X-ray crystallography is the most powerful approach for determining the structures of RNAs, DNAs, and nucleic acid-protein complexes to reveal their functional insights. However, the difficulties related to crystallization and to heavy atom derivatization for phase determination are the major limiting factors in nucleic acid X-ray crystallography. To address the latter problem, this project will develop methodologies for selective replacement of nucleotide oxygen with selenium. This derivatization is expected to maintain functional and structural properties of nucleic acids, although the size of Se atom (radius: 1.16 angstroms) is much larger than that of O atom (radius: 0.73 angstroms). Se and O elements are in the same family in periodic table. The project will test if this derivatization can create stable Se-labeled nucleic acids that are structurally and chemically isomorphous to the native molecules. Unlike conventional halogen derivatization (Br or I), where halogens are primarily introduced to the 5-position of deoxyuridine (a mimic of thymidine) and the 5-position of uridine, Se can be introduced to a variety of positions via oxygen replacement (e.g., 2'- or 3'-ribose oxygen, non-bridging phosphate oxygen, or oxygen on nucleobases). Selective incorporation of Se can avoid disruption of structure and function. Because the selenomethionine replacement has revolutionized protein phase and structure determination using Multiwavelength (or Single-Wavelength) Anomalous Dispersion (MAD or SAD), Se should also be able to serve as an ideal anomalous scatterer in nucleic acid crystallography. In addition, Br derivatives are light sensitive, and long-time exposure to X-ray sources may cause decomposition. Therefore, this novel Se derivatization of nucleic acids should be a much better alternative to the conventional Br derivatization. The intellectual merit of this project is to develop general methods for systematic and site-specific replacement of O with Se in RNAs and DNAs to facilitate phase and structure determination by X-ray crystallography via MAD or SAD. The project involves development of routes to synthesize a variety of the Se-containing nucleoside phosphoramidites and triphosphates, and develop chemical and enzymatic procedures to prepare Se-RNAs and Se-DNAs on large scales (10 mg). Chemical and thermodynamic stabilities of Se-derivatized nucleotides will be studied, and the Se-derivatization strategy in X-ray crystal structure study will be evaluated. This Se strategy provides a novel approach to derivatize nucleic acid-protein complexes, where the nucleic acids instead of the protein counterparts will be derivatized.
Broader Impacts: Development of methods and reagents for structural analysis of nucleic acids will be a major contribution of this project to the research community. In addition, the project will involve substantial research training of students. In 2003, Georgia State University (GSU) was ranked #1 among non-historically black institutions in the nation and #7 overall in awarding Bachelor's degrees in the Chemistry and other Physical Sciences to African-American students. Therefore, the project will provide an excellent opportunity to train underrepresented undergraduate and graduate students in macromolecular structural research. The students will obtain hands-on skills and experimental research, especially in organic chemistry and biochemistry skills. As a long tradition of the Chemistry Department in educating and serving undergraduate and graduate students at GSU, the applicant and his department offer various extensive training programs. The research project will enhance the environment for these training activities.
