Intellectual Merit: X-ray crystallography is one of the best methods for the determination and study of 3-D structures of nucleic acids and protein-nucleic acid complexes at the atomic level. Such studies provide novel insights into nucleic acid functions and mechanisms. Via atom-specific substitution of nucleic acid oxygen with selenium, the research group on this project has pioneered and developed the selenium modification for nucleic acid heavy atom derivatization and crystallization, which are the two major challenges/bottlenecks in nucleic acid X-ray crystallography. This project has led to a successful demonstration of the utility of selenium derivatization for phasing, and to the discovery of crystallization facilitation by the 2'-Se derivatization of nucleic acids. The current research project involves further X-ray crystallographic studies on DNAs and RNAs in order to provide a viable rational solution for standard heavy atom derivatization and phase determination, as well as a potential solution for crystallization facilitation. The intellectual merit of this research project is in providing new insights into molecular interactions, packing, crystallization mechanisms, crystal growth facilitation, as well as structures of the Se-derivatized DNAs and RNAs and the corresponding natural nucleic acids. In addition, new synthetic routes to introduce Se into multiple positions of DNAs and RNAs will be developed for function and crystal structure studies.
Broader Impact of the Research: This research project will significantly enlarge scientific knowledge on nucleic acids, lead to novel materials and applications, allow broader access to these valuable new materials and unique molecules, as well as attract the attention of the general public. This research group will reach out to faculty members in research non-intensive colleges and high schools, and invite them to participate in this research project. The outreach and research opportunities will help to raise the awareness and scientific literacy of the general public. Moreover, many students, especially underrepresented minority students, will be trained through participation in this project in hands-on experimental research, and scientific communication skills. The Chemistry Department at Georgia State University (GSU) has a strong tradition of educating and serving undergraduate and graduate students, especially underrepresented minority students. This project will offer various extensive multi-disciplinary training opportunities and generate synergy in research training in both Chemistry and Biology Departments at GSU.
Research Outcome 3-D structure determination of nucleic acids and protein-nucleic acid complexes at the atomic level is essential for providing novel insights into their functions and mechanisms. X-ray crystallography is one of the best methods for this research exploration. However, nucleic acid crystallization and heavy atom derivatization for phase determination are the two major challenges/bottlenecks in nucleic acid X-ray crystallography, which slow down determination of new structures and folds of nucleic acids. Via atom-specific substitution of nucleic acid oxygen with selenium, the PI laboratory has pioneered and developed the selenium derivatization of DNAs and RNAs as an atomic probe for structural studies. Regarding intellectual merit, the PI and research group have developed new and scalable syntheses of Se-containing nucleosides, phosphoramidites, triphosphates, DNAs and RNAs for investigating intramolecular and intermolecular interactions, crystal growth, and structure study. Moreover, they have found that selenium derivatization of nucleic acids can facilitate crystallization, phase determination as well as high-resolution structure determination. The Se-modifications don't cause significant perturbations of the native DNA and RNA structures. They have also found that in general, the Se-modification may be used as an atomic probing system to investigation structures and functions of nucleic acids and protein-nucleic acid complexes, via the site-specific replacement of nucleic acid oxygen atoms with selenium atoms, since oxygen and selenium elements are in the same elemental family. Therefore, during this NSF support, in addition to the crystallization and structure determination studies, they have successfully pioneered the Se-atom-specific mutagenesis (SAM) strategy for structure and function studies of nucleic acids. Using selenium as an atomic probe, they have successfully obtained new insights into molecular interaction and packing, crystallization mechanisms, crystal growth facilitation, and structures of the Se-derivatizated DNAs and RNAs as well as the corresponding native nucleic acids. They have successfully demonstrated that the selenium derivatization has great potentials for rational crystallization, phase determination as well as high-resolution structure determination. Regarding the broad impacts of their work, their nucleic acid research has provided novel insights into nucleic acid biochemistry and structure, and also opened new unexpected areas of research, i.e., crystallization facilitation and high-resolution structure determination by SAM strategy. Their research results have significantly enlarged scientific knowledge on nucleic acids and lead to novel materials and applications. In addition, this research project has allowed more access to these valuable new materials and molecules. Currently, selenium-derivatized nucleic acids (SeNA) are available from the PI laboratory free of charge. Their exciting and novel discoveries have encouraged commercial entities to distribute SeNA in an affordable manner, allowing broad access to these new and unique materials for function and structure studies. Moreover, many students have been trained through participation in this project. The PI has established the outreach programs and many students (especially undergraduate students) have been actively recruited and trained through participation in this project. Their novel approaches include the organization and participation in the research conferences and research-and-presentation competition for undergraduate students. Their outreach programs have been used to encourage and teach students in research, especially undergraduate research. For example, the PI has traveled to the comprehensive colleges, including minority institutions (such as Miles College and Savannah State University), and recruited the undergraduate minority students into their research program. Participation in this research project allows the undergraduate and graduate students to get exposed to the cutting-edge research, while learning skills and knowledge in organic synthesis, biochemistry, molecular biology, and structural biology. GSU and the Chemistry Department (the PI's institution) have excellent research and training environments for undergraduate and graduate students. Each semester, two to three undergraduate students take independent research courses in the PI's laboratory, and many of them stay for more than two semesters. Many of them have published peer-reviewed research papers with the PI. This research activity has been used as an excellent vehicle for student training, which promotes both teaching and learning. As this research requires integration of multiple disciplines, including organic synthesis, nucleic acid chemistry, kinetics, thermodynamics, biochemistry, structural biology, molecular engineering, and nanotechnology, this research project has generated strong synergy in research training in both Chemistry Department and Biology Department at GSU.
