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

用于 X 射线晶体学的硒系统核酸衍生化

• 批准号: 0517092
• 负责人: Zhen Huang
• 金额: --
• 依托单位: Georgia State University Research Foundation, Inc.
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-12-01 至 2008-11-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0517092&HistoricalAwards=false
• 关键词: Systematic; Derivatization; Nucleic; Acids; Selenium; Systematic; Derivatization; Nucleic; Acids; Selenium

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.

X射线晶体学是确定RNA，DNA和核酸 - 蛋白质复合物的结构以揭示其功能见解的最强大方法。然而，与结晶和相关原子衍生化有关相确定的困难是核酸X射线晶体学的主要限制因素。为了解决后一种问题，该项目将开发用于选择性替代核苷酸氧的方法。尽管SE原子的大小（半径：1.16埃斯特罗姆）的大小远大于O原子（半径：0.73 Angstroms），但这种衍生化有望维持核酸的功能和结构特性。 SE和O元素在元素周期表中位于同一家庭中。该项目将测试该衍生化是否可以产生稳定的SE标记的核酸，这些核酸在结构和化学上与天然分子同构。与传统的卤素衍生化（BR或I）不同，在该卤素中主要引入脱氧尿苷的5位（胸苷的模仿）和尿苷的5位，可以通过氧气替代（例如2'-或3'- ribose氧气氧化的氧化物，氧化氧化物或氧化物）的多种位置引入各种位置。 SE的选择性合并可以避免破坏结构和功能。由于硒甲米汀的替代已使用多波长（或单波长）异常分散（MAD或SAD）彻底改变了蛋白质相和结构的测定，因此SE还应该能够作为核酸晶体学中理想的异常散射剂。此外，BR衍生物是光敏的，并且长期暴露于X射线源可能会导致分解。因此，这种新型核酸的Se衍生化应该是传统BR衍生化的更好替代方法。该项目的智力优点是开发用于通过RNA和DNA中SE进行系统和位点特异性替换的通用方法，以通过MAD或SAD通过X射线晶体学来促进相位和结构确定。该项目涉及开发途径，以合成各种含SE的核苷磷光矿和三磷酸盐，并开发化学和酶促程序，以在大尺度（10 mg）上制备SE-RNA和SE-DNA。将研究SE衍生核苷酸的化学和热力学稳定性，并将评估X射线晶体结构研究中的SE衍生策略。这种SE策略提供了一种新的方法来衍生核酸 - 蛋白质复合物，其中将衍生核酸而不是蛋白质对应物。更广泛的影响：用于核酸结构分析的方法和试剂的开发将是该项目对研究界的主要贡献。此外，该项目将涉及对学生进行大量研究培训。 2003年，佐治亚州立大学（GSU）在全国非历史性黑人机构中排名第一，在化学和其他物理科学授予非裔美国学生的学士学位和其他物理科学学士学位方面排名第一。因此，该项目将提供一个绝佳的机会，以培训大分子结构研究中代表性不足的本科生和研究生。学生将获得动手技能和实验研究，尤其是在有机化学和生物化学技能方面。作为GSU教育和服务本科生和研究生的化学系的悠久传统，申请人及其部门提供了各种广泛的培训计划。研究项目将改善这些培训活动的环境。