Systematic Derivatization of Nucleic Acids with Selenium for X-ray Crystallography

X射线晶体学是确定RNA、DNA和核酸-蛋白质复合物结构以揭示其功能的最有力方法。然而，与结晶和重原子衍生相测定有关的困难是核酸X射线晶体学中的主要限制因素。为了解决后一个问题，该项目将开发用硒选择性取代核苷酸氧的方法。尽管Se原子的尺寸（半径：1.16埃）比O原子的尺寸（半径：0.73埃）大得多，但预期这种衍生化保持核酸的功能和结构性质。Se和O元素在元素周期表中属于同一族。该项目将测试这种衍生化是否可以产生结构和化学上与天然分子同晶的稳定的Se标记核酸。与其中卤素主要被引入脱氧尿苷（胸苷的模拟物）的5-位和尿苷的5-位的常规卤素衍生化（Br或I）不同，Se可以经由氧置换被引入到各种位置（例如，2 ′-或3 ′-核糖氧、非桥磷酸氧或核碱基上的氧）。硒的选择性掺入可以避免结构和功能的破坏。由于硒代甲硫氨酸的取代彻底改变了蛋白质的相位和结构确定使用多波长（或单波长）异常色散（MAD或SAD），硒也应该能够作为一个理想的异常散射体在核酸晶体学。此外，Br衍生物是光敏的，长时间暴露于X射线源可能导致分解。因此，这种新的硒衍生化的核酸应该是一个更好的替代传统的溴衍生化。该项目的智力价值是开发用于RNA和DNA中的Se的系统和位点特异性的O替换的通用方法，以促进通过MAD或SAD的X射线晶体学的相和结构测定。该项目涉及开发合成各种含硒核苷亚磷酰胺和三磷酸的路线，并开发大规模（10 mg）制备Se-RNA和Se-DNA的化学和酶促程序。研究了硒衍生化核苷酸的化学和热力学稳定性，并对硒衍生化策略在X射线晶体结构研究中的应用进行了评价。这种硒策略提供了一种新的方法来衍生化核酸-蛋白质复合物，其中核酸而不是蛋白质对应物将被衍生化。更广泛的影响：开发核酸结构分析的方法和试剂将是该项目对研究界的主要贡献。此外，该项目还将对学生进行大量的研究培训。2003年，格鲁吉亚州立大学（GSU）在全国非历史黑人机构中排名第一，在授予化学和其他物理科学学士学位方面排名第七。因此，该项目将提供一个极好的机会，培养在大分子结构研究的代表性不足的本科生和研究生。学生将获得实践技能和实验研究，特别是在有机化学和生物化学技能。作为化学系在教育和服务本科生和研究生在GSU的悠久传统，申请人和他的部门提供各种广泛的培训计划。该研究项目将改善这些培训活动的环境。

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.