Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Vilnius LT-02241, Lithuania.
Nucleic Acids Res. 2012 Aug;40(14):6765-73. doi: 10.1093/nar/gks381. Epub 2012 May 7.
Biophysical and mechanistic investigation of RNA function requires site-specific incorporation of spectroscopic and chemical probes, which is difficult to achieve using current technologies. We have in vitro reconstituted a functional box C/D small ribonucleoprotein RNA 2'-O-methyltransferase (C/D RNP) from the thermophilic archaeon Pyrococcus abyssi and demonstrated its ability to transfer a prop-2-ynyl group from a synthetic cofactor analog to a series of preselected target sites in model tRNA and pre-mRNA molecules. Target selection of the RNP was programmed by changing a dodecanucleotide guide sequence in a 64-nt C/D guide RNA leading to efficient derivatization of three out of four new targets in each RNA substrate. We also show that the transferred terminal alkyne can be further appended with a fluorophore using a bioorthogonal azide-alkyne 1,3-cycloaddition (click) reaction. The described approach for the first time permits synthetically tunable sequence-specific labeling of RNA with single-nucleotide precision.
生物物理和机械研究 RNA 功能需要特异性地掺入光谱和化学探针，这在当前技术下很难实现。我们在体外从嗜热古菌 Pyrococcus abyssi 中重新构建了一个功能性 box C/D 小核仁 RNA 2'-O-甲基转移酶 (C/D RNP)，并证明了它能够将一个丙炔基从合成辅因子类似物转移到模型 tRNA 和 pre-mRNA 分子中一系列预选的靶位点。RNP 的靶标选择是通过改变 64 个核苷酸的 C/D 引导 RNA 中的一个十二核苷酸引导序列来编程的，这导致在每个 RNA 底物中的四个新靶标中的三个得到有效修饰。我们还表明，转移的末端炔烃可以使用生物正交的叠氮-炔烃 1,3-环加成（点击）反应进一步与荧光团连接。该方法首次允许以单核苷酸精度对 RNA 进行合成可调谐的序列特异性标记。
