【9.3.6】mRNA polyA的测定

这是2020年公开的诺华的一篇专利,通过用LC-MS来测mRNA polyA的长度。

下图是不同polyA的长度对应的MS的数值,区分plolyA的长度的能力还可以。

用来测量 polyA tail length of mRNA的方法:

  1. RNase H cleavage
  2. chromatographic methods
  3. PCR based assays like LM-PAT and ePAT,
  4. next generation sequencing methods such as TAIL-seq and PAL-seq.
  5. LC-MC

优缺点比较:

*下一代测序方法具有最高的分辨率(TAIL-seq报道为1个碱基) * 使用凝胶或色谱法的方法通常会在尾巴长度不同的种群中出现偏差(smears) * 但是,基于PCR的技术可能由于其测量的间接性质而变得复杂,序列差别足够长。 * 与其他方法不同,质谱法是一种直接测量技术,不需要标记,并具有通过质量差异区分单个核苷酸的能力。

本文所述的方法使用质谱法研究mRNA polyA尾巴长度分布。质谱可以提供有关已知PCR扩增序列的碱基组成的信息,从而可以识别单个核苷酸变化,并可以区分多个PCR产物。此外,本文描述的方法使用质谱法以单个核苷酸分辨率同时直接测量许多寡核苷酸序列。实施例中描述的结果使用用于寡核苷酸的标准LC-MS条件来测量mRNA组合物中的polyA链长度。通过MS系统中的通用软件程序对所得的多电荷谱进行反卷积。

Next generation sequencing approaches have the highest resolution (1 base reported for TAIL-seq), whereas those that use gels or chromatography usually show smears for a population of different tail lengths. However, PCR-based techniques can be complicated by the indirect nature of their measurement, which must amplify long stretches of adenosines.

Unlike other methods, mass spectrometry is a direct measurement technique that does not require labels and has the ability to distinguish between single nucleotides by their differences in mass. The methods described herein use mass spectrometry to study mRNA polyA tail length distributions. Mass spectrometry can provide information about the base composition of known PCR amplified sequence, which allows single nucleotide changes to be identified and multiple PCR products to be distinguished from one another. Furthermore, the methods described herein use mass spectrometry to directly measure many oligonucleotide sequences simultaneously with single nucleotide resolution. The results described in the EXAMPLES used standard LC-MS conditions for oligonucleotides to measure polyA chain lengths within an mRNA composition. The deconvolution of the resulting multiply charged spectra was done by a common software procedure in MS systems.

“5-methylcytidine” (5 mC) is s a modified nucleoside derived from 5-methylcytosine. 5-Methylcytosine is a methylated form of the DNA base cytosine that may be involved in the regulation of gene transcription. See, WO WO2013/052523 (Moderna Therapeutics).

参考资料

  • US2020325532A1 POLYA TAIL LENGTH ANALYSIS OF RNA BY MASS SPECTROMETRY
  • Salles et al., PCR Methods Appl. 4: 317-321 (1995); Meijer et al., Nucleic Acids Res. 35: e132 (2007); Murray & Schoenberg, Methods EnzymoL 448: 483-504 (2008); Janicke et al., RNA 18: 1289-1295 (2012); Chang et al., Mol. Cell 53: 1044-1052 (2014); Subtelny et al., 2014. Nature 508: 66-71.
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