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.

在这个更多教师发展奖的申请中，Norman S.Kondo博士， 哥伦比亚特区大学(UDC)化学教授 建议在保罗·S·米勒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 连续的夏天。通过这一努力，近藤博士计划利用 新获得的核酸生物化学知识和专业技能 在JHU获得，在UDC建立一个核酸合成实验室。 此外，合作研究关系也将是 建立并在授权期后继续与米勒博士合作。这个 拟议研究项目的总体目标是确定影响 抗原寡核苷酸与一条或两条链交叉/S 双链DNA对其转录有影响。抗原脱氧核苷酸 (富含嘧啶的)与富含嘌呤的(R)链互补 双链DNA将被合成，以使修饰的碱基，旨在结合 到R链上的嘧啶中断，都包括在内。5‘端 的抗基因寡核苷酸将用光反应衍生化 补骨脂素残基和氨基连接臂将连接到3‘-端 (以降低对3‘-核酸外切酶的易感性。选定的目标DNA 将起源于92-启动子上的嘌呤富集区- KDA形式的胶原酶IV基因。包含这些的DNA插入物 区域将由标准的亚磷酰胺化学和 然后插入载体pGEM-3Z中的T-7和SP6启动子之间。 在将质粒线性化后，反基因寡聚体将被定向 利用Hoogsteen型氢到DNA靶上选定的位置 结合相互作用。三联体的紫外线照射将会交叉- 将抗基因寡核苷酸与靶DNA连接。交联体 然后用T7或SP6 RNA聚合酶处理DNA以产生 径流RNA。用聚丙烯酰胺凝胶对径流RNA的进一步分析 电泳法将使研究人员能够将该区域进行关联 与RNA的长度形成了交联体。最后，分析使用 连接锚定-聚合酶链式反应对从质粒中取出的交联DNA 更直接地揭示DNA上发生交叉连接的位置。