ANTIBODY MODEL FOR RECOGNITION OF DNA HELICAL DIVERSITY

拟议的研究将扩展对抗体的研究作为例子。 识别特定螺旋核酸的蛋白质 结构。目标是确定以下方面的结构基础 这些特定的互动。在第一个层面上，对 将研究免疫反应性。最小尺寸和 与B-DNA不同，B-DNA是疫苗免疫原性所必需的 核酸将被确定。将检测的免疫原将 包括固定连接连接低聚物、弯曲的DNA 含RNA-DNA杂合和DNA-DNA双链切片的寡核苷酸 在一个分子中，带有骨架修饰的寡核苷酸和 碱基不匹配或不配对，以及聚(A)、聚(U)的变化 尺码。小鼠杂交瘤将用选定的免疫原制备， 并将对特殊性和相互作用进行详细研究 用纯化的单抗。当前的研究现状 抗Z-DNA的单抗将得到推广。主要 重链和轻链可变区的序列如下 确定，并且Fab片段将为尝试生长做准备 用于晶体和核磁共振(核磁共振)光谱。 核磁共振将被用来研究寡核苷酸构象。重点 会被放置在非均匀的短螺旋分子上 发散构象区域之间的结构和连接。 核磁共振波谱还将用于检测寡核苷酸- FAB相互作用，反映在寡核苷酸或 蛋白质(或两者)在结合时产生共振。作为对角色的考验 抗体结合部位的特定氨基酸，其变体 产生抗体且抗Z-DNA活性降低的杂交瘤 将被选择并确定其可变区序列。 此外，杂交瘤中表达的免疫球蛋白基因将 被分离、突变并转移到突变的骨髓瘤细胞中 修饰抗体的表达和生长。目标将是 关于寡核苷酸-蛋白质相互作用的测试假说 站点，并修改 抗体。

The proposed research will extend studies of antibodies as examples of proteins that recognize specific helical nucleic acid structures. The goals are to determine the structural basis for these specific interactions. At the first level, recognition for immune responsiveness will be studied. The minimal size and difference from B-DNA that is required for immunogenicity of nucleic acids will be determined. Immunogens to be tested will include ligated oligomers of immobile junctions, bent DNA, oligonucleotides with RNA-DNA hybrid and DNA-DNA duplex sections in one molecule, oligonucleotides with backbone modifications and with mismatched or unpaired bases, and poly(A).poly(U) of varying size. Murine hybridomas will be prepared with selected immunogens, and detailed studies of specificity and interaction will be carried out with purified monoclonal antibodies. Current studies of monoclonal anti-Z-DNA antibodies will be extended. Primary sequences of the heavy and light chain variable regions will be determined, and Fab fragments will be prepared for attempts to grow crystals and for nuclear magnetic resonance (NMR) spectroscopy. NMR will be used to study oligonucleotide conformation. Emphasis will be placed on short helical molecules with non-uniform structures and junctions between regions of divergent conformation. NMR spectrometry will also be used to examine the oligonucleotide- Fab interaction, reflected in changes in either oligonucleotide or protein (or both) resonances upon binding. As tests of the role of specific amino acids in the antibody binding sites, variants of hybridomas producing antibodies with reduced anti-Z-DNA activity will be selected and their variable region sequences determined. In addition, expressed immunoglobulin genes of the hybridomas will be isolated, mutated and transferred to mutated myeloma cells for expression and growth of modified antibodies. The aims will be to test hypotheses about the oligonucleotide-protein interacting sites, and to modify the specificity and affinity of the antibodies.