Directed Evolution of Nucleic Acid Enzymes

描述(申请人提供)：拟议的研究涉及具有理想功能特性的核酸酶的体外进化。这项研究的目标有两个：第一，开发将在生物合成和生物医学中应用的新型RNA和DNA酶；第二，利用体外进化作为在分子水平上理解达尔文进化过程的一种手段。这些目标将通过五个研究项目来实现，这些项目扩展了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。其中两个项目旨在设计新的扩增方法，这些方法将在分子诊断和生物传感中应用。其中一种是DNA催化的连接酶链式反应，可以用来扩增特定的DNA序列，同时避免了目前蛋白质催化方法的一些限制。另一种是依赖配体的指数放大，作为检测特定小分子或蛋白质的一种手段。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方式催化交叉Aldol反应的能力。剩下的两个项目将探索功能大分子定向进化的新方法。其中之一涉及一种新的基于微流控的系统，用于核酶的持续体外进化。它将被用来解决进化群体如何摆脱局部最优的问题，以及在进化优化过程中突变频率与选择压力之间的关系。最后一个项目涉及纳米结构生物材料。它的目标是通过将功能核酸锚定在由单链DNA折叠成正八面体形状的刚性大分子支架上，为功能核酸提供精确的三维定位。DNA八面体的12个支柱中的每一个都有不同的序列，这些序列将被用来在特定位置定位功能DNA。然后，这些修饰的纳米结构将受到体外进化的影响，根据它们的形式和附加DNA的功能进行选择。

DESCRIPTION (provided by applicant): 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 molecular level. 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 in molecular diagnostics and biosensing. One of these involves a DNA-catalyzed version of the ligase chain reaction, which could be used to amplify specific DNA sequences, while avoiding some of the limitations of 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, especially with the ability to 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 acids by 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.