Beverly Michael, Hagen Caitlin, Slack Olga
Novartis Institutes of Biomedical Research, 700 Main Street, Cambridge, MA, 02139, USA.
Anal Bioanal Chem. 2018 Feb;410(6):1667-1677. doi: 10.1007/s00216-017-0840-6. Epub 2018 Jan 8.
The 3'-polyadenosine (poly A) tail of in vitro transcribed (IVT) mRNA was studied using liquid chromatography coupled to mass spectrometry (LC-MS). Poly A tails were cleaved from the mRNA using ribonuclease T1 followed by isolation with dT magnetic beads. Extracted tails were then analyzed by LC-MS which provided tail length information at single-nucleotide resolution. A 2100-nt mRNA with plasmid-encoded poly A tail lengths of either 27, 64, 100, or 117 nucleotides was used for these studies as enzymatically added poly A tails showed significant length heterogeneity. The number of As observed in the tails closely matched Sanger sequencing results of the DNA template, and even minor plasmid populations with sequence variations were detected. When the plasmid sequence contained a discreet number of poly As in the tail, analysis revealed a distribution that included tails longer than the encoded tail lengths. These observations were consistent with transcriptional slippage of T7 RNAP taking place within a poly A sequence. The type of RNAP did not alter the observed tail distribution, and comparison of T3, T7, and SP6 showed all three RNAPs produced equivalent tail length distributions. The addition of a sequence at the 3' end of the poly A tail did, however, produce narrower tail length distributions which supports a previously described model of slippage where the 3' end can be locked in place by having a G or C after the poly nucleotide region. Graphical abstract Determination of mRNA poly A tail length using magnetic beads and LC-MS.
使用液相色谱-质谱联用（LC-MS）技术对体外转录（IVT）mRNA的3'-聚腺苷酸（poly A）尾进行了研究。使用核糖核酸酶T1从mRNA上切割下poly A尾，然后用dT磁珠进行分离。接着通过LC-MS对提取的尾进行分析，该分析以单核苷酸分辨率提供尾长信息。这些研究使用了一种2100个核苷酸的mRNA，其质粒编码的poly A尾长度分别为27、64、100或117个核苷酸，因为酶促添加的poly A尾显示出明显的长度异质性。在尾中观察到的A的数量与DNA模板的桑格测序结果紧密匹配，甚至检测到了具有序列变异的少量质粒群体。当质粒序列在尾中包含特定数量的poly A时，分析显示存在一种分布，其中包括比编码尾长更长的尾。这些观察结果与T7 RNA聚合酶（RNAP）在poly A序列内发生的转录滑动一致。RNAP的类型并没有改变观察到的尾分布，对T3、T7和SP6的比较表明，这三种RNAP产生的尾长分布相当。然而，在poly A尾的3'端添加一个序列确实产生了更窄的尾长分布，这支持了先前描述的滑动模型，即在多核苷酸区域之后有一个G或C可以将3'端锁定在适当位置。图形摘要 使用磁珠和LC-MS测定mRNA poly A尾长度。
