A NOVEL METHOD FOR THE DETERMINATION OF POSTTRANSCRIPTIONAL MODIFICATION IN RNA BY MASS-SPECTROMETRY
A NOVEL METHOD FOR THE DETERMINATION OF POSTTRANSCRIPTIONAL MODIFICATION IN RNA BY MASS-SPECTROMETRY
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DOI：
10.1093/nar/21.19.4577
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发表时间：
1993-09-25
影响因子：
14.9
通讯作者：
MCCLOSKEY, JA
中科院分区：
文献类型：
--
作者：
KOWALAK, JA;POMERANTZ, SC;MCCLOSKEY, JA
描述了一种RNA中转录后修饰核苷酸的检测、化学表征和序列放置的方法。用电喷雾质谱仪可以测量RNaseT1水解产生的寡核苷酸的分子质量，误差小于1Da，提供了准确的碱基组成，并识别了质量增加引起的修饰。与液-质联用和基因序列数据结合使用，修饰残基可以在核苷水平上完全表征，并被分配到由选择性RNase切割定义的寡核苷酸中的序列位置。使用E.coli5S rRNA，其中从rDNA序列预测的所有RNaseT1片段仅根据它们的分子质量进行鉴定，并使用E.coli16S rRNA分析转录后修饰，包括将3-甲基尿苷放置在1498位。所描述的原理一般适用于产生质量变化的RNA的其他共价结构修饰，例如由编辑、光化学交联或异源修饰引起的修饰。
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.