Direct Nanopore Sequencing and Structural Profiling of Non-Polyadenylated RNA

项目摘要/摘要 大约2%的人类基因组编码蛋白质，而绝大多数细胞RNA是非编码的 (NcRNA)。越来越多的证据表明，ncRNA可以折叠成复杂的结构，并发挥关键作用 在细胞生理学和疾病方面。在耗尽丰富的ncRNA后，如转移RNA和 核糖体RNA，许多细胞内的ncRNA被发现带有Polya尾巴的3‘修饰(PA NcRNA)，类似于 信使核糖核酸。然而，细胞内ncRNA的很大一部分被发现缺乏Polya尾巴(非PA NcRNA)或被认为是双晶型的(以Pa和非Pa的形式存在)。该序列、结构和 非pA ncRNA和双态ncRNA的生物学功能在很大程度上仍不清楚，而且技术限制 是科学家探索这一基本上未知的人类转录组部分的主要障碍。 在这项提案中，肖博士的目标是开发一种新的纳米孔测序技术，使直接 Non-PAncRNA全长序列测定及二级结构检测这项技术将利用小说 细菌和真核生物逆转录酶(RTS)捕获天然非PAncRNA，并谨慎地穿线 通过污垢分枝杆菌孔蛋白A(MSPA)纳米孔测序仪捕获的RNA用于并发 RNA序列和RNA二级结构检测。这项技术的发展和应用 将进一步扩展目前的RNA测序工具箱，并使科学家更接近完全 了解人类转录组的功能。这项提议的具体目标是：(目标1)Dr。 Shaw将建立RTS单分子表征所需的实验框架， 核糖核酸的纳米孔测序，以及精确转化纳米孔离子所需的计算工具 当前为RNA序列。(AIM 2)Shaw博士将进行细菌和真核RT的筛选 以最小的棘轮缺陷通过纳米孔实现最强健的RT 对RNA二级结构的敏感性。(目标3)在他的独立研究阶段，肖博士将首先 通过对具有明确定义的序列的RNA混合物进行测序来验证他的新技术的稳健性 二次结构。然后，他将应用他的技术来描述非PAncRNA的序列和结构 从HeLa-S3细胞系中提取。最后，他将进一步调整他的技术，使其与化学物质兼容 可以直接探测体内天然RNA二级结构的方法，如硫酸二甲酯- 测序。在K99职业发展阶段，肖博士将在导师的指导下进行研究 和来自卡洛斯·布斯塔曼特博士(分子马达的单分子研究)、苏珊·马尔库塞博士的支持 (单分子生物物理学)、凯瑟琳·柯林斯博士(逆转录酶工程学)和延斯博士 Gundlach(纳米孔测序)这个由经验丰富的顾问和杰出的 加州大学伯克利分校的科学环境将为肖博士提供实现所有 建议的目的，并确立他作为首席调查员的独立研究生涯。

Project Summary/Abstract About 2% of the human genome encodes proteins, and the vast majority of cellular RNA is non-coding (ncRNA). Mounting evidence indicate that ncRNA could fold into complex structures, and play critical roles in cellular physiology and diseases. After depletion of the abundant ncRNA such as transfer RNA and ribosomal RNA, many cellular ncRNA was found to be 3’ modified with a polyA tail (pA ncRNA), similar to messenger RNA. However, a significant portion of cellular ncRNA was found to lack the polyA tail (non-pA ncRNA) or considered to be bimorphic (exist in both pA and non-pA form). The sequence, structure and biological function of non-pA ncRNA and bimorphic ncRNA remain largely unknown, and technical limitations are the main roadblocks for scientists to explore this largely uncharted fraction of the human transcriptome. In this proposal, Dr. Shaw aims to develop a novel nanopore sequencing technique that enables the direct sequencing and secondary structure detection of full-length non-pA ncRNA. This technique will utilize novel bacterial and eukaryotic reverse transcriptases (RTs) to capture native non-pA ncRNA, and discreetly thread the captured RNA through a Mycobacterium smegmatis porin A (MspA) nanopore sequencer for concurrent RNA sequence and RNA secondary structure detection. The development and application of this technique will further extend the current RNA sequencing tool box and bring scientists one step closer to fully understanding the function of the human transcriptome. The specific aims of this proposal are: (Aim 1) Dr. Shaw will establish the experimental framework necessary for the single molecule characterization of RTs, nanopore sequencing of RNA, and the computational tools needed to accurately transform nanopore ion current to RNA sequence. (Aim 2) Dr. Shaw will perform screening of bacterial and eukaryotic RTs in search for the most robust RT to ratchet RNA through nanopore with minimal ratcheting defects and optimal sensitivity to RNA secondary structures. (Aim 3) In his independent research phase, Dr. Shaw will first validate the robustness of his novel technique by sequencing RNA mixtures with well-defined sequences and secondary structures. He will then apply his technique to profile the sequence and structure of non-pA ncRNA extracted from HeLa-S3 cell line. Finally, he will further adapt his technique to be compatible with chemical methods that can directly probe native RNA secondary structures in vivo, such as dimethyl sulfate- sequencing. During the K99 career development stage, Dr. Shaw will conduct research under the mentorship and support from Dr. Carlos Bustamante (single molecule studies of molecular motors), Dr. Susan Marqusee (single molecule biophysics), Dr. Kathleen Collins (reverse transcriptase engineering), and Dr. Jens Gundlach (nanopore sequencing). This multidisciplinary group of experienced advisors and the outstanding scientific milieu of UC Berkeley will provide Dr. Shaw with the comprehensive training needed to achieve all aims of the proposal, and to establish his independent research career as principle investigator.