Sequencing of Single and Double Stranded RNA Oligonucleotides by Acid Hydrolysis and MALDI Mass Spectrometry
Sequencing of Single and Double Stranded RNA Oligonucleotides by Acid Hydrolysis and MALDI Mass Spectrometry
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DOI：
10.1021/ac900100x
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发表时间：
2009-04-15
影响因子：
7.4
通讯作者：
Karas, Michael
中科院分区：
文献类型：
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作者：
Bahr, Ute;Ayguen, Hueseyin;Karas, Michael
在pH 1-2下用强酸处理RNA寡核苷酸迅速导致核糖5 ′-位磷酸二酯键水解。通过与Orbitrap高分辨率质谱仪偶联的MALDI对所得降解产物进行分析，显示核苷酸两侧几乎完全的质量梯，没有来自碱基丢失或内部片段的干扰片段。从质量阶梯的相邻峰之间的质量差，可以确定序列。末端的低切割效率导致可以通过MS/MS鉴定的2 mer和3 mer。以这种方式，可以验证不同siRNA 2 - 1 mer单链和双链的完整序列。这种简单快速的方法可用于控制合成寡聚物的序列，以及从头测序。此外，该方法适用于定位和鉴定RNA修饰，如使用具有硫代磷酸酯骨架的寡核苷酸和含有2 '-甲氧基核糖修饰的寡核苷酸的实例所证明的。
Treatment of RNA oligonucleotides with strong acids at pH 1-2 rapidly leads to hydrolysis of the phosphodiester bonds at the 5'-position of ribose. Analysis of the resulting degradation products by MALDI coupled to an Orbitrap high resolution mass spectrometer shows almost complete mass ladders from both sides of the nucleotides without interfering fragments from base losses or internal fragments. From the mass differences between adjacent peaks of a mass ladder, the sequence can be determined. Low cleavage efficiency at the termini leads to 2mers and 3mers which can be identified by MS/MS. In this way the complete sequences of different siRNA 2 1 mer single and double strands could be verified. This simple and fast method can be applied for controlling sequences of synthetic oligomers, as well as for de-novo sequencing. Moreover, the method is applicable for localization and identification of RNA modifications as demonstrated using the examples of an oligonucleotide with phosphorothioate backbone and of one containing 2'-methoxyribose modifications.