Combined ultrahigh-resolution optical tweezers and single-molecule fluorescence

超高分辨率光镊与单分子荧光相结合

• 批准号: 7943010
• 负责人: Yann R. Chemla
• 金额: $ 24.59万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7943010
• 关键词: Address; Benchmarking; Biological Models; Biological Process; Collaborations; Color; Complex; Confocal Microscopy; Cruciform DNA; Cysteine; DNA; DNA biosynthesis; DNA-Directed RNA Polymerase; Defect; Detection; Development; Device or Instrument Development; E coli rep helicase; Energy Transfer; Engineering; Fluorescence; Fluorescence Resonance Energy Transfer; Freedom; Generations; Genetic Transcription; Goals; Housing; Hybrids; Illinois; Kinesin; Kinetics; Label; Laboratories; Malignant Neoplasms; Measurement; Measures; Medical; Metabolism; Methodology; Molecular; Molecular Conformation; Molecular Machines; Molecular Motors; Monitor; Motion; Motor; Myosin ATPase; Names; Nucleic Acids; Protein Dynamics; Proteins; Research Personnel; Resolution; Spectrum Analysis; System; Techniques; Technology; Testing; Time; Universities; Work; base; design; helicase; human disease; instrument; instrumentation; interest; laser tweezer; mutant; nanoscale; next generation; novel; novel strategies; optical traps; protein complex; public health relevance; recombinational repair; single molecule; tool; ultra high resolution

DESCRIPTION (provided by applicant): Single molecule techniques have developed into a powerful tool to study the molecular machines involved in many fundamental biological processes. Techniques such as fluorescence localization, F"rster resonance energy transfer (FRET), and optical tweezers have been instrumental in deciphering the mechanism of molecular motors such as myosin and kinesin, DNA and RNA polymerases, and helicases, to name just a few examples. Recently, the development of ultrahigh-resolution optical tweezers has made possible, for the first time, the direct observation of molecular motion on the scale of 1 basepair of DNA (3.4¿). Despite such advances, single molecule techniques have had important limitations. Although the conformation changes involved in molecular machines are inherently three-dimensional, such techniques typically project all motion onto a single axis and thus cannot capture the full complexity of molecular motion. Furthermore, these techniques have largely been limited to simple systems involving very few components examined in isolation, whereas, in the cellular context, molecular machines consist of highly coordinated multi-component protein assemblies. To address these limitations, we propose to 1) develop the new generation of single molecule instrumentation combining multi-color fluorescence detection and ultrahigh-resolution optical tweezers. Although instruments merging fluorescence and optical traps have been developed previously, achieving basepair resolution remains a grand challenge that will require a new approach. These capabilities nevertheless will be essential to understand the molecular complexes involved in DNA metabolism-transcription, replication, recombination, and repair-that have great biomedical significance. The hybrid instrument we propose will have the ability to measure multiple observables simultaneously, such as internal protein dynamics by FRET or the assembly kinetics of protein complexes by fluorescence localization, combined with detection of motor displacement at basepair resolution by optical tweezers. As a demonstration of this technique, we will 2) monitor translocation and duplex unwinding by E. coli Rep helicase at basepair resolution, simultaneously with conformational changes by FRET and oligomeric state by fluorescence detection. This proposed work involves the collaboration of experts in ultrahigh-resolution optical tweezers (Y. Chemla, PI) and single-molecule fluorescence (T. Ha, co-PI) at the University of Illinois, Urbana-Champaign. Public Health Relevance Statement: We are proposing to develop an instrument that will combine two powerful cutting-edge technologies: single-molecule fluorescence and ultrahigh-resolution optical trapping. Our goal is to study the dynamics of proteins and protein complexes involved in DNA replication, transcription, recombination, and repair at ¿ngstrom level resolution. This proposed technique has the potential to reveal the detailed mechanism of these molecular machines, a problem of great medical interest as defects in their activity have been implicated in a number of human diseases, specifically cancer.

描述（由适用提供）：单分子技术已发展为一种强大的工具，用于研究参与许多基本生物过程的分子机器。诸如荧光定位，f“ rster共振能量转移（FRET）和光学镊子）的技术在解释了分子电机（例如肌球蛋白和驱动蛋白和驱动蛋白，DNA和RNA聚合酶）等分子电机的机制方面，仅需几个示例，就可以进行了几个型号。在1个DNA的基础上（3.4校）的运动，尽管这种分子技术具有重要的局限性。而在细胞环境中，分子机器由高度协调的多组分蛋白质组件组成。 为了解决这些局限性，我们建议1）开发新一代的单分子仪器，结合了多色荧光检测和超高分辨率光学镊子。尽管仪器以前已经开发了荧光和光学陷阱，但实现基地分辨率仍然是一个巨大的挑战，需要一种新的方法。然而，这些能力对于了解与DNA代谢转录，复制，重组和修复有关的分子复合物至关重要 - 具有极大的生物医学意义。我们提出的杂种仪器将具有简单测量多个观察结果的能力，例如通过荧光定位的fret内部蛋白质动力学或蛋白质复合物的组装动力学，并通过光学镊子在碱基分辨率下检测到运动型替代。为了证明这项技术，我们将在基epair分辨率下监测大肠杆菌rep旋转酶放松的转运和双链体，仅通过FRET和寡聚状态的构象变化，通过荧光检测。这项提出的工作涉及在Urbana-Champaign伊利诺伊大学的超高分辨率光学镊子（Y. Chemla，PI）和单分子荧光（T. Ha，Co-Pi）的合作。 公共卫生相关性声明：我们建议开发一种将结合两种强大的尖端技术的工具：单分子荧光和超高分辨率光学诱捕。我们的目标是研究与NGSTROM水平分辨率下有关DNA复制，转录，重组和修复的蛋白质和蛋白质复合物的动力学。这项提出的技术有可能揭示这些分子机器的详细机制，这是医学兴趣的问题，因为在许多人类疾病（特别是癌症）中隐含了其活性缺陷。