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Poly A tail length analysis of in vitro transcribed mRNA by LC-MS

Michael Beverly1, Caitlin Hagen2, Olga Slack2

  • 1Novartis Institutes of Biomedical Research, 700 Main Street, Cambridge, MA, 02139, USA. Michael.beverly@novartis.com.

Analytical and Bioanalytical Chemistry
|January 10, 2018
PubMed

Insights

This study precisely measures messenger RNA (mRNA) poly-adenosine (poly A) tail lengths using liquid chromatography-mass spectrometry (LC-MS). Results reveal transcriptional slippage during mRNA synthesis, impacting tail length accuracy.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Analytical Chemistry

Background:

  • The polyadenosine (poly A) tail is crucial for mRNA stability and translation.
  • Accurate determination of poly A tail length is essential for understanding mRNA regulation.
  • In vitro transcribed (IVT) mRNA synthesis can lead to variations in poly A tail length.

Purpose of the Study:

  • To develop and validate a high-resolution method for determining mRNA poly A tail length.
  • To investigate the impact of RNA polymerase (RNAP) and plasmid sequence on poly A tail length distribution.
  • To explore the phenomenon of transcriptional slippage during IVT mRNA synthesis.

Main Methods:

  • Poly A tails were enzymatically cleaved from IVT mRNA using ribonuclease T1.
  • Poly A tails were isolated using paramagnetic oligo(dT) beads.
  • Tail lengths were precisely quantified using liquid chromatography coupled to mass spectrometry (LC-MS) at single-nucleotide resolution.

Main Results:

  • LC-MS analysis accurately determined poly A tail lengths, correlating closely with DNA template sequences.
  • Transcriptional slippage by T7 RNA polymerase (RNAP) was observed, leading to poly A tails longer than encoded.
  • Different RNAPs (T3, T7, SP6) produced similar tail length distributions, but 3' end modifications narrowed the distribution.

Conclusions:

  • LC-MS provides a robust method for single-nucleotide resolution of mRNA poly A tail lengths.
  • Transcriptional slippage is a significant factor influencing poly A tail length in IVT mRNA.
  • Understanding slippage mechanisms can optimize IVT mRNA production for therapeutic applications.
Keywords:
In vitro transcriptionMass spectrometryPoly ASlippageTail lengthmRNA

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