RNA Targeted Small Molecules: Connecting Binding Kinetics to Sequence Selectivity

描述(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型选择性RNA结合化合物的能力。这在很大程度上是由于该领域在选择性的基本决定因素方面的知识差距。这一建议试图检验这样的假设，即RNA结合化合物的序列选择性直接与其在所需结合部位的动力学关闭速度或“停留时间”有关。虽然这是蛋白质和酶识别领域中普遍接受的原理，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与RNA结合的化合物。这一假设将通过三个目标进行检验。首先，将使用经过良好验证(但吞吐量较低)的技术来分析一系列已知的RNA靶标化合物的结合特性。其次，我们实验室开发的一种名为阵列成像反射法的新分析方法将在RNA结合动力学常数的多路(高通量)评估的背景下进行测试。这还将涉及开发新的统计方法，用于分析随时间变化的阵列数据。第三，我们将研究系统的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，该化合物针对的是对HIV生命周期至关重要的病毒RNA。拟议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例，并为高通量表征RNA结合和针对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.