Directed Evolution of Nucleic Acid Enzymes

拟议的研究涉及具有理想功能的核酸酶的体外进化。 属性。这项研究的目标有两个：第一，开发新的RNA和DNA酶 在生物合成和生物医学方面有应用；第二，利用体外进化作为一种手段 在分子水平上理解达尔文进化的过程。这些目标将通过 五个研究项目，扩展了在前一个项目期间开展的工作，并寻求 为功能大分子的定向进化开发新的方法。 其中两个项目的目标是设计新的扩增方法，这些方法将在分子生物学中得到应用 诊断和生物传感。其中之一涉及DNA催化的连接链式反应， 它可以用来扩增特定的DNA序列，同时避免了电流的一些限制 蛋白质催化的方法。另一种是关于配体依赖的指数扩增作为一种手段 检测特定的小分子或蛋白质。第三个项目在生物合成方面有潜在的应用。 它涉及具有醛缩酶活性的核酸酶的体外进化，特别是具有 以底物特异性的方式催化交叉羟醛反应。剩下的两个项目将探索新的 功能大分子定向进化的方法。其中之一涉及一种新的 基于微流控技术的核酶体外连续进化系统。它将用于解决 进化中的种群如何摆脱深度局部最优的问题，以及它们之间的关系 进化优化过程中突变频率与选择压力之间的关系。这个 最后一个项目涉及纳米结构生物材料。它的目标是赋予精确的三维定位 功能核酸通过将它们锚定在一个刚性的大分子支架上，由一个单一的- 折叠成正八面体形状的搁浅的DNA。DNA八面体的12根支柱中的每一根 具有不同的序列，将用于在特定位置定位功能DNA。这些 然后，装饰的纳米结构将接受体外进化，根据它们的形式进行选择 以及附加的DNA的功能。

The proposed research concerns the in vitro evolution of nucleic acid enzymes with desirable functional properties. The goal of this research is two-fold: first, to develop novel RNA and DNA enzymes that will have applications in biosynthesis and biomedicine;and second, to employ in vitro evolution as a means to understand the processes of Darwinian evolution at the molecularlevel.These goals will be addressed by five research projects that extend work carried out during the previous project period and that seek to develop new approaches for the directed evolution of functional macromolecules. Two of the projects aim to devise novel amplification methods that will have applications inmolecular diagnostics and biosensing. One of these involves a DNA-catalyzedversion of the ligase chain reaction, which could be used to amplify specific DNA sequences, while avoiding some of the limitationsof current protein-catalyzed methods. The other concerns ligand-dependent exponential amplification as a means for detecting particular small molecules or proteins. A third project has potential applications in biosynthesis. It involves the in vitro evolution of nucleic acid enzymes with aldolase activity, especiallywith the abilityto catalyze crossed aldol reactions in a substrate-specific manner. The remaining two projects will explore new approaches for the directed evolution of functional macromolecules.One of these involves a novel microfluidic-based system for the continuous in vitro evolution of ribozymes. It will be used to address the question of how an evolving population escapes from a deep local fitness optimum, and the relationship between mutation frequency and selection pressure during the course of evolutionary optimization. The final project concerns nanostructured biomaterials.It aims to confer precise three-dimensional positioning to functional nucleic acidsby anchoring them to a rigid macromolecular scaffold, composed of a single- stranded DNA that folds into the shape of a regular octahedron. Each of the 12 struts of the DNA octahedron has a different sequence, which will be used to position functional DNAs at specific locations. These decorated nanostructures then will be subject to in vitro evolution, selecting on the basis of both their form and the function of the appended DNAs.