Ligand Binding and Catalysis in RNA Aptamers

核酸经常被用作化学、生物化学和材料科学的工具。DNA、RNA及其衍生物是生物传感器中的识别元件，是纳米结构的构件，也是药物。本文概述的研究的长期目标是提高我们对RNA-小分子复合体中分子识别的理解，并为开发可用作生物传感器的部分功能构件或在显微镜下对RNA进行活体成像提供合理的基础。为此，必须详细了解核酸中的配体结合和催化作用与序列和结构的关系。为了获得这些信息，我的研究项目重点是核酸的结构和功能研究，重点是RNA。我们将用于这些研究的模型系统包括有机染料(如荧光素或磺胺类染料B)的RNA适配子。此外，我们将在我的实验室中选择识别非荧光荧光素-二乙酸酯(FDA)的适配子，并继续筛选能够催化其转化为荧光素的核酶。我们将通过高分辨核磁共振波谱来确定这些分子的三维结构。等温滴定量热法将用于研究结合热力学。结合和折叠的动力学将用光谱学方法和表面等离子体共振技术来研究。实验工作将通过使用分子动力学和从头计算对结构和相互作用进行理论研究来补充。在需要的情况下，将采用合成化学方法来生产配体衍生物。这些研究目标的结合应用将提供对RNA适配子如何识别和改变其配体的分子性质的更好的理解。然后，这些基本知识将被用来开发基于核酸的传感器的构建块，这些构建块可以与现有的识别模块(DNA和RNA适配子)、核酶或纳米结构相结合。

Nucleic acids are frequently used as tools in chemistry, biochemistry and material sciences. DNA, RNA and their derivatives serve as recognition elements in biosensors, building blocks for nano-structures, and as pharmaceuticals. The long-term goals of the research outlined here are to improve our understanding of molecular recognition in RNA-small molecule complexes and to develop a rational basis for the development of functional building blocks that can be used as part of biosensors or for in vivo imaging of RNA in microscopy. Toward this end a detailed understanding of how ligand binding and catalysis in nucleic acids are related to sequence and structure must be obtained. In order to acquire this information, my research program focuses on structural and functional studies of nucleic acids with an emphasis on RNA. Model systems we will use for these studies include RNA aptamers to organic dyes such as fluorescein or sulforhodamine B. In addition we will select aptamers in my laboratory that recognize the non-fluorescent fluorescein-diacetate (FDA) and pursue the selection of ribozymes that can catalyze its conversion to fluorescein. We will determine the three-dimensional structures of these molecules by high resolution NMR spectroscopy. Isothermal titration calorimetry will be used to study binding thermodynamics. The kinetics of binding and folding will be studied using optical spectroscopy methods and surface plasmon resonance techniques. The experimental work will be supplemented by theoretical studies of both structure and interactions using molecular dynamics and ab initio calculations. Synthetic chemistry approaches will be employed for the production of ligand derivatives where needed. The combined application of these research objectives will provide an improved understanding of how RNA aptamers recognize and change the molecular properties of their ligands. This basic knowledge will then be employed to develop building blocks for nucleic acid based sensors that can be combined with existing recognition modules (DNA and RNA aptamers), ribozymes, or nanostructures.**