BIOCHEMICAL ANALYSIS OF A NOVEL U1 RNA-ANTIBODY COMPLEX

这项建议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 抗体-RNA形成过程中的特定氨基酸和核苷酸 将使用与U1的茎环II结合的抗体来描绘复合体 核糖核酸。抗U1核糖核酸作用下的RNA-蛋白质相互作用研究 抗体将允许相对简单地评估rna结合。 由于抗原结合区面积较小，已知的 几种抗体的结构。这项提案中的实验是 旨在：1)表征自然产生的U1 RNA-茎环II 抗体并产生基因工程U1 RNA结合抗体 片段(Fabs)；2)分析包含在 氨基酸评价法测定天然和重组纤维蛋白 序列、抗体特异性和结合特性；3)确定 特异性氨基酸在分子结合U1 RNA中的作用 建模和定点突变；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) 启动核磁共振和结晶学研究，长期目标是 阐明RNA抗体和/或抗体-RNA复合体的结构。 U1 RNA和许多其他细胞RNA一样，以 核糖核蛋白在RNA加工和基因调控中的作用。 这些核糖核蛋白通常是免疫系统的靶点 自身免疫性疾病患者，如系统性红斑狼疮 (SLE)、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。 对这些过程的更好理解将有助于识别细胞 与自身免疫性疾病相关的调节失调。此外，信息 所获得的结果将影响我们对RNA-反应结构的认识 并将在新型抗体的设计中有应用前景 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.