AQUEOUS SOLUTION DERIVATIZATION OF UNPROTECTED RNA/DNA

未受保护的 RNA/DNA 的水溶液衍生化

• 批准号: 3282329
• 负责人: LESLIE E ORGEL
• 金额: $ 16.15万
• 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 1992-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3282329
• 关键词: DNA; RNA; nucleic acid sequence; phosphamide; phosphates; water solution

We have devised a very simple procedure that allows us to attach a wide variety of organic molecules to the terminal phosphate of an unprotected oligonucleotide or polynucleotide. During the next grant period we plan to exploit this chemistry to develop a number of techniques that have broad general application. 1. We will attach a variety of porphyrins to the 5' terminus of an oligonucleotide to bring about the hybridization- dependent, specific and efficient cleavage of complementary DNA or RNA. Reagents of this kind would have many applications. They could, for example, be used as 'restriction enzymes' for single strand RNA (or DNA) or as probes to explore the tertiary structure of RNA molecules. 2. We will develop methods for the attachment of molecules that combine irreversibly with proteins to the 3'-terminus of oligonucleotides. We anticipate that, if we hybridize these reagents to a messenger RNA, the reactive groups will attack a protein at the messenger-entry site of the ribosome during protein synthesis. We hope to learn how to inhibit intracellular protein synthesis in a highly specific manner by inactivating ribosomes. Only the ribosomes in cells containing an RNA complementary to an oligomer should be affected by a protein-modifying adduct of that oligomer. In the long run, when adequate in vivo delivery methods become available, adducts of this type might find many applications in medicine. 3. We will develop a hybridization assay for DNA that makes use of short RNA substrates that are replicated exponentially by QB RNA polymerase as reporters. We have prepared an adduct in which QB-Midivariant I RNA is joined to avidin by a cleavable linker. This adduct replicates normally, with a doubling time of 34 seconds, thus producing about 10-12 descendents in 25 minutes. By joining this adduct to an oligodeoxynucleotide, we plan to develop a non-radioactive probe assay at least 100 times more sensitive than the best presently available non-radioactive assays. A sensitive assay of this kind would have many important applications in diagnostic medicine.

我们设计了一个非常简单的程序，允许我们附加 多种有机分子的末端磷酸基 未受保护的寡核苷酸或多核苷酸。 在接下来的时间里 资助期间我们计划利用这种化学来开发 许多具有广泛普遍应用的技术。 1. 我们将多种卟啉连接到 5' 末端 产生杂交的寡核苷酸- 依赖性、特异性和有效的裂解 互补DNA或RNA。 此类试剂将 有很多应用。 例如，它们可以用于 作为单链 RNA（或 DNA）的“限制性酶”或 作为探索RNA三级结构的探针 分子。 2.我们将开发分子附着的方法 与蛋白质不可逆地结合到 寡核苷酸。 我们预计，如果我们将这些杂交 信使 RNA 的试剂，反应基团将 攻击核糖体信使进入位点的蛋白质 在蛋白质合成过程中。 我们希望学习如何抑制 细胞内蛋白质以高度特异性的方式合成 使核糖体失活。 只有细胞内的核糖体 含有与寡聚体互补的RNA应该是 受到该寡聚物的蛋白质修饰加合物的影响。 在 从长远来看，当有足够的体内递送方法时 变得可用，这种类型的加合物可能会发现很多 在医学上的应用。 3. 我们将开发一种 DNA 杂交检测方法，使 使用可复制的短RNA底物 QB RNA 聚合酶作为报告基因呈指数增长。 我们有 制备了 QB-Midivariant I RNA 连接的加合物 通过可裂解的接头连接至抗生物素蛋白。 该加合物复制 通常情况下，倍增时间为 34 秒，从而产生 25分钟内大约有10-12个后代。 通过加入这个 寡脱氧核苷酸的加合物，我们计划开发 非放射性探针检测至少100倍以上 比目前最好的非放射性物质更敏感 化验。 这种灵敏的检测有很多 在诊断医学中的重要应用。