A Study of Site-Specifically Modified RNAs

描述(由申请人提供)：建议继续研究特定部位修饰的RNA，其中将采用合成和生物物理方法相结合的方法。这项工作的长期目标是了解RNA中修饰的核苷的功能意义以及它们在调节RNA相互作用中的作用。新的RNA靶向药物或基于RNA的疗法的设计最终将依赖于对RNA结构-功能关系的基本了解。短期目标是获得关于假性尿苷等天然修饰碱基的作用及其对RNA结构和稳定性的贡献的详细信息。将被合成和结构表征的修饰RNA是在E.Coli23S rRNA的IV区域暴露的发夹和真核生物中类似的大亚基rRNA发夹。这些RNA含有高度保守的假尿苷修饰和核苷酸序列，已被证明在核糖体组装中发挥作用。建议的结构研究结果将与RIVE功能研究中的结果进行比较。该研究计划的具体目的如下：1)将确定来自E.Coli23S rRNA螺旋69(残基1906-1924)的修饰的19-NT RNA发夹的生物物理性质和三维结构2)将确定完全修饰的19-NT人类类似物的结构和性质，3)将通过与过渡金属络合物和有机试剂的反应来比较19-NT、完全修饰的1920-区域RNA构建体和全长的、生物相关的大亚基rRNA的结构，4)对细菌1920-区RNA结构具有特异性的配体将被识别并用于研究1920-RNA的功能。综上所述，拟议的实验将用于确定假尿苷或3-甲基假尿苷等修饰碱基是否对局部RNA结构产生足够的影响，从而为选择性药物靶向提供合适的识别元件。

DESCRIPTION (provided by applicant): A continued study of site-specifically modified RNAs is proposed in which a combination of synthetic and biophysical approaches will be taken. The long-term goal of the work is to understand the functional significance of modified nucleosides in RNA and their roles in regulation of RNA interactions. The design of new RNA-targeting drugs or RNA-based therapeutics will ultimately rely on a fundamental understanding of RNA structure-function relationships. The short-term goal is to obtain detailed information regarding the roles of natural modified bases such as pseudouridine and their contributions to RNA structure and stability. Modified RNAs that will be synthesized and structurally characterized are an exposed hairpin in domain IV of E. coli 23S rRNA and the analogous large subunit rRNA hairpin in eukaryotes. These RNAs contain highly conserved pseudouridine modifications and nucleotide sequences that have been shown to play a role in ribosome assembly. Results of the proposed structure studies will be compared to in rive functional studies. The specific aims of the research plan are as follows: 1) the biophysical properties and three-dimensional structures of modified 19-nt RNA hairpins from helix 69 (residues 1906-1924) of E. coli 23S rRNA will be determined 2) the structure and properties of a fully modified 19-nt human analogue of the 1920-region RNA from E. coli will be determined, 3) the structures of the 19-nt, fully modified 1920-region RNA constructs and full-length, biologically relevant, large subunit rRNAs will be compared by reactions with transition metal complexes and organic reagents, and 4) ligands that have specificity for the bacterial 1920-region RNA structure will be identified and used to study the 1920 RNA function. Taken together, the proposed experiments will be used to determine whether modified bases such as pseudouridine or 3-methylpseudouridine influence local RNA structure enough to provide suitable recognition elements for selective drug targeting.