TRITIUM LABELLING OF DNA & RNA BY CHEMICAL & ENZYMATIC SYNTHESIS

DNA 氚标记

• 批准号: 6119713
• 负责人: DAVID E WEMMER
• 金额: $ 10.91万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6119713
• 关键词: biological products; biomedical resource; proteins; technology /technique

Tritiated ATP has been used as an essential component of several studies at the NTLF over the past few years. In initial studies [2-3H]-ATP of high specific activity was synthesized from 2-Bromo-ATP by heterogeneous tritiodehalogenation using tritium gas and PdO in aqueous solution. Availability of this labelled material allowed development of an enzymatic synthesis of highly tritiated RNA. This overall approach will be repeated and improved. Several synthetic efforts are underway to support the tritiated ATP project. Large quantities of 2-bromoadenosine have been prepared, ready for conversion to 2-bromo-ATP and subsequent use in RNA synthesis. Specific improvements over the past year have included the synthesis of 2-iodoadenosine as an alternative to 2-bromoadenosine. The two critical products share common TITLE: Tritium Labelling of DNA and RNA by Chemical and Enzymatic Synthesis (Continued) chemistry for all the previous synthetic steps. It remains to be seen whether the 2-iodoadenosine structure will survive the phosphorylation chemistry. Nevertheless, this precursor proved very useful for the labelling of [2-3H]-adenosine at high specific activity, for use in an adenosyl-cobalamin project. Similar chemistry was used recently to facilitate the production of [2-3H]-Deoxyadenosine for derivatization to the phosphoramidite, and subsequent incorporation into a DNA fragment. This overall process will also be repeated and improved. A particular improvement in the DNA sub-project involves moving away from chemical synthesis, and we will pursue the enzymatic synthesis of tritiated DNA by incorporation of tritiated dATP. To aid this approach we have embarked on a large scale synthesis of 2-Br-dATP to be used in an analogous tritiation reaction to 2-Br-ATP for RNA synthesis. We are also looking into synthesis of the analogous 2-iododeoxyadenosine compound, and its subsequent phosphorylation. The ability to synthesize specifically labelled RNA and DNA molecules will allow some very specific macromolecular NMR experiments to be conducted, addressing the interactions between DNA, proteins, RNA and other ligands. The importance of ribo- and deoxyribonucleoside-5'-triphosphates in biological systems as building units for RNA and DNA, have led to investigation for the development of a number of methods for their synthetic phosphorylation. Most of these methods involve the synthesis of the 5'-monophosphate of the nucleoside and its conversion to the triphosphate through a reactive intermediate or a barium salt derivative in two or more steps. Due to these lengthy, low yield and tedious methods a number of research groups have been exploring a one-pot, short and efficient synthetic method. Recently, a new method was reported for the conversion of nucleosides to nucleoside-5'-triphosphates in a convenient and quick route at relatively large scale, in which none of the functional groups sensitive to phosphorylation such as N-NH2 or secondary -OH need to be protected. This method appeared to be useful for the synthesis of a number of halogenated nucleosides as precursors for tritiation reactions in our RNA/DNA project. Through exploratory experiments we modified the method to work at mini-scale with adenosine and 2-deoxyadenosine for one-pot synthesis of ATP and dATP in good yield. A particularly important development was an HPLC method to monitor the monophosphorylation and triphosphorylation. Large differences in the rate of pyrophosphorylation were observed when comparing halogenated and non-halogenated bases, and between ribo and deoxyribomonophosphates. After purification, the nucleotides were converted to the tetrasodium salt and were finally analyzed by HPLC, 1H and 31P NMR, and mass spectrometry. The general procedure is as follows: Nucleoside (0.1-0.2 mmole) was pre-dried in a vacuum oven, dissolved in dried trimethyl phosphate, and the solution was kept at 0oC for 5 min. Freshly distilled phosphorus oxychloride was then added to the reaction vessel and the mixture was stirred at 0oC for 3 hrs. A solution of tributylammonium pyrophosphate in anhydrous DMF was separately made and kept at 0oC, after which dried tributylamine was added. This solution was combined with the nucleoside solution and was stirred for 1 min at 0oC. The reaction was then quenched with tributylammonium bicarbonate buffer and was stirred at 0oC for 3-6 hr. All solvents and volatiles were then removed under nitrogen gas to give a dry residue. The crude reaction was purified over DEAE Sephadex-25 using a linear gradient of 0 to 1M TEAB buffer. The appropriate fractions that showed a UV absorption at 260 nm for the triphosphate derivative were identified and combined for HPLC analysis of the nucleoside triphosphate. After the analysis, the residue was dried under nitrogen gas, and using a solution of anhydrous sodium perchlorate the tetrasodium salt was prepared for final HPLC, and 1H and 31P NMR analysis. We are in the process of applying this method to the synthesis of 2-halogenated nucleosides (2-bromoadenosine, 2-bromodeoxyadenosine and 2-iodoadenosine) and preparing the corresponding triphosphates as precursors for final heterogeneous catalytic dehalogenation with tritium gas to synthesize [2-3H]ATP and [2-3H]-dATP.

