Non-coding RNA Structure through a Mutate-and-Map Strategy

通过突变和映射策略研究非编码 RNA 结构

基本信息

批准号：
8345532
负责人：
Rhiju Das
金额：
$ 29.83万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-30 至 2017-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8345532
关键词：
Adenine Adenosine Adoption Algorithms Alkylation Anti-Bacterial Agents Antiviral Agents Antiviral Therapy Award Bacteria Base Pairing Benchmarking Binding Biological Biological Process Biophysics Chemicals Chemistry Code Collaborations Communities Computer Analysis Computing Methodologies Conflict (Psychology)Coupling Crystallography Data Databases Deposition Development Disease Distant Docking Drug Delivery Systems Elements Flavin Mononucleotide Foundations Functional RNA Funding Future Genetic Genome Glycine Grant HIV Hydroxyl Radical In Vitro Length Libraries Life Ligand Binding Ligand Binding Domain Ligands Literature Maps Measures Medicine Methods Minor Modeling Modification Molecular Biology Molecular Conformation Monitor Mutate Mutation NMR Spectroscopy NOESY Nucleotides Operative Surgical Procedures Organism Participant Phylogenetic Analysis Play Property RNA RNA Folding Resolution Retroviridae Role Seeds Signal Transduction Site Software Tools Solutions Spectrum Analysis Structure System Technology Testing Therapeutic Validation Viral Work X-Ray Crystallography base dimethyl sulfate in vivo mutant nanoengineering neoplastic cell new technology novel oxidation receptor research study restoration single molecule structural biology success three dimensional structure three-dimensional modeling tool two-dimensional

项目摘要

DESCRIPTION (provided by applicant): The continuing discoveries of non-coding RNAs (ncRNAs) and their critical roles in cellular and viral machinery are inspiring novel antibacterial antitumor, and antiviral therapies based on disabling or manipulating the RNAs involved. Unfortunately, our poor biophysical understanding of "how RNAs work" hinders the development of these potentially life-saving efforts. A critical bottleneck has been the inapplicability of crystallography, NMR, phylogenetic analysis, and current chemical methods to determine the partly ordered 3D conformations of non-coding RNAs in all their functional states. To resolve this bottleneck, we have recently invented and benchmarked a two-dimensional "mutate-and-map" (M2) technology. This strategy rapidly and comprehensively determines how every single mutation of an RNA perturbs the 2'-hydroxyl chemical accessibility of every other nucleotide, giving rich information on RNA secondary and tertiary structure. We aim here to more precisely reveal both canonical base pairs and pervasive A-minor tertiary interactions by coupling M2 to two additional chemistries, flavin-mononucleotide-induced photo-oxidation (M2-FMN) and dimethyl-sulfate alkylation (M2-DMS). We propose a high-throughput M2-rescue approach to validate the resulting inferences through "surgical" double-mutant/rescue experiments. Finally, we will apply these technologies to determine structures of mysterious states and regions in two paradigmatic systems in RNA biophysics, the add adenine-binding riboswitch and the FN double-glycine riboswitch; this critical information is not obtainable with any other approach. We will evaluate success through benchmarks on six ncRNA domains of known structure; through M2-rescue validation; and through adoption of our methods and software tools by the broader biological community. In the same way that 2D spectroscopy transformed NMR approaches to small biomolecule structure, we propose that 2D mutate-and-map technology will transform our understanding of structure in long non-coding RNAs, full-length RNA messages, and entire retroviral genomes targeted for biomedical activation or disruption. PUBLIC HEALTH RELEVANCE: RNA molecules play fundamental roles in transmitting and regulating genetic information in all living systems, including disease-causing bacteria, retroviruses like HIV, and tumor cells. New potentially life-saving therapies that target these RNAs are being hindered by the slow rate of determining RNA folds and conformational changes. Our work aims to resolve this critical bottleneck by advancing a new chemical/computational paradigm for high-throughput RNA structure determination.

描述（由申请人提供）：非编码RNA（NCRNA）的持续发现及其在细胞和病毒机械中的关键作用激发了新型的抗菌抗肿瘤，以及基于残疾或操纵RNA的抗病毒疗法。不幸的是，我们对“ RNA如何工作”的生物物理不良理解阻碍了这些潜在的挽救生命的努力的发展。临界瓶颈是晶体学，NMR，系统发育分析和当前化学方法的不适用性，以确定其所有功能状态下非编码RNA的部分有序的3D构象。为了解决这种瓶颈，我们最近发明并基准了二维“突变和映射”（M2）技术。该策略迅速，全面地确定RNA的每个突变如何渗透到其他所有核苷酸的2'-羟基化学可及性，从而提供有关RNA次级和高等教育结构的丰富信息。我们的目的是通过将M2耦合到另外两种化学物质，黄素单核苷酸诱导的光氧化（M2-FMN）和二甲基 - 硫酸盐 - 硫酸盐烷基化（M2-DMS），旨在更精确地揭示出典型的基础对和普遍的A-Minor第三纪相互作用（M2-FMN）。我们提出了一种高通量M2响应方法，以通过“手术”双突/救援实验来验证所得的推论。最后，我们将应用这些技术来确定RNA生物物理学两个范式系统中神秘状态和区域的结构，添加腺嘌呤结合核糖开关和FN双甘氨酸核糖开关；使用任何其他方法都无法获得此关键信息。我们将通过基准在已知结构的六个NCRNA结构域上评估成功；通过M2响应验证；以及通过更广泛的生物界采用我们的方法和软件工具。就像2D光谱法将NMR方法转化为小型生物分子结构一样，我们建议2D突变和映射技术将改变我们对长期非编码RNA结构的理解，全长RNA信息，全长RNA信息以及针对整个用于针对的逆转录病毒基因组生物医学激活或破坏。公共卫生相关性：RNA分子在传输和调节所有生物系统中的遗传信息中起着基本作用，包括引起疾病的细菌，HIV等逆转录病毒和肿瘤细胞。针对这些RNA的新型潜在挽救生命的疗法受到确定RNA折叠和构象变化的缓慢速度的阻碍。我们的工作旨在通过推进用于高通量RNA结构确定的新化学/计算范式来解决这种关键瓶颈。