Engineering a packaging nanomotor for delivery of RNA and other molecules

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成分来执行特定任务。本应用程序的目标是设计噬菌体T4 DNA包装马达来转运双链RNA和RNA:DNA杂交分子。改变马达易位特异性的能力将大大提高其作为递送纳米机器的潜力；调整现有的机制，将感兴趣的治疗分子输送到细胞中。最近确定了T4噬菌体包装马达的x射线和低温电镜结构。马达蛋白gp17由几个部分组成；atp酶（发动机），转位酶（车轮）和精氨酸手指（火花塞）。易位特异性是由DNA结合槽决定的，它与马达的其余部分分离得很好。凹槽的形状和大小符合双链DNA分子，并且沿凹槽排列的带正电荷残基的分布遵循双螺旋的螺距；与主磷酸盐相互作用。这些特点表明，包装电机是可以修改设计新的电机与改变的特异性。gp17易位槽将通过易出错和重叠延伸PCR诱变，每个槽引入约3个突变。数百种（如果不是数千种的话）变异将被筛选，以获得高通量形式转运RNA的能力。他标记的突变体将在大肠杆菌中过表达，并使用96孔Ni-Sepharose板通过亲和层析纯化。由纯化的前体、包装马达、ATP和核酸组成的已建立定义的体外包装试验将用于快速筛选RNA易位突变体。该试验将包装机组装在溶液中，通过琼脂糖凝胶电泳后核酸酶保护RNA的存在来评估前体的易位。该系统的简单性使其非常适合高通量格式，允许测试数百种突变蛋白和几种不同的核酸。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atp酶、效率和底物特异性。突变易位槽的结构建模将由已经确定的马达蛋白及其结构域的晶体结构指导。单分子研究将使用光学镊子来分析运动动力学；滑动或停顿的力度、威力和速度以及频率；识别DNA和RNA易位之间微妙的机制差异。总之，这些发现将为进一步工程马达建立设计原则，以提高RNA易位的特异性和/或效率。该应用产生的原理证明数据将为噬菌体T4封装电机作为多功能纳米机器的应用提供更广泛的基础。

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.