询问。我们提议开发技术，以公正、高通量的方式研究这些RNA，应该会使未来的临床应用成为现实，并推动比较组织疾病图谱的研究，进一步解锁转录调控。

DESCRIPTION (provided by applicant): Protein-RNA interactions make a profound contribution to the regulatory logic of post- transcriptional control of gene expression. Tools to decipher this intricate layer of molecular interactions on a global scale are coming to the forefront of molecular biology and genomics. The most wide spread methodology to examine interactions of RNA binding proteins (RBPs) with their targets is an IP-microarray plagued by several experimental pitfalls. The cross-linking immunoprecipitation (CLIP) method overcomes many of these pitfalls while facilitating identification of RNA targets as well as the in situ bindng sites. Developing a novel tool to decipher this intricate layer of molecular interaction is a focusof this proposal. During development, we will identify noise arising from clonal reads and improve the sensitivity of the procedure. Analytical techniques will be developed to allow modeling of ligation efficiency and estimate clonal biases accurately. The ultimate goal of the project is to create minimally biased CLIP-Seq based libraries that are easily and fairly interrogated. Our proposal to develop technology to study these RNAs in an unbiased, high-throughput manner should make future clinical applications a reality and propel research in comparative tissue disease profiles, further un-locking transcriptional regulation.