BIOCHEMICAL ANALYSIS OF A NOVEL U1 RNA-ANTIBODY COMPLEX

该提案概述了一项研究工作，以加深我们对 决定蛋白质-RNA 相互作用的结构规则。 的作用 形成抗体-RNA 的特定氨基酸和核苷酸 将使用与 U1 茎环 II 结合的抗体描绘复合物 核糖核酸。 抗 U1 RNA 背景下 RNA-蛋白质相互作用的研究 抗体将允许对 RNA 结合进行相对简单的评估 位点归因于抗原结合区域的小尺寸和已知的 几种抗体的结构。 该提案中的实验是 旨在：1) 表征天然存在的 U1 RNA 茎环 II 抗体并产生基因工程 U1 RNA 结合抗体 片段（Fab）； 2) 分析包含的RNA结合位点 通过氨基酸评估天然存在和重组的 Fab 序列、抗体特异性和结合特征； 3）确定 通过使用分子结合特定氨基酸在结合 U1 RNA 中的作用 建模和定点诱变； 4) 阐明核糖核苷酸 使用快速选择技术对抗体结合至关重要； 5） 启动核磁共振和晶体学研究，长期目标是 阐明RNA抗体和/或抗体-RNA复合物的结构。 U1 RNA 以及许多其他细胞 RNA 在细胞中以如下形式存在： 核糖核蛋白在 RNA 加工和基因调控中发挥作用。 这些核糖核蛋白通常是免疫系统的目标 患有系统性红斑狼疮等自身免疫性疾病的患者 （系统性红斑狼疮）、干燥综合征和类风湿性关节炎。 这项研究将 提高我们对 RNA 蛋白质识别和基因调控的理解。 更好地理解这些过程将有助于辨别细胞 与自身免疫性疾病相关的失调。 此外，信息 所获得的结果将影响我们对RNA反应结构的认识 抗体，并将在未来的新型药物设计中得到应用 治疗性抗体和核酸自身抗体抑制剂。

This proposal outlines a research effort to further our understanding of the structural rules that dictate protein-RNA interaction. The role of specific amino acids and nucleotides in the formation of an antibody-RNA complex will be delineated using antibodies that bind to stem-loop II of U1 RNA. The study of RNA-protein interactions in the context of anti-U1 RNA antibodies will allow for relatively simple assessment of the RNA binding site due to the small size of antigen binding regions and the known structures of several antibodies. Experiments in this proposal are designed to: 1) characterize naturally occurring U1 RNA-stem loop II antibodies and produce genetically engineered U1 RNA-binding antibody fragments (Fabs); 2) analyze the RNA binding sites contained in the naturally occurring and recombinant Fabs by evaluation of amino acid sequence, antibody specificity and binding characteristics; 3) determine the role of specific amino acids in binding U1 RNA by the use of molecular modeling and site-directed mutagenesis; 4) elucidate the ribonucleotides critical to antibody binding using a rapid selection technique and; 5) initiate NMR and crystallographic studies with the long-term goal of elucidating the structure of an RNA antibody and/or antibody-RNA complex. U1 RNA, as well as many other cellular RNAs, exist in the cell as ribonucleoproteins functioning in RNA processing and gene regulation. These ribonucleoproteins are often targeted by the immune system in patients with autoimmune diseases such as systemic lupus erythematosus (SLE), Sjogren's syndrome, and rheumatoid arthritis. This research will improve our understanding of RNA-protein recognition and gene regulation. A better understanding of these processes will help discern the cellular disregulation associated with autoimmune disease. In addition, information gained will impact on our knowledge of the structure of RNA-reactive antibodies and will have future application in the design of novel therapeutic antibodies and nucleic acid autoantibody inhibitors.