Karolinska Institutet, Department of Biosciences and Nutrition, Huddinge, Sweden.
Curr Protoc Nucleic Acid Chem. 2020 Mar;80(1):e102. doi: 10.1002/cpnc.102.
An efficient method for attachment of a variety of reporter groups to oligonucleotides (ONs) is copper (I) [Cu(I)]-catalyzed Huisgen azide-alkyne 1,3-dipolar cycloaddition ("click reaction"). However, in the case of ONs with phosphorothioate modifications as internucleosidic linkages (PS-ONs), this conjugation method has to be adjusted to be compatible with the sulfur-containing groups. The method described here is adapted for PS-ONs, utilizes solid-supported ONs, and implements the Cu(I) bromide dimethyl sulfide complex (CuBr × Me S) as a mediator for the click reaction. The solid-supported ONs can be readily transformed into "clickable ONs" by on-line addition of an alkyne-containing linker that subsequently can react with an azido-containing moiety (e.g., a peptide) in the presence of CuBr × Me S. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Conjugation on solid support Support Protocol: Removal of 4,4'-dimethoxytrityl group from amino linker Basic Protocol 2: Removal of protecting groups and cleavage from solid support Basic Protocol 3: HPLC purification.
一种将各种报告基团连接到寡核苷酸（ONs）的有效方法是铜（I）[Cu（I）]催化的叠氮化物-炔烃 1,3-偶极环加成（“点击反应”）。然而，对于具有硫代磷酸酯修饰作为核苷间键（PS-ONs）的 ONs，该连接方法必须进行调整以与含硫基团兼容。此处描述的方法适用于 PS-ONs，利用固载 ONs，并采用铜（I）溴化二甲硫复合物（CuBr × Me S）作为点击反应的介质。固载 ONs 可以通过在线添加含有炔烃的接头很容易地转化为“可点击的 ONs”，随后在 CuBr × Me S 的存在下与含有叠氮化物的部分（例如肽）反应。© 2019 年由 John Wiley & Sons， Inc. 基本方案 1：在固体载体上的缀合 支持方案：从氨基接头上去除 4,4'-二甲氧基三苯甲基基团 基本方案 2：从固体载体上去除保护基团和切割 基本方案 3：HPLC 纯化。
