Label-free analysis of mRNA capping efficiency using RNase H probes and LC-MS
Label-free analysis of mRNA capping efficiency using RNase H probes and LC-MS
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DOI：
10.1007/s00216-016-9605-x
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发表时间：
2016-07-01
影响因子：
4.3
通讯作者：
Houghton, Leslie
中科院分区：
文献类型：
--
作者：
Beverly, Michael;Dell, Amy;Houghton, Leslie
描述了一种用于确定信使RNA（mRNA）5 '端帽身份和方向的无标记方法。与靶mRNA的5'端互补的生物素标记的探针与RNA酶H一起使用以切割mRNA的5'端。使用链霉亲和素包被的磁珠分离切割的末端序列，然后通过LC-MS分析。从分离的切割序列的独特质量确定5'帽的定量和定性信息。该方法与5' RNA焦磷酸水解酶的使用相结合，也用于确定5'帽的方向。该测定法在检测封端反应杂质时显示出低皮摩尔灵敏度。加标至100 pmol加帽mRNA中的未加帽三磷酸mRNA可在0.5 - 25%的检测范围内检出，线性响应。根据mRNA的修饰类型和长度，几种牛痘加帽的mRNA制剂的加帽效率被确定为88 - 98%。2.2K和9 K核苷酸长度和含有修饰的核苷酸假尿苷和5-甲基胞苷的mRNA都被成功地分析，证明了该技术用于研究mRNA加帽的实用性。
A label-free method for determining the 5'-end cap identity and orientation of a messenger RNA (mRNA) is described. Biotin-tagged probes that were complementary to the 5' end of target mRNA were used with RNase H to cleave the 5' end of the mRNA. The cleaved end sequence was isolated using streptavidin-coated magnetic beads and then analyzed by LC-MS. Quantitative and qualitative information on the 5' cap was determined from the unique mass of the isolated cleaved sequence. This approach, combined with the use of 5' RNA pyrophosphohydrolase, was also used to ascertain the orientation of the 5' cap. The assay showed low-picomole sensitivity for detecting capping reaction impurities. Uncapped triphosphate mRNA, spiked into 100 pmol of capped mRNA, could be detected over the tested range of 0.5 to 25 % with a linear response. The capping efficiency of several vaccinia-capped mRNA preparations was determined to be between 88 and 98 % depending on the modification type and length of the mRNA. mRNA of 2.2K and 9K nucleotides in length and containing the modified nucleotides pseudouridine and 5-methylcytidine were all successfully analyzed, demonstrating the utility of the technique to study mRNA capping.