ANTIBODY MODEL FOR RECOGNITION OF DNA HELICAL DIVERSITY

拟议的研究将以抗体为例扩展研究 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： 这些具体的互动。 在第一个层面， 将研究免疫应答。 最小的尺寸和 与B-DNA的差异是免疫原性所需的 将测定核酸。 待检测的免疫原将 包括固定连接点连接寡聚物、弯曲DNA 具有RNA-DNA杂交和DNA-DNA双链体部分的寡核苷酸 在一个分子中，具有骨架修饰和 与错配或不配对的碱基，和聚（A），聚（U）的变化 尺寸 将用选定的免疫原制备鼠杂交瘤， 并对特异性和相互作用进行详细的研究 纯化的单克隆抗体。 目前的研究 单克隆抗Z-DNA抗体将被延伸。 初级 重链和轻链可变区的序列将被 确定，并将准备Fab片段用于尝试生长 晶体和核磁共振（NMR）光谱。 NMR将用于研究寡核苷酸构象。 重点 将被放置在短螺旋分子与非均匀 不同构象区域之间的结构和连接。 NMR光谱法也将用于检查寡核苷酸- Fab相互作用，反映在寡核苷酸或 蛋白质（或两者）在结合时共振。 作为角色的测试 抗体结合位点中的特定氨基酸， 产生抗Z-DNA活性降低的抗体的杂交瘤 并确定它们的可变区序列。 此外，杂交瘤表达的免疫球蛋白基因将 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。 其目的是 检验关于磷脂-蛋白质相互作用的假设 位点，并修改的特异性和亲和力， 抗体的

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.