氚化 ATP 已被用作多种物质的重要组成部分 过去几年在NTLF学习。 在初步研究中 由2-Bromo-ATP合成高比活度的[2-3H]-ATP 使用氚气和 PdO 进行非均相三硫脱卤反应 水溶液。 允许使用此标记材料 高氚化RNA酶法合成的发展。 这 总体方法将被重复和改进。 几种合成的 目前正在努力支持氚化 ATP 项目。 大的 2-溴腺苷的量已准备好，可用于 转化为 2-溴-ATP 并随后用于 RNA 合成。 过去一年的具体改进包括综合 2-碘腺苷作为2-溴腺苷的替代品。 两人 关键产品具有共同的标题：DNA 和的氚标记 化学和酶法合成 RNA（续）适用于所有人的化学 之前的合成步骤。 是否有待观察 2-碘腺苷结构将在磷酸化化学过程中保留下来。 然而，该前体被证明对于标记非常有用 [2-3H]-腺苷具有高比活性，用于 腺苷钴胺素项目。 最近使用类似的化学方法 促进 [2-3H]-脱氧腺苷的衍生化生产 亚磷酰胺，然后掺入 DNA 分段。 这个整体过程也将被重复和改进。 一个 DNA子项目的特别改进包括搬走 从化学合成开始，我们将追求酶法合成 通过掺入氚化 dATP 来进行氚化 DNA。 为了帮助这个 我们已经开始大规模合成 2-Br-dATP 用于与 2-Br-ATP 进行类似的 RNA 氚化反应 合成。 我们也在研究类似的合成 2-碘脱氧腺苷化合物及其随后的磷酸化。 合成特异标记的 RNA 和 DNA 分子的能力 将允许一些非常具体的大分子核磁共振实验 进行，解决 DNA、蛋白质、RNA 和 其他配体。 核糖和核糖核酸的重要性 生物系统中的脱氧核糖核苷-5'-三磷酸作为建筑 RNA 和 DNA 单位，引发了对开发的调查 其合成磷酸化的多种方法。 大部分 这些方法涉及5'-单磷酸的合成 核苷及其通过反应性转化为三磷酸 中间体或钡盐衍生物在两个或更多个步骤中。 由于 这些冗长、低产且繁琐的方法需要大量研究 研究小组一直在探索一种一锅法、短时间且高效的合成方法 方法。 最近，报道了一种新的转换方法 方便快捷地将核苷转化为核苷-5'-三磷酸 规模较大的路线，其中没有任何功能性 对磷酸化敏感的基团，例如 N-NH2 或仲-OH 需要受到保护。 这个方法似乎对 合成许多卤代核苷作为前体 我们的 RNA/DNA 项目中的氚化反应。 通过探索 实验中我们修改了方法以在小规模下工作 腺苷和 2-脱氧腺苷一锅法合成 ATP 和 dATP 产量良好。 一个特别重要的发展是 HPLC 监测单磷酸化和三磷酸化的方法。 观察到焦磷酸化率存在巨大差异 当比较卤化碱基和非卤化碱基以及核糖核酸之间时 和脱氧核单磷酸。 纯化后，将核苷酸 转化为四钠盐并最终通过HPLC进行分析， 1H 和 31P NMR 以及质谱。 一般程序如下 如下：核苷（0.1-0.2毫摩尔）在真空烘箱中预干燥， 溶解于干燥的磷酸三甲酯中，并将溶液保持在 0℃ 5 分钟。 然后将新鲜蒸馏的三氯氧磷 添加到反应容器中并将混合物在0℃下搅拌3 小时。 焦磷酸三丁基铵的无水 DMF 溶液 单独制备并保存在0℃，然后干燥三丁胺 已添加。 将该溶液与核苷溶液合并 并在0℃下搅拌1分钟。 然后用以下物质猝灭反应 三丁基碳酸氢铵缓冲液并在0℃下搅拌3-6小时。 然后在氮气下除去所有溶剂和挥发物以 得到干燥的残渣。 粗反应物经 DEAE 纯化 Sephadex-25 使用 0 至 1M TEAB 缓冲液的线性梯度。 这 在 260 nm 处显示出 UV 吸收的适当级分 鉴定三磷酸衍生物并合并用于 HPLC 分析 三磷酸核苷。 分析后，残留物为 在氮气下干燥，并使用无水钠溶液 制备高氯酸盐四钠盐用于最终 HPLC，并 1H 和 31P NMR 分析。 我们正在应用这个方法 2-卤代核苷（2-溴腺苷， 2-溴脱氧腺苷和2-碘腺苷）并制备 相应的三磷酸盐作为最终异质的前体 氚气催化脱卤合成[2-3H]ATP [2-3H]-dATP。