Investigation of deoxyribozyme structure, function and mechanism by DNA nucleotide analog interference mapping and biophysical characterization

Deoxyribozymes are catalytically active DNAs that are not known in nature but have been identified by in vitro selection from random-sequence DNA pools. Although very little is known about the molecular details of the complex three-dimensional structures of deoxyribozymes, they have been developed into practical tools for basic and applied research. This proposal addresses the analysis of fundamental questions of DNA catalysis by characterizing different classes of deoxyribozymes using a variety of chemical and biophysical approaches. DNA nucleotide analog interference mapping (D-NAIM) will be developed as a new and reliable tool for the simultaneous chemical probing of individual functional groups of single-stranded DNAs. Chemically modified deoxyribonucleotide analogs bearing functional group deletions or substitutions at their nucleobase moieties will be randomly incorporated into DNA by chemical and enzymatic methods. The statistical distribution of nucleotide analogs in the catalytic DNA region will dramatically reduce the number of experiments required for analysis of individual functional group mutations, compared to the conventional approach in which separate DNAs with single-nucleotide modifications are investigated. It is specifically proposed to equip the DNA nucleotide building blocks with phosphorothioate or 2`- hydroxyl chemical tags to enable site-specific cleavage of the phosphate backbone at nucleotide analog positions. The comparison of cleavage patterns of active and inactive deoxyribozyme variants will permit the identification of functional group requirements for DNA catalysis. The power of the D-NAIM method will be illustrated by characterizing several RNA-ligating deoxyribozymes. Of particular interest are DNA enzymes that catalyze the site-specific formation of 2`,5`-branched and lariat RNA, as well as RNA-ligating deoxyribozymes that form linear 3`-5`-linked RNA. The D-NAIM results will be integrated with chemical probing and photocrosslinking data to develop mechanistic frameworks for DNA-catalyzed RNA ligation. Biophysical methods including nuclear magnetic resonance spectroscopy, fluorescence resonance energy transfer and comparative gel electrophoresis will be applied to shed light on key structural features of the complex between the 7S11 deoxyribozyme and its branched RNA product, for which a three-helix junction architecture has been proposed. The insights from these studies will enable a deeper understanding of nucleic acid catalysis and are potentially useful for the development of rational design strategies for improved biocatalysts.