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: 7712849
• 负责人: Venigalla B. Rao
• 金额: $ 18.66万
• 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 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7712849
• 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）：纳米生物技术中最大的挑战之一是设计生物成分来执行特定任务。该应用的目标是设计噬菌体 T4 DNA 包装马达，以移位双链 RNA 和 RNA:DNA 杂合分子。改变马达易位特异性的能力将大大增强其作为递送纳米机器的潜力；调整现有的机器将感兴趣的治疗分子输送到细胞中。噬菌体 T4 包装马达的 X 射线和冷冻电镜结构最近已确定。运动蛋白 gp17 由几个部分组成； ATP酶（发动机）、转位酶（车轮）和精氨酸指（火花塞）。易位特异性由与电机其余部分充分分离的 DNA 结合槽决定。凹槽的形状和大小适合双链DNA分子，凹槽内带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。这些特征表明封装电机可以修改以设计具有改变的特异性的新型电机。 gp17 的易位槽将通过易错和重叠延伸 PCR 进行诱变，以在每个槽中引入约 3 个突变。将筛选数百个（如果不是数千个）变体，以确定其以高通量形式易位 RNA 的能力。带组氨酸标签的突变体将在大肠杆菌中过表达，并使用 96 孔 Ni-Sepharose 板通过亲和层析进行纯化。由纯化的前头、包装马达、ATP 和核酸组成的已确定的体外包装测定将用于快速筛选 RNA 易位突变体。该测定在溶液中组装包装机，并通过琼脂糖凝胶电泳后核酸酶保护的 RNA 的存在来评估易位到前头中。该系统的简单性非常适合高通量形式，允许测试数百种突变蛋白和几种不同的核酸。高通量筛选中鉴定出的 gp17 RNA 易位突变体将被纯化并分析包装 ATP 酶、效率和底物特异性。突变易位槽的结构建模将由已确定的马达蛋白及其结构域的晶体结构指导。将使用光镊进行单分子研究以分析运动动力学；力、功率和速率，以及滑倒或停顿的频率；识别 DNA 和 RNA 易位之间细微的机制差异。总之，这些发现将为进一步工程设计电机建立设计原则，以提高 RNA 易位的特异性和/或效率。该应用生成的原理数据证明将为噬菌体 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。 公共健康相关性：纳米生物技术的一个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。当前的应用程序旨在使用诱变、生物化学、结构分析和生物物理测量的既定方案，并将其应用于从明确的病毒包装马达设计新型纳米机器。该项目将为将该系统开发为多功能输送工具提供原理数据证明，将 DNA、RNA、肽或药物治疗输送到细胞中。