AQUEOUS SOLUTION DERIVATIZATION OF UNPROTECTED RNA/DNA

未受保护的 RNA/DNA 的水溶液衍生化

• 批准号: 3282332
• 负责人: LESLIE E ORGEL
• 金额: $ 8.13万
• 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 1987-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3282332
• 关键词: DNA; RNA; nucleic acid sequence; phosphamide; phosphates; water solution

The primary aim of this research is to develop a general method for covalently linking amine-containing molecules to the 5'-terminal phosphate of DNA or RNA without affecting the ability of the nucleic acid to hybridize with its complement. We will activate the unprotected nucleic acid in aqueous solution at pH 6 and room temperature with a water-soluble carbodiimide in an imidazole buffer. The 5' -phosphorimidazolide formed in this way will be isolated and then treated with a concentrated solution of the amine. This will lead to the formation of a stable covalent phosphoramidate adduct. In other experiments the 5' -terminal phosphate of DNA or RNA will be converted to a phosphoramidate without the isolation of an intermediate. The condensation reaction will be carried out in a 1-MeImidazole buffer. The very reactive phosphorimidazolium ion that is formed will immediately react with amines to give the corresponding phosphoramidates. These reactions will be used to prepare non-radioactive DNA probes, to prepare reagents that break DNA in a sequence-specific fashion, and to develop a non-enzymatic ligation reaction for DNA.

本研究的主要目的是开发一种通用方法， 将含胺分子共价连接至5 ′-末端磷酸 不影响核酸的能力， 与其互补物杂交。 我们会激活未受保护的核酸 在pH 6和室温下的水溶液中的酸与水溶性 在咪唑缓冲液中的碳二亚胺。 5' -磷咪唑形成于 这种方式将被分离，然后用浓缩的 胺。 这将导致形成稳定的共价键 氨基磷酸酯加合物。 在其他实验中，DNA或RNA的5' -末端磷酸将是 转化为氨基磷酸酯而不分离中间体。 缩合反应将在1-MeImidazole缓冲液中进行。 形成的非常活泼的磷咪唑离子将立即 与胺反应得到相应的氨基磷酸酯。 这些反应将用于制备非放射性DNA探针， 制备以序列特异性方式破坏DNA的试剂，以及 开发一种DNA的非酶连接反应。