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

描述（由申请人提供）：非编码rna （ncRNAs）及其在细胞和病毒机制中的关键作用的不断发现，激发了基于禁用或操纵相关rna的新型抗菌、抗肿瘤和抗病毒治疗。不幸的是，我们对“rna如何工作”的生物物理学理解不足，阻碍了这些可能挽救生命的努力的发展。一个关键的瓶颈是晶体学、核磁共振、系统发育分析和目前的化学方法无法确定非编码rna在所有功能状态下部分有序的3D构象。为了解决这个瓶颈，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。这种策略快速而全面地确定了RNA的每一个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RNA二级和三级结构的丰富信息。我们的目标是通过将M2与黄素-单核苷酸诱导的光氧化（M2- fmn）和硫酸二甲基烷基化（M2- dms）这两种化学反应耦合，更精确地揭示典型碱基对和普遍的a -次要叔级相互作用。我们提出了一种高通量m2拯救方法，通过“外科”双突变/拯救实验来验证由此得出的推论。最后，我们将应用这些技术来确定RNA生物物理学中两个典型系统的神秘状态和区域结构，即添加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。我们将通过六个已知结构的ncRNA结构域的基准来评估成功；通过m2救援验证；通过更广泛的生物界采用我们的方法和软件工具。就像二维光谱将核磁共振方法转化为小生物分子结构一样，我们提出二维突变和图谱技术将改变我们对长链非编码RNA、全长RNA信息和整个逆转录病毒基因组的结构的理解，这些逆转录病毒基因组的目标是生物医学激活或破坏。

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.