Ortoleva-Donnelly L, Kronman M, Strobel S A
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
Biochemistry. 1998 Sep 15;37(37):12933-42. doi: 10.1021/bi980723j.
Nucleotide analogue interference mapping (NAIM) is a general biochemical method that rapidly identifies the chemical groups important for RNA function. In principle, NAIM can be extended to any nucleotide that can be incorporated into an in vitro transcript by an RNA polymerase. Here we report the synthesis of 5'-O-(1-thio)-N2-methylguanosine triphosphate (m2GalphaS) and its incorporation into two reverse splicing forms of the Tetrahymena group I intron using a mutant form of T7 RNA polymerase. This analogue replaces one proton of the N2 exocyclic amine with a methyl group, but is as stable as guanosine (G) for secondary structure formation. We have identified three sites of m2GalphaS interference within the Tetrahymena intron: G22, G212, and G303. All three of these guanosine residues are known to utilize their exocyclic amino groups to participate in tertiary hydrogen bonds within the ribozyme structure. Unlike the interference pattern with the phosphorothioate of inosine (IalphaS, an analogue that deletes the N2 amine of G), m2GalphaS substitution did not cause interference at positions attributable to secondary structural stability effects. Given that the RNA minor groove is likely to be widely used for helix packing, m2GalphaS provides an especially valuable reagent to identify RNA minor groove tertiary contacts in less well-characterized RNAs.
核苷酸类似物干扰图谱法（NAIM）是一种通用的生化方法，可快速鉴定对RNA功能至关重要的化学基团。原则上，NAIM可扩展至任何能被RNA聚合酶掺入体外转录本的核苷酸。在此，我们报道了5'-O-(1-硫代)-N2-甲基鸟苷三磷酸（m2GαS）的合成，并使用T7 RNA聚合酶的突变形式将其掺入嗜热四膜虫I组内含子的两种反向剪接形式中。这种类似物用一个甲基取代了N2环外胺的一个质子，但对于二级结构形成而言，其稳定性与鸟苷（G）相同。我们已在嗜热四膜虫内含子中鉴定出m2GαS干扰的三个位点：G22、G212和G303。已知这三个鸟苷残基均利用其环外氨基参与核酶结构内的三级氢键形成。与肌苷的硫代磷酸酯（IαS，一种删除G的N2胺的类似物）的干扰模式不同，m2GαS取代在归因于二级结构稳定性效应的位置未引起干扰。鉴于RNA小沟可能广泛用于螺旋堆积，m2GαS为鉴定特征较少的RNA中的RNA小沟三级接触提供了一种特别有价值的试剂。
