Molecular engineering of nucleic acid enzymes with controllable catalytic activity

The discovery of catalytic RNAs (ribozymes) and the subsequent in vitro selection of DNA enzymes (DNAyzmes) have revolutionized conceptions about biological catalysis and enzyme function. In this thesis, we describe a number of strategies that are used to introduce an element of regulation into the existing simple catalytic nucleic acid enzymes.;A rational design approach was utilized to generate allosteric hammerhead ribozymes that respond to protein ligands by incorporating an ‘aptamer’ RNA module to the catalytic module of the hammerhead ribozyme. The resulting hammerhead ribozyme variant undergoes structural stabilization upon ligand binding to enhanced the catalytic power of the hammerhead catalytic module by 2- to 3-fold.;A novel strategy is also described for controlling the activity of an RNA-cleaving DNAzyme. This strategy has been demonstrated to be a very general method that can be used to control the catalytic activity of other nucleic acid enzymes in which Watson-Crick base-pairing is used as a means for substrate recognition. This strategy is designed to severely weaken the complementarity between the enzyme and substrate such that catalysis does not take place. The addition of a third oligonucleotide (Regulator) that is partly complementary to the enzyme and partly complementary to the substrate restores the enzyme-substrate complementarity by forming a three-way junction between the enzyme, substrate and regulator and therefore activates the catalysis.;Further rational designs were utilized to re-engineer expansively-controlled DNAzymes as well as ribozymes to be responsive to small organic molecules such as adenosine and flavin mononucleotide (FMN). This was achieved by incorporating aptamer motifs into the substrate-binding arms of the enzymes. In the absence of ligand, aptamer motifs are unstructured and consequently the substrate-binding arms cannot stably bind to the substrate. The addition of ligands stabilizes the aptamer motifs, which in turn allow the stable association between the enzyme and substrate, and therefore, allow the catalysis to proceed.;In vitro selection is a more comprehensive approach towards obtaining controllable DNAzymes and ribozymes. This technology will permit the isolation of controllable DNAzymes and ribozymes from a vast combinatorial pool. With this allosteric selection strategy, highly specific and responsive nucleic acid enzymes have been identified. The basis of allosteric selection is discussed to conclude this thesis.