Engineering RNA switches that respond to dopamine/analog

RNA酶可以被设计成精确的变构核酶，或由特定效应器调节的“分子开关”。由RNA制成的分子开关有许多应用，从构建生物传感器到开发新型遗传开关。我们已经开始努力产生RNA分子开关，这些开关受到具有最高生物学和治疗重要性的化合物的调节。在这项研究中，我们试图产生对多巴胺和几种相关化合物做出反应的核酶，最终整合到体外和体内的诊断和遗传控制系统中。我们建议 鉴定和优化一系列需要多巴胺存在以发挥催化功能的变构核酶。此外，我们还将继续构建由多巴胺类似物触发的变构核酶。特别令人感兴趣的是核酶，它是由一种我们假设介导DA神经元氧化损伤的分子特异性激活的。这种分子的形成是由胞液中的DA自动氧化成多巴胺邻苯二酚(DA-邻苯二酚)，然后与L-半胱氨酸加成生成5-S-半胱氨基 多巴胺(5-S-半胱氨酸-DA)。需要5-S-半胱氨酸-DA来激活的核酶的产生将 允许构建新的生物传感器和基因控制系统，这些系统可能诊断帕金森氏病的致病分子过程。我们还打算继续分离其他靶标依赖的核酶。该项目的具体目标包括：(I)制备用于变构选择的核酶构建物；(Ii)使用非立体选择来创建靶标依赖的RNA开关种群；(Iii)通过随机突变和变构选择来优化原型RNA开关；(Iv)最佳RNA开关变体的动力学表征；以及(V)将新开关整合到生物传感器和基因工程中 用于测试的表达系统。

RNA enzymes can be engineered to perform as precision allosteric ribozymes, or "molecular switches", that are modulated by specific effectors. Molecular switches made of RNA have numerous applications ranging from the construction of biosensors to the development of novel genetic switches. We have embarked on an effort to generate RNA molecular switches that are modulated by compounds of highest biological and therapeuticimportance. In this study, we seek to generate ribozymes that respond to dopamine and several related compounds for ultimate integration into in vitro and in vivo diagnostic and genetic control systems. We propose to identify and optimize a series of allosteric ribozymes that require the presence of dopamine for catalytic function. In addition, we will pursue the construction of allosteric ribozymes that are triggered by analogs of dopamine. Of particular interest are ribozymes that are activated specifically by a molecule that we hypothesize mediates oxidative injury in DA neurons. This molecule's formation is initiated by the auto-oxidation of cytosolic DA to dopamine-o-quinone (DA-o-quinone) followed by its subsequent addition to L-cysteine to generate 5-S-Cysteinyl dopamine (5-S-CyS-DA). The generation of ribozymes that require 5-S-CyS-DA for activation will permit the construction of novel biosensor and genetic control systems that might be diagnostic of causative molecular processes of Parkinson's disease. We intend to pursue the isolation of other target-dependent ribozymes as well. The specific aims of this project include: (I) The preparation of ribozyme constructs for allosteric selection; (II) The use of aUosteric selection to create populations of target-dependent RNA switches; (III) Optimization of prototype RNA switches by random mutagenesis and allosteric selection; (IV) Kinetic characterization of optimal RNA switch variants; and (V) Integration of new switches into biosensor and genetic expression systems for testing.