Structure and Function of Aptamer based Biosensor Building Blocks

近年来，核酸适体作为工具在化学、生物化学和材料科学中得到越来越多的应用。DNA、RNA及其衍生物在生物传感器中用作识别和报告元件，用于纳米结构的构建块，以及用作药物。本文概述的研究计划的长期目标是提高对适体-小分子复合物中分子识别的理解，并为开发可用作生物传感器或其他核酸应用的一部分的功能性构建块奠定合理的基础。为此，必须详细了解核酸中配体结合与序列和结构的关系。为了获得这些信息，我的研究计划集中在核酸适体的结构和功能研究。用于这些研究的模型系统包括有机染料如Hoechst 33342或其衍生物的RNA和DNA适体。此外，我的研究小组已经通过体外选择确定了一种DNA适体，该适体识别强荧光分子噻唑橙子，其性质与其早期选择和表征的RNA等价物芒果适体相似。我们将通过高分辨率核磁共振光谱确定这些分子的三维结构。此外，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使用光谱方法和表面等离子体共振技术进行研究。差示迁移率光谱技术将用于探索适体与配体、离子和溶剂的相互作用。在需要时，将采用合成化学方法生产配体衍生物。这些研究工具的联合应用将提供这些适体如何识别和改变其配体的分子特性的更好的理解。通过这项研究计划获得的基本知识将为科学家和工程师提供工具，使用更合理和系统的方法开发基于核酸的传感和可视化应用的构建模块。

In recent years, nucleic acid aptamers were increasingly employed as tools in chemistry, biochemistry and material sciences. DNA, RNA and their derivatives serve as recognition and reporter elements in biosensors, building blocks for nanostructures, and as pharmaceuticals. The long-term goals of the research program outlined here are to improve understanding of molecular recognition in aptamer - 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 other nucleic acid applications. Toward this end a detailed understanding of how ligand binding in nucleic acids is 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 acid aptamers. Model systems used for these studies include RNA and DNA aptamers to organic dyes such as Hoechst 33342 or its derivatives. In addition, my research group has identified a DNA aptamer by in vitro selection that recognizes the strongly fluorogenic molecular thiazole Orange with properties similar to its earlier selected and characterized RNA equivalent, the Mango Aptamer. We will determine the three-dimensional structures of these molecules by high resolution NMR spectroscopy. In addition, 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. Differential Mobility Spectrometry techniques will be used to explore the interactions of the aptamers with ligands, ions, and solvents. Synthetic chemistry approaches will be employed for the production of ligand derivatives where needed. The combined application of these research tools will provide an improved understanding of how these aptamers recognize and change the molecular properties of their ligands. The basic knowledge gained by this research program will provide scientists and engineers with the tools to develop building blocks for nucleic acid based sensing and visualization applications using a more rational and systematic approach.