Engineering a packaging nanomotor for delivery of RNA and other molecules

设计用于递送 RNA 和其他分子的包装纳米马达

• 批准号: 7904763
• 负责人: Venigalla B. Rao
• 金额: $ 16.18万
• 依托单位: CATHOLIC UNIVERSITY OF AMERICA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7904763
• 关键词: ATP phosphohydrolase; Affinity Chromatography; Agar Gel Electrophoresis; Arginine; Bacteriophage T4; Biological; Biological Assay; Cells; Charge; Computer Systems Development; DNA; DNA Groove Binding; DNA Packaging; Data; Devices; Double-Stranded RNA; Engineering; Fingers; Foundations; Frequencies; Goals; Hybrids; In Vitro; Infection; Measurement; Motor; Mutagenesis; Mutation; Nucleic Acids; Peptides; Pharmacotherapy; Proteins; RNA; Rest; Roentgen Rays; Scheme; Sepharose; Shapes; Solutions; Specificity; Structural Biochemistry; Structural Models; Structure; Substrate Specificity; System; Testing; Therapeutic; Variant; Vertebral column; Viral Packaging; design; ds-DNA; high throughput screening; improved; inorganic phosphate; interest; laser tweezer; mutant; nanobiotechnology; nanomachine; novel; nuclease; overexpression; public health relevance; single molecule; translocase

DESCRIPTION (provided by applicant): One of the biggest challenges in nanobiotechnology is to engineer a biological component to carry out a specific task. The goal of this application is to engineer the bacteriophage T4 DNA packaging motor to translocate double stranded RNA and RNA:DNA hybrid molecules. The ability to alter the translocation specificity of the motor will greatly enhance its potential as a delivery nanomachine; adapting the existing machinery to transport a therapeutic molecule of interest into cells. The X-ray and cryo-EM structures of the phage T4 packaging motor have been recently determined. The motor protein, gp17, consists of several parts; ATPase (the engine), translocase (the wheel) and arginine finger (the spark plug). The translocation specificity is determined by a DNA binding groove that is well- separated from the rest of the motor. The shape and size of the groove fit a double stranded DNA molecule, and the distribution of positively charged residues lining the groove follows the pitch of the double helix; interacting with the backbone phosphates. These features suggest that the packaging motor is amendable to design novel motors with altered specificity. The translocation groove of gp17 will be mutagenized by error-prone and overlap extension PCR to introduce ~3 mutations per groove. Hundreds, if not thousands, of variants will be screened for the ability to translocate RNA in a high-throughput format. The his-tagged mutants will be overexpressed in E.coli and purified by affinity chromatography using 96-well Ni-Sepharose plates. An established defined in vitro packaging assay consisting of purified proheads, the packaging motor, ATP, and nucleic acid will be used to rapidly screen for RNA translocation mutants. This assay assembles the packaging machine in solution, and translocation into proheads is assessed by the presence of nuclease-protected RNA following agarose gel electrophoresis. The simplicity of this system lends itself well to the high-throughput format, allowing hundreds of mutant proteins and several different nucleic acids to be tested. The gp17 RNA translocation mutants identified in the high-throughput screen will be purified and analyzed for packaging ATPase, efficiency, and substrate specificity. Structural modeling of the mutant translocation grooves will be guided by the already determined crystal structures of the motor protein and its domains. Single molecule studies will be performed using optical tweezers to analyze motor dynamics; force, power, and rate, as well as frequency of slips or pauses; identifying subtle mechanistic differences between DNA and RNA translocation. Together, these findings will establish the design principles for further engineering of the motor to improve specificity and/or efficiency of RNA translocation. The proof of principle data generated from this application will provide a broader foundation for the application of the phage T4 packaging motor as a versatile nanomachine. PUBLIC HEALTH RELEVANCE: A major challenge in nanobiotechnology is the ability to engineer new or improved biological devices for a specific purpose. The current application aims to use an established scheme of mutagenesis, biochemistry, structural analysis, and biophysical measurements and apply it to the design of a novel nanomachine from a well-defined viral packaging motor. This project will provide proof of principle data for the development of this system into a versatile delivery vehicle, delivering DNA, RNA, peptide, or drug therapy into the cell.

描述（由申请人提供）：纳米生物技术的最大挑战之一是设计一种生物组件来执行特定的任务。该应用的目的是设计噬菌体T4 DNA包装电机以易位双链RNA和RNA：DNA混合分子。改变电动机的易位特异性的能力将大大提高其作为输送纳米机械的潜力。调整现有机械以将兴趣的治疗分子传输到细胞中。最近已经确定了噬菌体T4包装电机的X射线和冷冻EM结构。运动蛋白GP17由几个部分组成。 ATPase（发动机），易位酶（车轮）和精氨酸手指（火花塞）。易位特异性由DNA结合凹槽确定，该凹槽与电动机的其余部分分开。凹槽的形状和尺寸适合双链DNA分子，凹槽内衬有正电荷的残基的分布遵循双螺旋的螺距；与主链磷酸盐相互作用。这些功能表明，包装电动机是可以修改的，以设计具有改变特异性的新型电动机。 GP17的易位凹槽将通过容易出错的和重叠的延伸PCR进行诱变，以引入每个凹槽的3个突变。将筛选数百种（即使不是数千个）的变体，以使其具有高通量格式的RNA转移RNA。 His标记的突变体将在大肠杆菌中过表达，并使用96孔Ni-Sepharose板通过亲和色谱法纯化。已建立的定义的体外包装测定法，该测定法包括纯化的Prohead，包装电机，ATP和核酸将用于快速筛选RNA易位突变体。该测定法在溶液中组装了包装机，并通过琼脂糖凝胶电泳后的受核酸酶保护的RNA的存在来评估易位。该系统的简单性非常适合高通量格式，可以测试数百种突变蛋白和几种不同的核酸。在高通量屏幕中鉴定出的GP17 RNA易位突变体将被纯化并分析以包装ATPase，效率和底物特异性。突变体易位凹槽的结构建模将由运动蛋白及其结构域的已经确定的晶体结构指导。单分子研究将使用光学镊子进行分析运动动力学。力，功率和速率，以及滑动或暂停的频率；确定DNA和RNA易位之间的细微机械差异。这些发现将共同建立用于进一步工程电动机的设计原理，以提高RNA易位的特异性和/或效率。从本应用程序生成的原理数据证明将为将噬菌体T4包装电机作为多功能纳米机器的应用提供更广泛的基础。 公共卫生相关性：纳米生物技术的主要挑战是为特定目的设计新的或改进的生物设备的能力。当前的应用旨在使用既定的诱变，生物化学，结构分析和生物物理测量方案，并将其应用于从明确定义的病毒包装运动中的新型纳米机器的设计中。该项目将提供原理数据的证明，以将该系统开发为多功能输送车辆，将DNA，RNA，肽或药物治疗输送到细胞中。