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

• 批准号: 8899593
• 负责人: Rhiju Das
• 金额: $ 29.83万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-30 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8899593
• 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 Targeting; Elements; Flavin Mononucleotide; Foundations; Funding; Future; 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; Untranslated RNA; Validation; Viral; Work; X-Ray Crystallography; base; dimethyl sulfate; genetic information; in vivo; mutant; nanoengineering; neoplastic cell; new technology; novel; oxidation; receptor; research study; restoration; single molecule; structural biology; success; targeted treatment; three dimensional structure; three-dimensional modeling; tool; two-dimensional

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.

描述（由申请人提供）：非编码 RNA (ncRNA) 的不断发现及其在细胞和病毒机制中的关键作用正在激发基于禁用或操纵相关 RNA 的新型抗菌抗肿瘤和抗病毒疗法。不幸的是，我们对“RNA 如何发挥作用”的生物物理学了解不足，阻碍了这些潜在的挽救生命的努力的发展。一个关键瓶颈是晶体学、NMR、系统发育分析和当前的化学方法无法用于确定非编码 RNA 在其所有功能状态下的部分有序 3D 构象。为了解决这个瓶颈，我们最近发明了二维“变异和映射”（M2）技术并对其进行了基准测试。该策略快速、全面地确定 RNA 的每个突变如何干扰其他每个核苷酸的 2'-羟基化学可及性，从而提供有关 RNA 二级和三级结构的丰富信息。我们的目标是通过将 M2 与另外两种化学物质黄素单核苷酸诱导的光氧化 (M2-FMN) 和硫酸二甲酯烷基化 (M2-DMS) 偶联，更准确地揭示经典碱基对和普遍的 A 小三级相互作用。我们提出了一种高通量 M2 救援方法，通过“外科手术”双突变/救援实验来验证由此产生的推论。最后，我们将应用这些技术来确定RNA生物物理学中两个范例系统中神秘状态和区域的结构，即add腺嘌呤结合核糖开关和FN双甘氨酸核糖开关；通过任何其他方法都无法获得此关键信息。我们将通过对已知结构的六个 ncRNA 域进行基准评估成功；通过M2-救援验证；并通过更广泛的生物界采用我们的方法和软件工具。就像 2D 光谱将 NMR 方法转变为小生物分子结构一样，我们建议 2D 突变和图谱技术将改变我们对长非编码 RNA、全长 RNA 信息和针对生物医学激活或破坏的整个逆转录病毒基因组的结构的理解。