CROSS-LINKED ANTIGENE OLIGOMERS EFFECT ON TRANSCRIPTION

交联抗原寡聚物对转录的影响

• 批准号: 2444413
• 负责人: NORMAN S KONDO
• 金额: $ 2.82万
• 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/2444413
• 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.

在这项更高的教师发展奖申请中，诺曼·康多（Norman S. Kondo）博士 哥伦比亚特区化学教授（UDC） 建议在Paul S. Miller博士的实验室进行研究 约翰·霍普金斯大学（JHU）生物化学教授五 连续的夏天。 通过这项努力，Kondo博士计划利用 核酸生物化学的新知识和专业知识 在JHU获得以在UDC建立核酸合成实验室。 此外，协作研究关系也将是 在赠款期内建立并继续与米勒博士一起。 这 拟议的研究项目的总体目标是确定效果 抗原寡核苷酸交联至链/s的抗原寡核苷酸 双链DNA在转录上会有。 抗原脱氧核苷酸 （富含嘧啶的）补充嘌呤富含嘌呤（R） 双链DNA将合成，以使旨在结合的修改碱 包括R链中的嘧啶中断。 5'-end 将用光电反应性衍生化抗基因寡核苷酸的 牛coral子残留物和氨基链臂将连接到3'末端 （降低对3'-核酸酶的敏感性。选定的目标DNA 将源自92-启动子部位上富含嘌呤的区域 胶原酶IV基因的KDA形式。 DNA插入包含这些的DNA 区域将由标准磷光化化学和 然后在载体中的T-7和SP6启动子PGEM-3Z中插入。 质粒线性化后，将定向抗基因寡聚物 利用Hoogsteen型氢的DNA目标上的选定位点 粘结相互作用。 三元的紫外线辐射将跨 将抗基因寡核苷酸与靶DNA联系起来。 交联 然后将用T7或SP6 RNA聚合酶处理DNA以产生 径流RNA。 通过聚丙烯酰胺凝胶对径流RNA的进一步分析 电泳将使研究人员能够将区域关联 与形成的RNA长度交联。 最后，分析使用 从质粒切除的交联DNA上的连接锚定PCR将 更直接地揭示了DNA上的交联发生位置。