Effects of Vicinal Surface Chemistry on DNA Base-Pairing using Single-Molecule RE

描述（由申请人提供）：这项工作的总体目标是开发能够提供有关附近表面的化学功能影响核酸碱基配对和杂交方式的机制信息的方法，并使用这些方法来了解如何使用表面化学来控制DNA自组装。基于dna的生物医学纳米技术直接依赖于沃森-克里克碱基配对来实现分子识别，能够指导各种诊断和靶向应用的自组装。虽然碱基配对在溶液阶段相对来说已经被很好地理解，但在许多DNA纳米技术中，碱基配对/杂交是在一个或多个界面/表面附近发生的，由于特定的邻近表面化学，这些界面/表面可能会促进与核酸的竞争性非特异性相互作用。更好地了解杂交的表面效应将最终导致广泛的基于dna的技术的改进。特别是，它将使设计和制备改进的表面改性策略成为可能。我们建议开发先进的单分子跟踪方法，使用单分子分辨率的全反射荧光显微镜（TIRFM），其中共振能量转移（RET）用于同时获得构象信息。这种方法可以识别构象和动态界面事件（如吸附/解吸、界面迁移率、构象波动、杂交）之间的直接分子间的相关性，提供了对邻近表面化学如何影响特异性和非特异性DNA相互作用的机制理解。这些方法将用于研究DNA动力学和模型表面上的碱基配对，这些模型表面代表了竞争非共价相互作用的最重要例子。这个为期两年的计划侧重于了解(1)可以自杂交形成茎环二级结构的寡核苷酸的界面动力学行为，以及(2)互补的寡核苷酸对。

DESCRIPTION (provided by applicant): The overall objective of this work is to develop methods that can provide mechanistic information about the ways in which the chemical functionality of a nearby surface affects nucleic acid base- pairing and hybridization, and to use these methods to understand how surface chemistry can be used to control DNA self-assembly. DNA-based biomedical nanotechnologies rely directly on Watson-Crick base-pairing to achieve molecular recognition capable of directing self-assembly for a variety of diagnostic and targeting applications. While base-pairing in the solution phase is relatively well-understood, in many DNA nanotechnologies, base-pairing/hybridization is intended to occur in the vicinity of one or more interfaces/surfaces that may promote competitive non- specific interactions with nucleic acids due to the particular vicinal surface chemistry. A better understanding of surface effects on hybridization will ultimately lead to improvements in a wide range of DNA-based technologies. In particular, it will enable the design and preparation of improved surface-modification strategies. We propose to develop advanced single-molecule tracking methods using total internal reflection fluorescence microscopy (TIRFM) with single-molecule resolution, where resonance energy transfer (RET) is used to obtain simultaneous conformational information. This approach can identify direct molecule-by-molecule correlations between conformation and dynamic interfacial events (e.g. adsorption/desorption, interfacial mobility, conformational fluctuations, hybridization), providing mechanistic understanding of how vicinal surface chemistry affects both specific and non-specific DNA interactions. These methods will be used to study DNA dynamics and base- pairing on model surfaces that represent the most important examples of competing non-covalent interactions. This two-year plan focuses on understanding the interfacial dynamic behavior of (1) oligonucleotides that can self-hybridize to form stem-loop secondary structures, and (2) complementary oligonucleotide pairs. PUBLIC HEALTH RELEVANCE: DNA hybridization, the specific recognition of a DNA sequence by its complementary sequence, is the fundamental enabling phenomenon underlying a wide range of biomedical nanotechnologies, including molecular diagnostics, gene delivery, DNA sequencing, and many others. In these technologies, DNA molecules are required to exhibit specific recognition in the presence of foreign surfaces and materials that can interfere due to competitive chemical interactions. This work involves the development of novel single-molecule microscopy methods that will lead to a detailed understanding of these competitive interactions, permitting the design of improved materials and technologies.