Department of Chemistry, The University of Chicago, Chicago, IL, 60637, USA.
Department of Biochemistry & Molecular Biology, The University of Chicago, Chicago, IL, 60637, USA.
Angew Chem Int Ed Engl. 2017 Apr 24;56(18):5017-5020. doi: 10.1002/anie.201700537. Epub 2017 Mar 30.
The abundant Watson-Crick face methylations in biological RNAs such as N -methyladenosine (m A), N -methylguanosine (m G), N -methylcytosine (m C), and N ,N -dimethylguanosine (m G) cause significant obstacles for high-throughput RNA sequencing by impairing cDNA synthesis. One strategy to overcome this obstacle is to remove the methyl group on these modified bases prior to cDNA synthesis using enzymes. The wild-type E. coli AlkB and its D135S mutant can remove most of m A, m G, m C modifications in transfer RNA (tRNA), but they work poorly on m G. Here we report the design and evaluation of a series of AlkB mutants against m G-containing model RNA substrates that we synthesize using an improved synthetic method. We show that the AlkB D135S/L118V mutant efficiently and selectively converts m G modification to N -methylguanosine (m G). We also show that this new enzyme improves the efficiency of tRNA sequencing.
生物 RNA 中存在大量的 Watson-Crick 面甲基化，如 N6-甲基腺嘌呤（m6A）、N2-甲基鸟嘌呤（m2G）、N4-甲基胞嘧啶（m4C）和 N6，N2-二甲基鸟嘌呤（m2G），这些甲基化会通过损害 cDNA 合成，对高通量 RNA 测序造成严重阻碍。克服这一障碍的一种策略是在 cDNA 合成之前使用酶去除这些修饰碱基上的甲基。野生型大肠杆菌 AlkB 及其 D135S 突变体可以去除 tRNA 中大多数 m6A、m2G、m4C 的修饰，但对 m2G 的作用效果较差。在这里，我们报告了一系列针对含有 m2G 的模型 RNA 底物的 AlkB 突变体的设计和评估，我们使用改进的合成方法合成了这些底物。我们表明，AlkB D135S/L118V 突变体能够高效且选择性地将 m2G 修饰转化为 N6-甲基鸟嘌呤（m6G）。我们还表明，这种新酶提高了 tRNA 测序的效率。
