Jahn Kasper, Olsen Eva Maria, Nielsen Morten Muhlig, Tørring Thomas, MohammadZadegan Reza, Andersen Ebbe Sloth, Gothelf Kurt Vesterager, Kjems Jørgen
Danish National Research Foundation, Center for DNA Nanotechnology at Interdisciplinary Nanoscience Center, Aarhus University, DK-8000 Aarhus, Denmark.
Bioconjug Chem. 2011 Jan 19;22(1):95-100. doi: 10.1021/bc100422k. Epub 2010 Dec 7.
Site-specific labeling of RNA molecules is a valuable tool for studying their structure and function. Here, we describe a new site-specific RNA labeling method, which utilizes a DNA-templated chemical reaction to attach a label at a specific internal nucleotide in an RNA molecule. The method is nonenzymatic and based on the formation of a four-way junction, where a donor strand is chemically coupled to an acceptor strand at a specific position via an activated chemical group. A disulfide bond in the linker is subsequently cleaved under mild conditions leaving a thiol group attached to the acceptor-RNA strand. The site-specific thiol-modified target RNA can then be chemically labeled with an optional group, here demonstrated by coupling of a maleimide-functionalized fluorophore. The method is rapid and allows site specific labeling of both in vitro and in vivo synthesized RNA with a broad range of functional groups.
RNA分子的位点特异性标记是研究其结构和功能的一种有价值的工具。在此，我们描述了一种新的位点特异性RNA标记方法，该方法利用DNA模板化学反应在RNA分子的特定内部核苷酸处连接一个标记。该方法是非酶促的，基于四链体连接的形成，其中供体链通过活化化学基团在特定位置与受体链化学偶联。随后，连接子中的二硫键在温和条件下裂解，留下一个连接到受体RNA链上的硫醇基团。然后，位点特异性硫醇修饰的靶RNA可以用一个任选基团进行化学标记，此处通过马来酰亚胺功能化荧光团的偶联进行了证明。该方法快速，并且能够用广泛的官能团对体外和体内合成的RNA进行位点特异性标记。
