Engineering RNA switches that respond to dopamine/analog

$163,636U54FY2002NSNIH

University Of Rochester, Rochester NY

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Abstract

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.

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