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

描述(申请人提供)：越来越多的人需要确定核酸和蛋白质-核酸复合体的三维结构，以便在分子水平上了解疾病机制，从而促进新药发现。X射线结晶学是确定这些大分子和配合物结构的最有力的工具之一。然而，结晶和相的确定一直是阻碍RNA和蛋白质-核酸复合体新结构和折叠结构确定的瓶颈问题。虽然通常采用核酸溴化的方法进行定相，但溴代衍生物往往存在稳定性问题、微扰、结晶性和衍生化位置限制等问题。因此，促进结晶和定相的新技术具有巨大的价值。硒取代了蛋白质中的硫，通过硒相变帮助蛋白质结晶学发生了革命性的变化。最近，申请人率先在结构和功能研究中用硒取代核酸中的氧。他们的研究基于他们的中心假设：硒可以稳定地取代核酸原子中的氧--特别是没有明显的扰动，因为硒和氧属于同一个元素家族。他们成功地证明了核酸的硒衍生化可以解决相问题。令人兴奋的是，他们还发现，硒的衍生化可以促进RNA、DNA和蛋白质-核酸复合体的结晶。因此，这一拟议项目寻求创新性地改变目前蛋白质/核酸结晶学的范式，将硒衍生化纳入核酸和蛋白质-核酸复合体中，以便常规地解决结晶和分相问题。申请人还证明，多重Se衍生不会在核酸和蛋白质-核酸复合体中引起显著的结构扰动。因此，申请人建议合成通过多个Se修饰(“Se-簇”)衍生的新型亚磷酰胺和三磷酸盐，用于化学和酶法合成Se-DNA和Se-RNAs。多重Se修饰可以作为强大的“Se衍生团簇”进行晶化和晶化。他们还将对这些合成的硒核酸进行生物物理和结构研究，以进行结晶、定相和结构确定。此外，申请人还计划研究促进结晶的机制。其新颖的硒衍生技术对于高通量的核酸(如非编码RNA)和蛋白质-核酸复合体的结构测定具有极其重要的价值。他们的长期目标是建立将彻底改变核酸和蛋白质-核酸复合体的结晶、定相和结构确定的新技术。 与公共健康相关：硒衍生核酸(SENA)在促进结晶、合理定相和高通量测定核酸和蛋白质-核酸复合体的晶体结构方面具有巨大的潜力，这为深入了解分子水平的疾病机制提供了见解，并导致了新的药物发现和疾病治疗。

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.