Nano-FRET: Analysis of Nucleoprotein Complexes

Nano-FRET：核蛋白复合物的分析

• 批准号: 6596967
• 负责人: GEOFFREY F. STROUSE
• 金额: $ 12.42万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA BARBARA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2003-11-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6596967
• 关键词: biomaterial development /preparation; biotechnology; chemical kinetics; combinatorial chemistry; conformation; dyes; endonuclease; fluorescence resonance energy transfer; high throughput technology; image enhancement; intermolecular interaction; methyltransferase; molecular biology; molecular dynamics; nanotechnology; nucleic acid structure; nucleoproteins; protein binding; thermodynamics

DESCRIPTION (provided by applicant): We propose to develop and analyze water-soluble nanomaterials as reporters in FRET optical bioassay technologies to probe conformational dynamics for nucleo-protein interactions. The ability to interrogate bimolecular interactions in the context of binding and dissociation kinetics and thermodynamics is essential to obtain a molecular understanding of the interplay between structure and function. Using variations on existing organic dye based bioassays, we propose to develop the chemistry, probe the properties, and demonstrate the applicability of nanomaterials in the bio-nano conjugates, while providing a foundation for interfacing more complex and diverse protein-DNA-systems. The selected protein "systems" include the well studied EcoRV endonuciease (R.ECORV), the EcoRi DNA methyltransferase (M.ECORI), and the Papl regulon that controls the expression of pill-related proteins in pathogenic E.coli bacteria. Each of the three systems involves dramatic conformation changes, ranging from base flipping (M.ECORi) and localized DNA bending (M.EcoRi and R.EcoRV) to large scale DNA looping (Papl regulon). The use of nanoscale materials in FRET based assays results in substantial enhanced photostabiiity in comparison to organic dyes (20x), and enhanced long range energy transfer (> 10 nm) for FRET applications. The nanomaterial bimolecular interface, which involves components of similar size scales, remains poorly understood and any application in bioassay technologies requires analysis of the biocompatibility of these materials. The development of long-range FRET assays based on nanomaterial bio-conjugates will allow a significant extension of the current technology into multiple protein binding assays for analysis of conformational changes in the nucleic acid structures. This information is needed for a basic understanding of protein ligand interactions, for the intelligent redesign of such systems, and for the development of modulators of such interactions (e.g., novel drugs).

描述（由申请人提供）：我们建议开发和分析水溶性纳米材料作为 FRET 光学生物测定技术中的报告分子，以探测核-蛋白质相互作用的构象动力学。在结合和解离动力学和热力学背景下探究双分子相互作用的能力对于获得结构和功能之间相互作用的分子理解至关重要。利用现有基于有机染料的生物测定的变化，我们建议开发化学方法，探索其特性，并证明纳米材料在生物纳米缀合物中的适用性，同时为连接更复杂和多样化的蛋白质-DNA 系统提供基础。选定的蛋白质“系统”包括经过充分研究的EcoRV核酸内切酶（R.ECORV）、EcoRi DNA甲基转移酶（M.ECORI）以及控制病原性大肠杆菌中pill相关蛋白质表达的Papl调节子。这三个系统均涉及显着的构象变化，范围从碱基翻转 (M.ECORi) 和局部 DNA 弯曲（M.EcoRi 和 R.EcoRV）到大规模 DNA 成环（Papl 调节子）。与有机染料 (20x) 相比，在基于 FRET 的测定中使用纳米级材料可显着增强光稳定性，并增强 FRET 应用的长距离能量转移 (> 10 nm)。纳米材料双分子界面涉及相似尺寸尺度的成分，但人们对它仍然知之甚少，生物测定技术中的任何应用都需要分析这些材料的生物相容性。基于纳米材料生物共轭物的长程 FRET 测定的发展将使当前技术显着扩展到多种蛋白质结合测定，以分析核酸结构的构象变化。这些信息对于对蛋白质配体相互作用的基本了解、此类系统的智能重新设计以及此类相互作用的调节剂（例如新药）的开发是必需的。