CHARACTERIZATION OF THE STRUCTURE OF A LEAD-DEPENDENT DNAZYME

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have previously characterized the structure of the 17E DNAzyme using Fluorescence Resonance Energy Transfer (FRET). This study revealed that in the presence of Zn2+ the DNAzyme undergoes a two-step folding process in order to adopt a more compact structure. We propose to use EXAFS to further study the three-dimensional structure of the 17E catalytic core when bound to Pb2+. Pb has core-shell levels that are accessible using EXAFS. The catalytic activity of the Pb DNAzyme and the dependence of activity on Pb concentration and buffer conditions have been thoroughly characterized. The chemical environment around the Pb binding site is expected to be complex. However, spatial correlations in complex, noncrystalline systems are routinely studied using EXAFS. We intend to investigate the EXAFS of Pb in order to determine the distances between the Pb metal ion cofactor and key atoms in the DNAzyme catalytic core. The DNAzyme structure will be investigated as a function of Pb concentration, monovalent salt concentration, and pH. This 3-D information will then be combined with existing knowledge of the sequence and structure of the DNAzyme in order to form a better understanding of the functional structure of the DNAzyme.