Harnessing Small Molecules to Probe the Structure and Function of Long Noncoding RNAs

项目摘要 PI的长期目标是开发高度特异的、以RNA为靶标的小分子配体来探测 长非编码RNA(LncRNAs)的动态结构、基本生物学和治疗潜力选择 已发现lncRNA在癌症进展中发挥关键作用，包括lncRNA HOTAIR，它是 与转移性乳腺癌有关，以及在几种癌症中过度表达的lncRNA Malat-1， 包括非小细胞肺癌。尽管提出了lncRNA的治疗潜力，但足够小的 分子靶向策略尚未实现。这种缓慢的进展部分是由于RNA AS的性质 一种动态结构，具有有限的化学功能，但也与引导方面的知识差距有关 小分子的原理和方法：RNA相互作用。我们的中心假设是 发现有利于分化的小分子化学空间和RNA拓扑空间将产生 对小分子的基本见解：可应用于快速发展的RNA识别 对于广泛的RNA靶标具有高亲和力和特异性的配体。在之前的工作中，PI已经确定 生物活性RNA配体的常见化学性质，阐述了RNA结合支架的改进 识别小的RNA靶标，揭示了可以区分的RNA二级结构 小分子，以及通过计算确定的靶向IncRNA HOTAIR的“热点”。在建议的 在工作中，我们将同时进行两条独立但互补的基础调查路线 并将开发的指导原则和技术应用于两个关键的LncRNA靶标。在区域1中，我们将 使用化学信息学分析、有机合成和快速筛选方法鉴定小分子 偏向于特定RNA识别的特性。在区域2中，我们将使用模式识别协议来 识别容易被小分子区分的RNA结构。在区域3中，我们将结合我们的RNA- 偏倚文库和优化的筛选试验以鉴定lncRNA三级结构的第一抑制物， 尤其是MALAT1的3‘-三螺旋。在区域4中，我们将使用广泛的计算和 识别抑制lncRNA的小分子的实验工具：蛋白质相互作用，即。HOTAIR及其智能交通系统 蛋白质结合伙伴，PRC2。这项研究的基本原理是我们的新的RNA特异性文库和 技术将使对RNA结构和功能的新研究成为可能，并为 RNA靶向治疗的未来发展。综上所述，我们的工作将(I)产生第一个广泛的 具有对RNA靶标的亲和力和特异性的可用的基于RNA的分子文库；(Ii)开发 一系列的计算、合成和筛选工具，以增强RNA配体的识别；以及(Iii)产生第一- 为lncRNA开发小分子配体的一流技术。这些截然不同但相辅相成 方法不仅将确保这项研究的成功和小分子领域的进步：RNA 靶向，但也将为一系列对人类健康至关重要的靶点提供铅小分子。

Project Summary The long-term goal of the PI is to develop highly specific, RNA-targeted, small molecule ligands to probe the dynamic structure, fundamental biology, and therapeutic potential of long noncoding RNAs (lncRNAs). Select lncRNAs have been found to play critical roles in cancer progression, including lncRNA HOTAIR, which is implicated in metastatic breast cancer, and lncRNA MALAT-1, which is over-expressed in several cancers, including non-small cell lung cancer. Despite the proposed therapeutic potential of lncRNAs, adequate small molecule targeting strategies have yet to be realized. This slow progress is due in part to the nature of RNA as a dynamic structure with limited chemical functionality but also to a gap in knowledge with respect to guiding principles and methods for small molecule:RNA interactions. Our central hypothesis is that the parallel discovery of small molecule chemical space and RNA topological space privileged for differentiation will yield fundamental insights into small molecule:RNA recognition that can be applied to the rapid development of ligands with high affinity and specificity for a wide range of RNA targets. In prior work, the PI has identified common chemical properties of biologically active RNA ligands, elaborated RNA binding scaffolds for improved recognition of small RNA targets, revealed RNA secondary structures that can be differentially recognized by small molecules, and computationally identified “hot spots” for targeting lncRNA HOTAIR. In the proposed work, we will simultaneously pursue two independent but complementary lines of fundamental investigation and apply the developed guiding principles and technologies to two critical lncRNA targets. In Area 1, we will use cheminformatic analysis, organic synthesis, and rapid screening methods to identify small molecule properties biased toward specific RNA recognition. In Area 2, we will use pattern recognition protocols to identify RNA structures that are readily differentiated by small molecules. In Area 3, we will combine our RNA- biased libraries and optimized screening assays to identify the first inhibitors of lncRNA tertiary structure, particularly the 3'-triple helix of MALAT1. In Area 4, we will use a wide range of computational and experimental tools to identify small molecules that inhibit lncRNA:protein interactions, viz. HOTAIR and its protein binding partner, PRC2. The rationale for this research is that our novel RNA-specific libraries and technologies will enable new investigations of RNA structure and function and serve as a rich platform for future development of RNA targeted therapeutics. In summary, our work will (i) produce the first widely available RNA-biased molecular library with demonstrated affinity and specificity for RNA targets; (ii) develop a range of computational, synthetic, and screening tools to enhance RNA ligand identification; and (iii) yield first- in-class technologies to develop small molecule ligands for lncRNAs. These distinct but complementary approaches will not only ensure success of this research and progress in the field of small molecule:RNA targeting, but will also provide lead small molecules for a range of critically important targets in human health.