Characterization of in vitro-transcribed (IVT) mRNA poly(A) tail by LC-HRAM-MS and BioPharma Finder 5.0 software

Significance of the topic

The 3' poly(A) tail is a critical feature of mature mRNA, influencing nuclear export, translational efficiency and transcript stability. In the context of in vitro‐transcribed (IVT) mRNA therapeutics and vaccines, accurate determination of poly(A) tail length is essential for product design, efficacy and quality control.

Goals and overview of the study

This application note describes the development of a sensitive and robust LC‐HRAM‐MS method for confident identification and sequence confirmation of 3' polyadenylated tails in IVT mRNA. The workflow integrates enzymatic digestion, oligo(dT) enrichment, ion‐pair reversed‐phase liquid chromatography and high‐resolution accurate mass spectrometry, followed by automated deconvolution in BioPharma Finder 5.0 software.

Methods and instrumentation

• Sample preparation: 100 pmol of IVT mRNA was digested with 1,000 U RNase T1 in presence of ZnCl₂ and purified on Dynabeads Oligo(dT)₂₅ magnetic beads.
• Chromatography: Thermo Scientific Vanquish Horizon UHPLC with DNAPac RP column (2.1×100 mm, 4 µm), gradient of 25 mM HFIP/15 mM DBA in water (mobile phase A) and acetonitrile (mobile phase B); column at 70 °C, autosampler at 5 °C, flow rate 400 µL/min.
• Mass spectrometry: Thermo Scientific Orbitrap Exploris 240 in negative ion mode; resolution 240,000 at m/z 200; scan range m/z 1,000–2,000; AGC target normalized to 200; maximum injection time 300 ms; data acquired in profile mode.
• Software: Chromeleon 7.3.1 CDS for LC control; Orbitrap Exploris Series ICSW for MS control; BioPharma Finder 5.0 Intact Mass Analysis workflow employing Xtract deconvolution and terminal truncation search.

Main results and discussion

RNase T1 cleaves IVT mRNA at guanosine residues, leaving poly(A) tails intact. Following oligo(dT) enrichment, the poly(A) pool produced a single chromatographic peak with improved shape and retention using DBA over TEA. HRAM spectra exhibited overlapping multiply charged ions across m/z 1,000–2,000. BioPharma Finder’s Xtract algorithm deconvoluted isotopically resolved charge states to monoisotopic masses with 3–6 ppm mass error. Deconvoluted masses were annotated via a terminal truncation search against a theoretical 140mer poly(A) sequence. Division of monoisotopic masses by 330 Da (AMP minus water) provided individual tail lengths, revealing a distribution centered around 120–130 adenosine residues.

Benefits and practical application of the method

• High confidence in poly(A) tail length measurement without amplification biases.
• Streamlined workflow integrating digestion, enrichment, UHPLC and HRAM‐MS.
• Automated deconvolution and annotation accelerate data analysis in vaccine and mRNA therapeutic development.
• Precise mass accuracy supports QC and regulatory compliance for critical quality attributes.

Future trends and potential applications

Ongoing advances may include:

• Automation of sample preparation and data processing to increase throughput.
• Extension to modified nucleotides and cap structure analysis in mRNA profiling.
• Integration with machine learning for real‐time QC and process monitoring.
• Application to other biopolymer tail characterization, such as poly(U) or unconventional 3’ modifications.

Conclusion

The combination of RNase T1 digestion, ion‐pair UHPLC separation, Orbitrap Exploris 240 HRAM‐MS and BioPharma Finder 5.0 software provides a powerful, accurate and efficient platform for poly(A) tail length determination in IVT mRNA. This method supports critical quality attribute analysis in mRNA vaccine and therapeutic development.

Reference

• Crick F. Central Dogma of Molecular Biology. Nature 1970, 227(5258), 561–563.
• Millevoi S.; Vagner S. Molecular Mechanisms of Eukaryotic Pre-mRNA 3' End Processing Regulation. Nucleic Acids Res. 2010, 38(9), 2757–2774.
• Edmonds M.; Abrams R. Formation of a Sequence of Adenylate Units by Thymus Polymerase. J. Biol. Chem. 1960, 235(4), 1142–1149.
• Edmonds M.; Vaughan M. H.; Nakazato H. Polyadenylic Acid Sequences in Nuclear RNA. Proc. Natl. Acad. Sci. U. S. A. 1971, 68(6), 1336–1340.
• Deo R. C.; Bonanno J. B.; Sonenberg N.; Burley S. K. Recognition of Poly(A) RNA by Poly(A)-Binding Protein. Cell 1999, 98(6), 835–845.
• Natalizio B. J.; Wente S. R. Designating Routes for Nuclear mRNA Export. Trends Cell Biol. 2013, 23(8), 365–373.
• Weill L.; Belloc E.; Bava F.-A.; Méndez R. Translational Control by Poly(A) Tail Changes. Nat. Struct. Mol. Biol. 2012, 19(6), 577–585.
• Gallie D. R. Cap and Poly(A) Tail Synergy in Translation. Genes Dev. 1991, 5(11), 2108–2116.
• Goldstrohm A. C.; Wickens M. Multifunctional Deadenylase Complexes. Nat. Rev. Mol. Cell Biol. 2008, 9(4), 337–344.
• Garneau N. L.; Wilusz J.; Wilusz C. J. The Highways and Byways of mRNA Decay. Nat. Rev. Mol. Cell Biol. 2007, 8(2), 113–126.
• Park J.-E.; Yi H.; Kim Y.; Chang H.; Kim V. N. Regulation of Poly(A) Tail and Translation during Cell Cycle. Mol. Cell 2016, 62(3), 462–471.
• Chang H.; Lim J.; Ha M.; Kim V. N. TAIL-Seq: Genome-Wide Poly(A) Tail Measurement. Mol. Cell 2014, 53(6), 1044–1052.
• Roussis S. G.; Pearce M.; Rentel C. Small Alkyl Amines as Ion-Pair Reagents. J. Chromatogr. A 2019, 1594, 105–111.
• Senko M. W.; Beu S. C.; McLaffertycor F. W. Monoisotopic Mass Determination from Isotopic Distributions. J. Am. Soc. Mass Spectrom. 1995, 6(4), 229–233.