Site-Specific Covalent Labeling of RNA by Enzymatic Transglycosylation
Site-Specific Covalent Labeling of RNA by Enzymatic Transglycosylation
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DOI：
10.1021/jacs.5b07286
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发表时间：
2015-10-14
影响因子：
15
通讯作者：
Devaraj, Neal K.
中科院分区：
文献类型：
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作者：
Alexander, Seth C.;Busby, Kayla N.;Devaraj, Neal K.
我们证明了位点特异性纳入核碱基衍生物轴承荧光团或亲和标记到一个短的RNA茎环识别基序的鸟嘌呤残基的交换。RNA-TAG（鸟苷转糖基化）由细菌（E.大肠杆菌）tRNA鸟嘌呤转糖基酶（TGT），其天然底物是含氮碱基PreQ（1）。值得注意的是，我们已经成功地通过连接到PreQ（1）的环外胺的聚乙二醇接头引入了大的官能团，包括生物素、BODIPY、噻唑橙子和Cy 7。较大的RNA，如mRNA转录物，如果它们具有17个核苷酸的发夹识别基序，则可以进行位点特异性标记：RNA-TAG方法可以通过使用简单的发夹识别元件直接掺入功能性人工核碱基来促进RNA分子的检测和操作。
We demonstrate the site-specific incorporation of nucleobase derivatives bearing fluorophores or affinity labels into a short RNA stem loop recognition motif by exchange of a guanine residue. The RNA-TAG (transglycosylation at guanosine) is carried out by a bacterial (E. coli) tRNA guanine transglycosylase (TGT), whose natural substrate is the nitrogenous base PreQ(1). Remarkably, we have successfully incorporated large functional groups including biotin, BODIPY, thiazole orange, and Cy7 through a polyethylene glycol linker attached to the exocyclic amine of PreQ(1). Larger RNAs, such as mRNA transcripts, can be site-specifically labeled if they possess the 17-nucleotide hairpin recognition motif: The RNA-TAG methodology could facilitate the detection and manipulation of RNA molecules by enabling the direct incorporation of functional artificial nudeobases using a simple hairpin recognition element.