RNA Targeted Small Molecules: Connecting Binding Kinetics to Sequence Selectivity

描述（由申请人提供）：具有潜在生物医学重要性的RNA序列的发现大大超过了化学家设计和合成新的选择性RNA结合化合物的能力。这在很大程度上是由于对选择性的基本决定因素这一领域的知识存在差距。本研究旨在验证这样一种假设，即rna结合化合物的序列选择性与其在所需结合位点的动力学关闭率或“停留时间”直接相关。虽然这是蛋白质和酶识别领域普遍接受的原则，并且也在DNA识别的背景下进行了测试，但据我们所知，这一概念尚未应用于结合RNA的化合物。这一假设将通过三个目标来验证。首先，经过验证（但低通量）的技术将用于分析一系列已知rna靶向化合物的结合特性。其次，我们的实验室开发了一种新的分析方法，称为阵列成像反射法，将在多重（高通量）评估rna结合动力学常数的背景下进行测试。这也将涉及发展新的统计方法来分析随时间变化的阵列数据。第三，我们将研究系统官能团修饰对我们实验室发现的一种新化合物的结合动力学和序列选择性的影响，该化合物靶向对HIV生命周期至关重要的病毒RNA。该研究的完成将为基于结合动力学的RNA靶向分子设计提供新的范式，并为RNA结合的高通量表征和靶向HIV的新先导化合物提供新的分析工具。

DESCRIPTION (provided by applicant): The discovery of RNA sequences of potential biomedical importance has dramatically outpaced chemists' ability to design and synthesize novel selective RNA-binding compounds. This is due largely to a gap in knowledge in the field with regard to fundamental determinants of selectivity. This proposal seeks to test the hypothesis that the sequence selectivity of an RNA-binding compound is directly related to its kinetic off rate or "residence time" in the desired binding site. While a generally accepted principle in the realm of protein and enzyme recognition, and tested also in the context of DNA recognition, to our knowledge this concept has not been applied to compounds binding RNA. This hypothesis will be tested via three Aims. First, well-validated (but low- throughput) techniques will be used to analyze the binding properties of a series of known RNA-targeted compounds. Second, a new analytical methodology developed in our laboratory termed Arrayed Imaging Reflectometry will be tested in the context of multiplex (high-throughput) assessment of RNA-binding kinetic constants. This will also involve the development of new statistical methods for the analysis of time-dependent array data. Third, we will examine the effect of systematic functional group modification on the binding kinetics and sequence selectivity of a novel compound discovered in our lab that targets a viral RNA critical to the HIV life cycle. Completion of the proposed research will provide a new paradigm for RNA-targeted molecular design based on consideration of binding kinetics, as well as a new analytical tool for high-throughput characterization of RNA binding, and new lead compounds targeting HIV. PUBLIC HEALTH RELEVANCE: RNA is rapidly emerging as an important target for drug development. However, in contrast to proteins and DNA, our understanding of the factors that lead to the successful design of an RNA-targeted small molecule are still at an early stage. In the proposed research, we will develop new tools that will dramatically increase the speed of analyzing small molecule - RNA interactions, while testing a novel hypothesis regarding RNA-binding selectivity. These methods will be applied in the context of new molecules targeting HIV.