Genome-Scale Analysis of RNA Virus Tertiary Structure

描述（由申请人提供）：RNA在生物学中作为信息传递的中心管道-在简单的复制实体如RNA病毒和复杂的多细胞生物学中。 有机体RNA是唯一能够发挥这一作用的，因为它在两个层面上编码信息：在其线性初级序列和在其形成功能关键的高阶折叠的能力。 迄今为止的工作主要集中在对一些特定调控基序的深入研究上，已经揭示了RNA的二级和三级结构调控剪接、翻译和蛋白质折叠，通过小配体和药物与蛋白质的结合，并崩溃成大规模的结构域。只有少数的例子，其中基因组规模的RNA结构已被表征。然而，在每个案例中都发现了许多新的生物学见解。RNA病毒是特别信息系统，因为它们紧凑的基因组具有功能重要的二级和三级结构元件的密集阵列。此外，病原性病毒中新的调节基序的每一次鉴定都为抗病毒治疗设计提供了独特的靶标。在几年的初步和探索性研究的指导下，我们准备进行非常高通量和高含量的RNA二级和三级结构分析。我们将应用新发明的大规模并行二级和三级结构约束生成技术，再加上新的分子动力学驱动的结构优化，以了解丙型肝炎病毒（HCV）RNA基因组中高阶结构的生物学作用。我们的特定目标旨在揭示RNA结构在HCV复制周期中的许多新作用，以可能告知许多生物学领域的方式做到这一点，使抑制病毒复制的新治疗策略成为可能，并创造可被非专业实验室广泛采用的工具，用于分析复杂的生物学上真实的RNA。目标1：使用SHAPE分析三个代表性HCV RNA基因组的结构，通过大规模平行测序检测，以确定保守的碱基配对和三级结构基序。目标二：创建并验证一种准确且可扩展的方法，用于使用实验碱基配对和空间三级约束来驱动大型RNA的三维折叠细化。目标3：整合这项工作中开发的技术，以完善三维结构模型，并发现正义HCV RNA基因组的新调控基序。

DESCRIPTION (provided by applicant): RNA functions as the central conduit for information transfer in biology - in simple replicating entities like RNA viruses and in complex multi-cellular organisms. RNA is uniquely able to play this role because it encodes information at two levels: in its linear primary sequence and in its ability to form functionally critical higher-order folds. Work to date, largely focused on intensive study of a few specific regulatory motifs, has revealed that RNA secondary and tertiary structures regulate splicing, translation and protein folding, binding by small ligands and drugs and proteins, and collapse into large-scale structural domains. There are only a small number of examples in which genome-scale RNA structures have been characterized. However, numerous new biological insights were uncovered in each case. RNA viruses are especially informative systems because their compact genomes feature a dense array of functionally important secondary and tertiary structure elements. Moreover, every identification of a new regulatory motif in a pathogenic virus presents a unique target for anti-virus therapeutic design. Guided by several years of preliminary and exploratory studies, we are poised to make very high- throughput and high-content RNA secondary and tertiary structure analysis straightforward. We will apply newly invented massively parallel secondary and tertiary structure constraint-generation technologies coupled with novel molecular dynamics-driven structural refinement to understand the biological roles of higher-order structure in the hepatitis C virus (HCV) RNA genome. Our Specific Aims are designed to reveal numerous new roles for RNA structure in the replication cycle of HCV, to do so in a way likely to inform many fields of biology, to make possible new therapeutic strategies for inhibiting viral replication, and to create tools that can be widely adopted by non-expert laboratories for analysis of complex, biologically authentic RNAs. Aim 1: Analyze the structure of three representative HCV RNA genomes using SHAPE, detected by massively parallel sequencing, to identify conserved base pairing and tertiary structure motifs. Aim 2: Create and validate an accurate and scalable approach for using experimental base pairing and through-space tertiary constraints to drive three-dimensional fold refinement for large RNAs. Aim 3: Integrate the technologies developed in this work to refine three-dimensional structure models and to discover new regulatory motifs for plus-sense HCV RNA genomes.