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关于附近表面的化学功能影响核酸碱基配对和杂交的方式的机械信息的方法，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靠沃森-克里克碱基配对来实现分子识别，能够指导自组装用于各种诊断和靶向应用。虽然溶液相的碱基配对相对容易理解，但在许多DNA纳米技术中，碱基配对/杂交旨在发生在一个或多个界面/表面的附近，由于特定的邻近表面化学，这些界面/表面可能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最终将导致广泛的基于DNA的技术的改进。特别是，它将使改进的表面改性策略的设计和准备成为可能。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内反射荧光显微镜(TIRFM)，其中使用共振能量转移(RET)来获得同时的构象信息。这种方法可以确定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)之间的直接逐个分子关联，从而提供对邻近表面化学如何影响特定和非特定DNA相互作用的机械理解。这些方法将被用于研究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的例子。这项为期两年的计划侧重于了解(1)能够自杂交形成茎环二级结构的寡核苷酸和(2)互补寡核苷酸对的界面动力学行为。 与公共卫生相关：DNA杂交，即通过互补序列对DNA序列的特定识别，是广泛的生物医学纳米技术的基本使能现象，包括分子诊断、基因传递、DNA测序等。在这些技术中，DNA分子需要在外来表面和材料存在的情况下表现出特定的识别能力，这些表面和材料可能会由于竞争性的化学相互作用而受到干扰。这项工作涉及开发新颖的单分子显微镜方法，这将导致对这些竞争相互作用的详细了解，从而允许设计改进的材料和技术。

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.