Real-time Single-Molecule Analysis for Transient Molec. Complex Characterization

描述（由申请人提供）：单分子荧光共振能量转移（smFRET）已经迅速发展到回答有关生物机制的基本问题，提供分子机器工作的详细视图。Nesher Technologies, Inc. （NTI）成立的目的是将一种创新的定量、超灵敏和特异性单分子生物检测技术商业化，该技术具有精湛的多路复用潜力和简化的样品处理程序。它是由加州大学洛杉矶分校的单分子生物物理实验室首创的，该实验室由Shimon Weiss教授领导。该技术基于荧光探针标记的单分子的交变激光激发（ALEX）。除了用于诊断之外，ALEX最近还被用于研究分子过程的动力学，例如使用smFRET进行转录起始和延伸。在双色(2c)-ALEX中，利用两束交变激光研究分子相互作用（通过探针化学比S和/或FRET效率E）和分子内距离分析构象和机理（通过E），分子被排序为S和E的二维直方图。在3c-ALEX中，分子被排序为三维S和E直方图，大大扩展了2c- ALEX的能力。ALEX可以用扩散分子进行，从而分析单个分子的平衡行为，也可以用毫秒级ALEX动态成像（ALEX- di）来固定分子。采用全内反射光学显微镜的ALEX- di方法比其姐妹方法共聚焦微秒尺度ALEX光谱学具有优势，可以建立非平衡时间轨迹来跟踪单个分子和复合物的动力学。还可以获得复杂组成和功能的详细随机分子信息。长时间监测单个分子的能力为从毫秒到分钟的动态信息增加了一个全新的维度。高阶FRET方案也可用于探测瞬态多组分相互作用或大分子复合物中不同构象变化之间的时空关系。NTI的长期目标是开发一种完全集成的基于alex5色的仪器，适用于瞬态分子复合物的表征。监测分子相互作用和构象动力学的高分辨率能力也将极大地帮助结构指导的合理药物设计努力。对于第一阶段，我们建议扩展2色alexi - di以实现3色和4色功能。具体目标是：1.；单分子全内反射光学显微镜四色交替激光激发动态成像仪（4c- alexi - di）的研制2. 荧光团标记细菌转录起始复合物组分的制备。3. 使用4c- alexi - di在3色和4色模式下检测和表征固定化RNA聚合酶(RNAP)-启动子初始转录复合物（RPitc）中的dna裂解，以及启动子逃逸。公共卫生相关性：提出的基于多色交替激光激发（ALEX）的单分子方法用于识别和表征瞬态分子复合物，将极大地提高收集分子信息的能力，这反过来将增强我们理解微妙突变和/或环境因素在疾病发生和进展中的复杂影响的能力。ALEX技术在基础研究和药物开发应用中非常有用，可以补充生物分子及其复合物的结构分析，特别是对于由于过度异质性、有限稳定性、大尺寸、存在柔性结构域和/或瞬态性而无法通过传统结构生物学方法解决的物种。例如，拟议的仪器将能够促进细菌转录过程的详细机制研究，从而允许快速开发下一代抗生素（例如，在转录机制的新位点上抑制细菌转录），这对于有效控制全球新出现的多重耐药细菌菌株至关重要，例如耐多药结核病（MDR TB）。广泛耐药结核病（XDR TB）、耐甲氧西林金黄色葡萄球菌（MRSA）和耐万古霉素肠球菌（VRE）。

DESCRIPTION (provided by applicant): Single-molecule fluorescence resonance energy transfer (smFRET) has rapidly developed to answer fundamental questions about biological mechanisms, providing detailed views of molecular machines at work. Nesher Technologies, Inc. (NTI) was established to commercialize an innovative quantitative, ultrasensitive and -specific single-molecule biodetection technology with exquisite multiplexing potential and simplified sample handling procedures. It was pioneered at the UCLA Single Molecule Biophysics Lab, which is headed by Prof. Shimon Weiss. The technology is based on alternating-laser excitation (ALEX) of single molecules labeled with fluorescent probes. In addition to its suitability for diagnostics, ALEX has been employed recently to study the dynamics of molecular processes such as transcription initiation and elongation using smFRET. In two-color (2c)-ALEX, employing two alternating lasers to study molecular interactions (through probe stoichiometry ratio S and/or FRET efficiency E) and intramolecular distances for analysis of conformation and mechanism (through E), molecules are sorted in a two-dimensional histogram of S and E. In 3c-ALEX, molecules are sorted in three-dimensional S and E histograms, substantially extending the capabilities of 2c- ALEX. ALEX can be performed with diffusing molecules, enabling analysis of equilibrium behaviors of single molecules, or with immobilized molecules using millisecond-scale ALEX dynamic imaging (ALEX-DI). The ALEX-DI methodology, employing total-internal-reflection optical microscopy, has the advantage over its sister methodology, confocal microsecond-scale ALEX spectroscopy, that non-equilibrium time trajectories can be established to follow kinetics of individual molecules and complexes. Detailed stochastic molecule information on complex composition and function can be obtained as well. The ability to monitor individual molecules for long stretches of time adds a whole new dimension with dynamic information ranging from milliseconds to minutes. Higher-order FRET schemes can also be applied to probe transient multi-component interactions or spatiotemporal relationships between different conformational changes in large molecular complexes. NTI's long-term goal is development of a fully integrated 5-color ALEX-based instrument suitable for transient molecular complex characterization. The high-resolution power to monitor molecular interactions and conformational dynamics will also greatly aid structure-guided rational drug design endeavors. For Phase I, we propose to extend 2-color ALEX-DI to enable 3-color and 4-color functionality. Specific aims are: 1. Construction of an instrument for single-molecule total-internal-reflection optical microscopy with 4-color alternating-laser-excitation and dynamic imaging (4c-ALEX-DI). 2. Preparation of fluorophore-labeled bacterial transcription initiation complex components. 3. Use of 4c-ALEX-DI in 3-color and 4-color mode to detect and characterize DNA-scrunching in immobilized RNA polymerase (RNAP)-promoter initial transcribing complexes (RPitc), as well as promoter escape. PUBLIC HEALTH RELEVANCE: The proposed multi-color alternating-laser excitation (ALEX)-based single-molecule method for identifying and characterizing transient molecular complexes will vastly improve the ability to gather molecular information, which in turn will enhance our ability to understand the complex effects that subtle mutations and/or environmental factors have in the development and progression of diseases. The ALEX technology, highly useful for basic research as well as drug development applications, can complement structural analysis of biomolecules and their complexes, especially for species intractable by conventional structural biology methods due to excessive heterogeneity, limited stability, large size, presence of flexible domains, and/or transient nature. As an example, the proposed instrumentation will be able to facilitate detailed mechanistic studies of the bacterial transcription process, allowing rapid development of next-generation antibiotics (e.g. inhibitors of bacterial transcription at new sites of the transcription machinery), which is of critical importance for effective control of globally emerging multi-drug resistant bacterial strains, such as multidrug-resistant tuberculosis (MDR TB), extensively drug-resistant tuberculosis (XDR TB), methicillin-resistant Staphylococcus aureus (MRSA),and vancomycin-resistant enterococci (VRE).