Biotin and fluorescent labeling of RNA using T4 RNA ligase.
Biotin and fluorescent labeling of RNA using T4 RNA ligase.
复制标题
使用 T4 RNA 连接酶对 RNA 进行生物素和荧光标记。
DOI：
10.1093/nar/11.18.6167
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发表时间：
1983
影响因子：
14.9
通讯作者：
Gumport,RI
中科院分区：
文献类型：
--
作者：
Richardson,RW;Gumport,RI
合成P1-（6-氨基己基）- p2 -（5′-腺苷）焦磷酸的生物素、荧光素和四甲基罗丹碱衍生物，并通过T4 RNA连接酶作为底物。在没有ATP的情况下，这些底物的非腺苷基部分被转移到RNA受体的3 ' -羟基上形成磷酸二酯键，AMP部分被释放。melanogaster5S RNA、酵母tRNAphe、（Ap）3C和（Ap）3A作为受体，产物产率从50到100Z不等。生物素标记的寡核苷酸选择性和定量地结合到亲和素琼脂糖上，可以用6 M盐酸胍（pH 2.5）洗脱。荧光素和四甲基罗丹明标记的寡核苷酸是高度荧光的，由于与受体的附着而没有猝灭。附加基团的不同结构以及受体RNA的链长和组成表明，T4 RNA连接酶将是一种有用的修饰试剂，用于在RNA分子的3 '端添加各种功能基团。
Biotin, fluorescein, and tetramethylrhodamlne derivatives of P1-(6-aminohex-l-yl)-P2-(5′-adenosine) pyrophosphate were synthesized and used as substrates with T4 RNA ligase. In the absence of ATP, the non-adenylyl portion of these substrates is transferred to the 3′-hydroxyl of an RNA acceptor to form a phosphodiester bond and the AMP portion is released.E.coliandD.melanogaster5S RNA, yeast tRNAphe, (Ap)3C, and (Ap)3A serve as acceptors with yields of products varying from 50 to 100Z. Biotin-labeled oligonucleotides are bound selectively and quantitatively to avidin-agarose and may be eluted with 6 M guanidine hydrochlorlde, pH 2.5. Fluorescein and tetramethylrhodamine-labeled oligonucleotides are highly fluorescent and show no quenching due to attachment to the acceptor. The diverse structures of the appended groups and of the chain lengths and compositions of the acceptor RNAs show that T4 RNA ligase will be a useful modification reagent for the addition of various functional groups to the 3′-terminus of RNA molecules.