CHARACTERIZATION OF THE STRUCTURE OF A LEAD-DEPENDENT DNAZYME

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们以前的特点是使用荧光共振能量转移（FRET）的17 E DNAzyme的结构。 本研究表明，在Zn 2+的存在下，DNA酶经历了两步折叠过程，以采用更紧凑的结构。 我们建议使用EXAFS进一步研究的三维结构的17 E催化核心时，绑定到Pb 2+。 铅的核-壳层水平可以用EXAFS来测量。 的铅DNA酶的催化活性和铅浓度和缓冲液条件下的活性的依赖性已被彻底表征。 铅结合位点周围的化学环境预计是复杂的。 然而，空间相关性在复杂的，非结晶系统的常规研究使用EXAFS。 我们打算研究铅的EXAFS，以确定铅金属离子辅因子和关键原子的DNAzyme催化核心之间的距离。 DNA酶的结构将作为铅浓度，单价盐浓度和pH值的函数进行研究。这3-D的信息，然后将结合现有的知识的DNA酶的序列和结构，以形成一个更好的理解的功能结构的DNA酶。

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.