CROSS-LINKED ANTIGENE OLIGOMERS EFFECT ON TRANSCRIPTION

交联抗原寡聚物对转录的影响

• 批准号: 6179222
• 负责人: NORMAN S KONDO
• 金额: $ 3.13万
• 依托单位: UNIVERSITY OF THE DISTRICT OF COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6179222
• 关键词: DNA; SDS polyacrylamide gel electrophoresis; chemical binding; crosslink; gel electrophoresis; genetic transcription; nucleic acid chemical synthesis; nucleic acid structure; oligonucleotides; polymerase chain reaction; protein structure function; ultraviolet radiation

In this MORE Faculty Development Award application, Dr. Norman S. Kondo, Professor of Chemistry, University of the District of Columbia (UDC) proposes to conduct research in the laboratory of Dr. Paul S. Miller, Professor of Biochemistry, Johns Hopkins University (JHU) for five consecutive summers. Through this effort Dr. Kondo plans to utilize newly acquired knowledge and expertise in nucleic acid biochemistry gained at JHU to establish a nucleic acid synthesis laboratory at UDC. In addition, a collaborative research relationship will also be established and continued with Dr. Miller beyond the grant period. The overall goal of the proposed research project is to determine the effect antigen oligonucleotides cross-linked to either or both strand/s of duplex DNA would have on its transcription. Antigen deoxynucleotides (pyrimidine-rich) complementary to the purine-rich (R) strand of the duplex DNA will be synthesized such that modified bases, designed to bind to pyrimidine interruptions in the R strand, are included. The 5'-end of the antigene oligonucleotide will be derivatized with a photoreactive psoralen residue and an amino-linker arm will be attached to the 3'-end (to reduce susceptibility to 3'-exonucleases. The selected target DNA will originate from purine-rich regions on the promoter site of the 92- kDa form of the collagenase IV gene. A DNA insert encompassing these regions will be synthesized by standard phosphoramidite chemistry and then inserted between T-7 and SP6 promoters in the vector, pGEM-3Z. After linearization of the plasmid, antigene oligomers will be directed to selected sites on the DNA target utilizing Hoogsteen type hydrogen bonding interactions. Ultraviolet irradiation of the triplex will cross- link the antigene oligonucleotide with the target DNA. The cross-linked DNA will then be treated with either T7 or SP6 RNA polymerase to produce runoff RNA. Further analysis of the runoff RNA by polyacrylamide gel electrophoresis will enable the investigators to correlate the region cross-linked with the length of RNA formed. Finally, analysis using ligation anchored-PCR on cross-linked DNA excised from the plasmid will more directly reveal where cross-linking occurred on the DNA.

在这个更多的教师发展奖申请，博士诺曼S。近藤 化学教授，哥伦比亚哥伦比亚特区大学（UDC） 建议在保罗·S博士的实验室进行研究。米勒， 生物化学教授，约翰霍普金斯大学（JHU）五 连续的夏天 通过这项努力，近藤博士计划利用 新获得的核酸生物化学知识和专业知识 在JHU获得在UDC建立核酸合成实验室。 此外，还将建立合作研究关系， 建立并继续与米勒博士超过赠款期。 的 拟议研究项目的总体目标是确定 抗原寡核苷酸交联至以下任一条或两条链： 双链体DNA对转录的影响 抗原脱氧核苷酸 （富含嘧啶的）互补的嘌呤（R）链。 双链体DNA将被合成，使得被设计成结合 到R链中的嘧啶中断。 5 '-端 的抗原寡核苷酸将用光反应性的 pepsilen残基和氨基-连接臂将连接到3 ′-末端 (to降低对3 '-核酸外切酶的敏感性。 选择的目标DNA 将起源于92- kDa形式的胶原酶IV基因。 DNA插入物包含这些 区域将通过标准亚磷酰胺化学合成， 然后插入到载体pGEM-3 Z的T-7和SP 6启动子之间。 质粒线性化后，将抗原寡聚体定向至 利用Hoogsteen型氢作用于DNA靶点上的选定位点 成键相互作用 紫外线照射三链体会交叉- 将所述抗基因寡核苷酸与所述靶DNA连接。 交联 然后用T7或SP 6 RNA聚合酶处理DNA， 径流RNA。 用聚丙烯酰胺凝胶进一步分析径流RNA 电泳将使研究人员能够将该区域 与形成的RNA长度交联。 最后，分析使用 对从质粒上切下的交联DNA进行连接扩增-PCR， 更直接地揭示了DNA上发生交联的位置。