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

描述（由申请人提供）：为了在分子水平上理解疾病机制，对核酸和蛋白质-核酸复合物的三维结构测定的需求越来越大，从而促进新药的发现。x射线晶体学是测定这些大分子和复合物结构的最有力工具之一。然而，结晶和相确定一直是瓶颈问题，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。虽然核酸溴化的方法通常用于相化，但溴衍生物经常遭受稳定性问题，微扰，结晶性和衍生化位点限制。因此，促进结晶和相化的新技术具有巨大的价值。硒在蛋白质中取代硫有助于通过硒的MAD相位彻底改变蛋白质晶体学。最近，申请人率先在核酸结构和功能研究中使用硒代替氧。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，他们还发现硒衍生化可以促进rna、dna和蛋白质-核酸复合物的结晶。因此，本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，以创新性地改变目前蛋白质/核酸晶体学的范式，以常规解决结晶和分相问题。申请人还证明了多重硒衍生物不会引起核酸和蛋白质-核酸复合物的显著结构扰动。因此，申请人建议合成具有多重硒修饰（“硒簇”）衍生的新型磷酰胺和三磷酸盐，用于化学和酶合成硒- dna和硒- rna。多重硒修饰可以作为强大的“硒衍生团簇”用于结晶和相化。他们还将对这些合成的硒核酸进行生物物理和结构上的研究，以进行结晶、分相和结构测定。此外，申请人计划研究结晶促进机制。他们的新型硒衍生化技术对核酸（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将彻底改变核酸和蛋白质-核酸复合物的结晶、分相和结构测定的新技术。

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.