m6A-SAC-seq在单核苷酸分辨率水平检测RNAm6A修饰

m6A-SAC-seq method detecting RNA m6A modification at single nucleotide resolution level

目的建立m6A特异性烯丙基标记测序(m6A-selective allyl chemical labeling and sequencing,m6A-SAC-seq)实验方法体系,在单核苷酸分辨率水平对RNA的N6-甲基腺苷(m6A)修饰进行检测。方法m6A烯丙基标记测序(m6A-SAC-seq)使用特异性甲基转移酶MjDim1对m6A添加烯丙基标记成为N6-烯丙基-甲基腺苷(am6A),使用I2处理产生N1,N6-环化腺苷(A)产物,在逆转录时引入突变,从而实现在单核苷酸分辨率水平的检测。通过HPLC法纯化甲基转移酶MjDim1,使用探针及液相色谱-质谱联用系统(LC-MS/MS)检测m6A的转换率,计算MjDim1活性作为质控。提取样品RNA,用烯丙基标记m6A,并用m6A-SAC-seq技术构建文库,测序后通过数据分析得到m6A位点信息,通过标准曲线校准计算得到m6A的含量。结果m6A-SAC-seq处理后,HIV逆转录酶识别环化的am6A位点,引入突变,但附近未修饰位点不发生突变。通过特定的A突变成T/C的突变位置(A to T/C)来识别m6A位点,并添加内参探针,用标准曲线来定量m6A含量。结论m6A-SAC-seq技术仅需30 ng的mRNA或去除了rRNA的总RNA样本就可以实现m6A位点在单核苷酸分辨率水平的检测。

Objective To establish a step-by-step protocol of m6A-selective allyl chemical labeling and sequencing(m6A-SAC-seq)for detection of N6-methyladenosine(m6A)modifications of RNA at single nucleotide resolution level.Methods m6A-SAC-seq used specific methyltransferase MjDim1 to add allyl group to m6A,generating am6A.Then I2 was added to produce N1,N6-cyclized adenine(A)products.Mutations were introduced during reverse transcription to achieve single-nucleotide resolution detection.Methyltransferase MjDim1 was purified by HPLC.The conversion rate of m6A was calculated by probe and liquid chromatography-mass spectrometry(LC-MS/MS)to determine the activity of MjDim1 as quality control.Sample RNA was extracted and m6A sites were labeled with allyl group.The DNA library was constructed with m6A-sac-seq technology and sent for high-throughput sequencing.After sequencing,m6A sites were inferred via data analysis.The stoichiometry of m6A was calculated by standard curve calibration.Results After m6A-SAC-seq treatment,HIV reverse transcriptase recognized the cyclized am6A sites and introduced mutations during reverse transcription while the unmodified sites nearby remain unmutated.m6A sites could be identified by the specific mutation of A to T/C.Stoichiometry of m6A could be quantified by addition of spike-in probes and calibrating the mutation rate with the standard curve.Conclusion By using m6A-SAC-seq method,only 30 ng of mRNA or rRNA depleted total RNA samples and is capable to detect RNA m6A sites at single-nucleotide resolution.