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

使用单分子 RE 邻位表面化学对 DNA 碱基配对的影响

• 批准号: 8280932
• 负责人: DANIEL SCHWARTZ
• 金额: $ 17.59万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8280932
• 关键词: Adsorption; Affect; Base Pairing; Behavior; Characteristics; Chemicals; Chemistry; Complex; Contrast Media; DNA; DNA Sequence; Development; Diagnostic; Diffusion; Electrostatics; Energy Transfer; Environment; Equilibrium; Event; Exhibits; Fluorescence Microscopy; Gene Delivery; Glass; Hydrocarbons; Hydrogen Bonding; Hydrophobic Interactions; Image; Lead; Methods; Microscopy; Modeling; Modification; Molecular; Molecular Conformation; Nanotechnology; Nanotubes; Nucleic Acids; Oligonucleotides; Phase; Polyethylene Glycols; Polymers; Population; Preparation; Process; Property; Quantum Dots; Resolution; Silicon; Solid; Solutions; Structure; Surface; Techniques; Technology; Time; Work; aqueous; base; capsule; design; improved; interfacial; member; molecular assembly/self assembly; molecular dynamics; molecular recognition; nanoparticle; novel; particle; research study; residence; self assembly; single molecule; stem

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.

描述（由申请人提供）：这项工作的总体目的是开发可以提供有关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，并使用这些方法来了解如何使用表面化学来控制DNA自组件。基于DNA的生物医学纳米技术直接依赖于Watson-Crick碱基配对来实现能够为各种诊断和靶向应用指导自组装的分子识别。虽然在溶液阶段的碱基配对相对充分理解，但在许多DNA纳米技术中，碱生成/杂交的目的是出现在一个或多个接口/表面的附近，这些界面/表面的附近可能会促进由于特定情况表面化学而引起的竞争性非特异性与核酸的竞争性相互作用。更好地了解表面对杂交的影响最终将导致广泛的基于DNA的技术的改善。特别是，它将能够设计和准备改进的表面修饰策略。我们建议使用具有单分子分辨率的总内反射荧光显微镜（TIRFM）开发高级单分子跟踪方法，其中使用共振能传递（RET）来获得同时构象信息。这种方法可以鉴定构象与动态界面事件之间的直接分子相关性（例如吸附/解吸，界面迁移率，构象波动，杂交），从而提供了对综合表面化学如何影响特定和非特异性DNA相互作用的机械理解。这些方法将用于研究模型表面上的DNA动力学和基础配对，这些模型表面代表了竞争非共价相互作用的最重要示例。这个为期两年的计划着重于理解（1）可以自脑型以形成茎环二级结构的（1）寡核苷酸的界面动态行为，以及（2）互补的寡核苷酸对。 公共卫生相关性：DNA杂交是DNA序列通过其互补序列对DNA序列的特定识别，是基本的促成现象，其基础是广泛的生物医学纳米技术，包括分子诊断，基因递送，DNA测序以及许多其他现象。在这些技术中，需要DNA分子在存在外国表面和材料的情况下表现出特定的识别，这些材料可能会因竞争性化学相互作用而干扰。这项工作涉及开发新型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，从而设计改进的材料和技术。