Characterization of poly(A) and poly(T) tail lengths in plasmid DNA by liquid chromatography high-resolution mass spectrometry

Rapid advances in messenger RNA (mRNA) technology necessitate effective analytical methods. This study describes the development of a novel in vitro method using ion-pair reversed-phase liquid chromatography coupled to high-resolution mass spectrometry (IP-RP-LC-HRMS) for assessing the poly(A) and complementary poly(T) tail lengths directly into the DNA template used to manufacture mRNA. Briefly, after the validation of poly(A) tail length in the plasmid by Sanger sequencing, double-stranded DNA fragments containing these tails in the plasmid of interest were amplified by the polymerase chain reaction (PCR), purified on silica column, and digested with restriction enzymes ClaI and HindIII. Gel or capillary electrophoresis confirmed sample quality and enzymatic digestion efficiency. Subsequently, poly(A) and complementary poly(T) tails were extracted and analyzed by LC-MS to determine their length and heterogeneity at a single-nucleotide resolution. Three DNA templates containing poly(A) tail lengths of 60A-G, 95A, or 108A were studied. LC-MS results correlated well with Sanger sequencing, identifying major populations of 60A-G, 95A, or 108A. Surprisingly, unlike Sanger sequencing, LC-MS analysis revealed minor poly(A) populations with lengths longer or shorter than the theoretically encoded poly(A) tail length. This finding could be explained by (i) the slippage of bacterial DNA polymerase I during plasmid replication in bacterial culture, which occurs on repeat mononucleotide sequences, or (ii) the slippage of Q5 (R) High-Fidelity DNA Polymerase during PCR amplification. In conclusion, the method is easy, rapid, and accurate and could replace Sanger sequencing to assess the poly(A) and complementary poly(T) tail lengths in plasmid DNA.