Selenium-Derivatized RNAs and DNAs for High-Throughput Protein/Nucleic Acid Cryst

描述（由申请人提供）：对于核酸和蛋白质-核酸复合物的3D结构测定的需求日益增长，以在分子水平上理解疾病机制，从而促进新药发现。X射线晶体学是测定这些大分子和复合物结构的最有力的工具之一。然而，结晶和相确定一直是瓶颈问题，这在很大程度上减缓了RNA和蛋白质-核酸复合物的新结构和折叠的结构确定。尽管核酸溴化的方法常规用于定相，但溴衍生物通常遭受稳定性问题、扰动、结晶性和衍生化位点限制。因此，促进结晶和定相的新技术具有巨大的价值。硒取代蛋白质中的硫有助于通过硒MAD定相彻底改变蛋白质晶体学。最近，本申请人开创了用于结构和功能研究的核酸中的氧的硒替代。他们的研究是基于他们的中心假设：硒可以用来稳定地取代核酸中的氧原子特异性地没有显着的扰动，因为硒和氧是在同一个元素家族。他们已经成功地证明了核酸的硒衍生化可以解决相位问题。令人兴奋的是，他们还发现硒衍生化可以促进RNA、DNA和蛋白质-核酸复合物的结晶。因此，该项目旨在通过将硒衍生化纳入核酸和蛋白质-核酸复合物中，以常规解决结晶和定相问题，从而创新性地改变当前蛋白质/核酸晶体学的范式。申请人还证明了多次Se-衍生化不会引起核酸和蛋白质-核酸复合物的显著结构扰动。因此，申请人提出合成用多Se修饰衍生的新型亚磷酰胺和三磷酸酯（“Se簇”），用于Se-DNA和Se-RNA的化学和酶促合成。多硒修饰可以作为强有力的“硒衍生簇”用于结晶和定相。他们还将研究这些合成的硒核酸的生物学和结构的结晶，定相和结构测定。此外，申请人计划研究结晶促进的机制。他们的新型硒衍生化技术对于核酸（如非编码RNA）和蛋白质-核酸复合物的高通量结构测定非常有价值。他们的长期目标是建立新的技术，这将彻底改变核酸和蛋白质-核酸复合物的结晶，定相和结构测定。 公共卫生相关性：硒衍生核酸（SeNA）在核酸和蛋白质-核酸复合物的结晶促进、合理定相和高通量晶体结构测定方面具有巨大的潜力，可为深入了解分子水平的疾病机制和发现新药和治疗疾病提供帮助。

DESCRIPTION (provided by applicant): There are growing demands for 3D structure determination of nucleic acids and protein-nucleic acid complexes for understanding disease mechanisms at the molecular level, thus facilitating new drug discoveries. X-ray crystallography is one of the most powerful tools for structure determination of these macromolecules and complexes. However, crystallization and phase determination have been the bottleneck problems that largely slow down structural determination of new structures and folds of RNAs and protein-nucleic acid complexes. Though the approach of the nucleic acid bromination is routinely used for phasing, the bromo-derivatives often suffer from the stability issue, perturbation, crystallizability, and derivatization site limitation. Therefore, the novel technologies that facilitate crystallization and phasing are of tremendous value. The selenium replacement of sulfur in proteins has helped to revolutionize protein crystallography via selenium MAD phasing. Recently the applicant has pioneered the selenium replacement of oxygen in nucleic acids for structure and function studies. Their research is based on their central hypothesis: selenium can be used to stably replace oxygen of nucleic acids atom-specifically without significant perturbation, because selenium and oxygen are in the same elemental family. They have successfully demonstrated that the selenium derivatization of nucleic acids can solve the phase problem. Excitingly, they have also found that the Se-derivatization can facilitate crystallization of RNAs, DNAs, and protein-nucleic acid complexes. Thus, this proposed project seeks to innovatively shift the current paradigms on protein/nucleic acid crystallography by incorporating the selenium derivatization into nucleic acids and protein-nucleic acid complexes in order to routinely solve crystallization and phasing problems. The applicant has also demonstrated that the multiple Se-derivatizations do not cause significant structural perturbation in nucleic acids and protein-nucleic acid complexes. Thus, the applicant proposes to synthesize the novel phosphoramidites and triphosphates derivatized with the multi-Se-modifications ("Se-clusters") for chemical and enzymatic synthesis of Se-DNAs and Se-RNAs. The multi-Se-modifications can serve as the powerful "Se-derivatizing clusters" for the crystallization and phasing. They will also investigate these synthesized Se-nucleic acids biophysically and structurally for crystallization, phasing, and structure determination. Furthermore, the applicant plans to study the mechanisms of crystallization facilitation. Their novel Se-derivatization technology is extremely valuable to high-throughput structural determination of nucleic acids (such as non-coding RNAs) and protein-nucleic acid complexes. Their long-term goal is to establish the novel technologies that will revolutionize crystallization, phasing, and structure determination of nucleic acids and protein-nucleic acid complexes. PUBLIC HEALTH RELEVANCE: Selenium-derivatized nucleic acids (SeNA) have great potentials in crystallization facilitation, rational phasing, and high-throughput crystal structure determination of nucleic acids and protein-nucleic acid complexes, which provides insights into molecular-level disease mechanisms and leads to new drug discoveries and disease treatments.