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

描述(申请人提供)：不断发现的非编码RNA(NcRNAs)及其在细胞和病毒机制中的关键作用正在激发新的抗菌抗肿瘤药物，以及基于禁用或操纵所涉及的RNA的抗病毒疗法。不幸的是，我们对“RNA如何工作”的生物物理理解很差，阻碍了这些潜在挽救生命的努力的发展。一个关键的瓶颈是结晶学、核磁共振、系统发育分析和当前的化学方法不适用于确定非编码RNA在所有功能状态下的部分有序3D构象。为了解决这一瓶颈，我们最近发明了一种二维“突变和映射”(M2)技术，并对其进行了基准测试。这一策略快速而全面地确定了RNA的每一个单一突变是如何扰乱其他核苷酸的2‘-羟基化学可及性的，从而提供了关于RNA二级和三级结构的丰富信息。我们的目标是通过将M2与另外两个化学作用，即黄素-单核苷酸诱导的光氧化(M2-FMN)和硫酸二甲酯-烷基化(M2-DMS)耦合，更准确地揭示正则碱基对和普遍存在的A-次要三级相互作用。我们提出了一种高通量的M2救援方法，通过“外科”双突变/救援实验来验证得出的推论。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；这一关键信息是任何其他方法都无法获得的。我们将通过对六个已知结构的ncRNA结构域进行基准测试、通过M2救援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来评估成功。就像2D光谱学将核磁共振方法转变为小生物分子结构一样，我们认为2D突变和映射技术将改变我们对长非编码RNA、全长RNA消息和整个逆转录病毒基因组中用于生物医学激活或破坏的结构的理解。

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.