Kowalak J A, Pomerantz S C, Crain P F, McCloskey J A
Department of Biochemistry, University of Utah, Salt Lake City 84112.
Nucleic Acids Res. 1993 Sep 25;21(19):4577-85. doi: 10.1093/nar/21.19.4577.
A method is described for the detection, chemical characterization and sequence placement of post-transcriptionally modified nucleotides in RNA. Molecular masses of oligonucleotides produced by RNase T1 hydrolysis can be measured by electrospray mass spectrometry with errors of less than 1 Da, which provides exact base composition, and recognition of modifications resulting from incremental increases in mass. Used in conjunction with combined liquid chromatography-mass spectrometry and gene sequence data, modified residues can be completely characterized at the nucleoside level, and assigned to sequence sites within oligonucleotides defined by selective RNase cleavage. The procedures are demonstrated using E.coli 5S rRNA, in which all RNase T1 fragments predicted from the rDNA sequence are identified solely on the basis of their molecular masses, and using E.coli 16S rRNA for analysis of post-transcriptional modification, including placement of 3-methyluridine at position 1498. The principles described are generally applicable to other covalent structural modifications of RNA which produce a change in mass, such as those resulting from editing, photochemical cross-linking, or xenobiotic modification.
本文描述了一种用于检测、化学表征和确定RNA中转录后修饰核苷酸序列位置的方法。核糖核酸酶T1水解产生的寡核苷酸的分子量可以通过电喷雾质谱法测量，误差小于1道尔顿，这可以提供精确的碱基组成，并识别由于质量增加而产生的修饰。结合液相色谱-质谱联用和基因序列数据，修饰的残基可以在核苷水平上完全表征，并指定到由选择性核糖核酸酶切割定义的寡核苷酸内的序列位点。使用大肠杆菌5S rRNA证明了这些程序，其中从rDNA序列预测的所有核糖核酸酶T1片段仅根据其分子量进行鉴定，并使用大肠杆菌16S rRNA分析转录后修饰，包括1498位3-甲基尿苷的位置。所描述的原理通常适用于导致质量变化的RNA的其他共价结构修饰，例如由编辑、光化学交联或异生物质修饰产生的修饰。
