Engineering a packaging nanomotor for delivery of RNA and other molecules

描述(申请人提供)：纳米生物技术中最大的挑战之一是设计一个生物组件来执行特定的任务。这项应用的目标是设计噬菌体T4 DNA包装马达，以转移双链RNA和RNA：DNA杂化分子。改变马达易位特异性的能力将极大地增强其作为输送纳米机器的潜力；调整现有的机制，将感兴趣的治疗分子运送到细胞中。最近测定了噬菌体T4包装马达的X射线和低温电子显微镜结构。马达蛋白gp17由几个部分组成：ATPase(发动机)、转位酶(车轮)和精氨酸指(火花塞)。易位的特异性由DNA结合槽决定，该槽与发动机的其余部分很好地分开。凹槽的形状和大小符合双链DNA分子，凹槽内带正电的残基的分布遵循双螺旋的间距；与主链磷酸盐相互作用。这些特点表明，包装电机是可以修改的，以设计新的电机与改变的专属性。Gp17的易位槽用易错和重叠延伸的聚合酶链式反应进行突变，每个槽位引入~3个突变。将对数百个(如果不是数千个)的变种进行筛选，以确定是否有能力以高通量的形式转移RNA。His标记的突变体将在大肠杆菌中高效表达，并通过96孔Ni-Sepharose板亲和层析进行纯化。一种已建立的明确的体外包装试验将用于快速筛选RNA易位突变体，该方法由纯化的丙二醛、包装马达、ATP和核酸组成。这种方法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的存在来评估转位到探针中的存在。该系统的简单性非常适合高通量格式，允许测试数百种突变蛋白质和几种不同的核酸。在高通量筛选中鉴定的gp17 RNA易位突变体将被纯化并分析包装ATPase、效率和底物特异性。突变易位沟槽的结构建模将以已经确定的马达蛋白及其结构域的晶体结构为指导。单分子研究将使用光学镊子来分析运动动力学；力、功率和速率，以及滑倒或停顿的频率；识别DNA和RNA易位之间的细微机制差异。总之，这些发现将为进一步设计马达以提高RNA转位的特异性和/或效率奠定基础。本申请产生的原理数据的证明将为噬菌体T4包装马达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：纳米生物技术的一个主要挑战是为特定目的设计新的或改进的生物装置的能力。目前的应用旨在使用已建立的诱变、生物化学、结构分析和生物物理测量方案，并将其应用于从定义明确的病毒包装马达设计新型纳米机器。该项目将为该系统发展成为一种多功能的递送载体，向细胞内递送DNA、RNA、多肽或药物治疗提供原理数据证明。

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.