AQUEOUS SOLUTION DERIVATIZATION OF UNPROTECTED RNA/DNA

未受保护的 RNA/DNA 的水溶液衍生化

• 批准号: 3282330
• 负责人: LESLIE E ORGEL
• 金额: $ 20.11万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-04-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3282330
• 关键词: DNA; DNA binding protein; RNA; adduct; chemical aggregate; chemical binding; chloramphenicol acetyltransferase; crosslink; electron microscopy; eukaryote; metal complex; method development; nucleic acid hybridization; nucleic acid sequence; nucleic acid structure; platinum; protein structure; reagent /indicator; surface property; transcription factor; water solution

The primary objective of this research program is to develop simple, efficient, specific procedures for covalently crosslinking protein transcription factors to the short double-stranded DNA sequences to which they bind. We plan to use these procedures to control transcription in eukaryotic cells by decoying transcription factors, so we will develop DNA sequences that are resistant to nuclease digestion, and crosslinking reagents that can function in the intranuclear environment. We hope that this approach will, in the long run, lead to the synthesis of DNA- crosslinker adducts that can be used in whole animals and can, therefore, find a place in medicine. In much of our work we will use DNA sequences that fold into hairpin and dumbbell structures with the enhancer-binding sequence present in the double-helical stem. Such sequences bind enhancer proteins with undiminished affinity but do not undergo strand separation. The dumbbell sequences are also completely resistant to exonuclease digestion in serum. Hairpins and dumbbells, therefore, should prove superior to short double- stranded DNA's as decoys. Crosslinking reagents that we plan to use include a variety of metal complexes and a number of commercially available bifunctional alkylating and acylating agents. Usually we will attach the crosslinkers to DNA via modified nucleotides. The resulting adducts will first be used to crosslink to protein in vitro. Then the most effective adducts will be tested as transcription inhibitors in eukaryotic cells using a chloramphenicol acetyl transferase (CAT) assay system. The systems that we plan to study include the CREB-CRE system, the Jun-TRE system and the Zif268 protein with its consensus binding site. A second objective of our program is to develop methods for immobilizing polynucleotides stereospecifically on metal or mineral surfaces. This will facilitate studies of nucleic acid structure by electron tunnelling microscopy and atomic force microscopy. We will insert several functional groups into the nucleic acid in positions that can simultaneously contact a planar surface, for example, that of metallic gold. The tight attachment of modified nucleotides to gold should hold the nucleic acid in a fixed position and orientation on the surface.

这项研究计划的主要目标是开发简单， 共价交联蛋白质的有效、特异的方法 转录因子的短双链DNA序列， 他们绑定。 我们计划使用这些程序来控制转录， 真核细胞通过诱骗转录因子，所以我们将开发DNA 对核酸酶消化和交联具有抗性的序列 可以在核内环境中发挥作用的试剂。 我们希望 从长远来看，这种方法将导致DNA的合成， 交联剂加合物可用于整个动物，因此， 在医学界找到一席之地 在我们的大部分工作中，我们将使用折叠成发夹结构的DNA序列， 哑铃状结构，增强子结合序列存在于 双螺旋杆 这样的序列结合增强子蛋白， 亲和力不减弱，但不发生链分离。 哑铃 序列也完全抵抗血清中的核酸外切酶消化。 发夹和哑铃，因此，应证明上级短双- DNA链是诱饵 我们计划使用的交联剂包括各种金属 络合物和许多市售的双官能烷基化 和酰化剂。 通常，我们将通过以下方式将交联剂连接到DNA上： 修饰的核苷酸 所得加合物将首先用于 在体外与蛋白质交联。 那么最有效的加合物将是 在真核细胞中作为转录抑制剂进行了测试， 氯霉素乙酰转移酶（CAT）测定系统。 我们的系统 计划研究包括CREB-CRE系统，Jun-TRE系统和 Zif 268蛋白及其共有结合位点。 我们计划的第二个目标是开发固定 立体特异性地在金属或矿物表面上的多核苷酸。 这将 通过电子隧道效应促进核酸结构的研究 显微镜和原子力显微镜。 我们将插入几个函数 在核酸中的位置， 平面，例如金属金的平面。 紧密的联系 修饰的核苷酸的金应该保持核酸在一个固定的 在表面上的位置和方向。