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

描述（由申请人提供）：非编码RNA（ncRNA）及其在细胞和病毒机制中的关键作用的不断发现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如何工作”的生物物理理解不足，阻碍了这些潜在的拯救生命的努力的发展。一个关键的瓶颈是晶体学，NMR，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，我们最近发明了一种二维的“突变和映射”（M2）技术。这种策略快速而全面地确定了RNA的每一个单一突变如何干扰每一个其他核苷酸的2 '-羟基化学可及性，从而提供了关于RNA二级和三级结构的丰富信息。我们在这里的目标是更精确地揭示规范的碱基对和普遍的A-次要三级相互作用耦合M2到两个额外的化学反应，黄素单核苷酸诱导的光氧化（M2-FMN）和二甲基硫酸烷基化（M2-DMS）。我们提出了一个高通量的M2救援的方法来验证由此产生的推论，通过“手术”双突变/救援实验。最后，我们将应用这些技术来确定在RNA生物物理学的两个范例系统，添加腺嘌呤结合核糖开关和FN双甘氨酸核糖开关的神秘状态和区域的结构，这是不可能获得的关键信息与任何其他方法。我们将通过已知结构的六个ncRNA结构域的基准评估成功;通过M2拯救验证;并通过更广泛的生物界采用我们的方法和软件工具。以同样的方式，2D光谱学将NMR方法转化为小生物分子结构，我们提出，2D突变和映射技术将改变我们对长非编码RNA，全长RNA信息和整个逆转录病毒基因组的结构的理解，这些基因组被靶向用于生物医学激活或破坏。 公共卫生相关性：RNA分子在传递和调节所有生命系统中的遗传信息方面发挥着重要作用，包括致病细菌、逆转录病毒（如HIV）和肿瘤细胞。靶向这些RNA的新的可能挽救生命的疗法受到确定RNA折叠和构象变化的缓慢速率的阻碍。我们的工作旨在通过推进一种新的高通量RNA结构测定的化学/计算范式来解决这一关键瓶颈。

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.