RNA Structure and Dynamics In Vivo

知识价值。这项研究的总体愿景是创造一种技术，以单核苷酸分辨率分析活细胞内任意复杂性的RNA结构。这个目标是及时的，因为RNA在生物学的各个领域日益突出。这种突出部分反映了人们对核糖体和许多其他核糖核蛋白是高度动态和灵活的生物分子机器的认识。这也反映了许多具有关键功能的非翻译和调控rna的新角色被发现。因此，本研究计划的目标是将项目实验室发明的高通量SHAPE（通过引物延伸分析的选择性2'-羟基酰化）RNA结构分析技术扩展为一种强大的体内RNA结构分析工具。这项技术随后将被用于理解一些令人惊讶的发现，即初步研究揭示的大肠杆菌核糖体中的RNA结构，因为它们存在于健康的、生长的细菌细胞中。SHAPE技术也足够成熟，可以作为完全由本科生驱动的研究工作的基础，这将发展成为本科生转录组项目（UTP）。预计更广泛的影响将是多方面的。一种准确而高效的体内RNA结构分析技术有可能改变生物科学的许多领域。在这个研究项目下，体内SHAPE将连接RNA结构的分子信息，这些信息是通过高分辨率的结构和生化方法在高度纯化的系统中获得的，具有复杂的体内环境中的细胞功能和适应性。在这项工作中创造的技术将通过正式和非正式渠道广泛传播。大学生转录组计划（UTP）是一种新型的本科生驱动的研究经验。学生将被指导承担项目的大部分责任，并将发起、领导和保持其智力动力。UTP将侧重于二十面体植物病毒RNA基因组的结构分析。这类病毒是生物学中分布最广泛的植物、动物和农业病原体。该项目有几个独特的特点，其目标是激励本科生对科学研究进行深度投资和热情。

Intellectual Merit. The overarching vision of this research is to create a technology for analyzing RNA structure at single nucleotide resolution for RNAs of arbitrary complexity inside living cells. The goal is timely because of the growing prominence of RNA in all fields of biology. This prominence reflects, in part, the appreciation that the ribosome and many other ribonucleoproteins are highly dynamic and nimble biomolecular machines. It also reflects the many new roles being discovered for the numerous untranslated and regulatory RNAs with critical functions. The goal of this research program is therefore to extend the high-throughput SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) RNA structure analysis technology, invented in the project laboratory, into a robust tool for in vivo analysis of RNA structure. This technology will then be used to understand surprising discoveries that preliminary studies have revealed about RNA structure in E. coli ribosomes as they exist inside healthy, growing, bacterial cells. SHAPE technology is also sufficiently mature to serve as the basis for a completely undergraduate-driven research endeavor, which will be developed into the Undergraduate Transcriptome Project (UTP).Broader Impacts are envisioned to be multi-fold. An accurate and efficient technology for analyzing RNA structure in vivo has the potential to transform many fields in the biological sciences. When fully developed under this research program, in vivo SHAPE will interconnect molecular information about RNA structure as obtained from high-resolution structural and biochemical approaches in highly purified systems with cellular function and fitness in the complex in vivo environment. The technology created in this work will be disseminated widely via both formal and informal channels. The Undergraduate Transcriptome Project (UTP) is a new kind of undergraduate-driven research experience. Students will be mentored to assume a large part of the responsibility for the project and will initiate, lead, and maintain its intellectual momentum. The UTP will focus on structural analysis of the RNA genome of an icosahedral plant virus. Such viruses are among the most widely distributed plant, animal and agricultural pathogens in biology. The project has several distinctive features with the goal of inspiringundergraduates to become deeply invested in and passionate about scientific research.