Poly A tail length analysis of in vitro transcribed mRNA by LC-MS
The 3′-polyadenosine (poly A) tail of in vitro transcribed (IVT) mRNA was studied using liquid chromatography coupled to mass spectrometry (LC-MS). Poly A tails were cleaved from the mRNA using ribonuclease T1 followed by isolation with dT magnetic beads. Extracted tails were then analyzed by LC-MS which provided tail length information at single-nucleotide resolution. A 2100-nt mRNA with plasmid-encoded poly A tail lengths of either 27, 64, 100, or 117 nucleotides was used for these studies as enzymatically added poly A tails showed significant length heterogeneity. The number of As observed in the tails closely matched Sanger sequencing results of the DNA template, and even minor plasmid populations with sequence variations were detected. When the plasmid sequence contained a discreet number of poly As in the tail, analysis revealed a distribution that included tails longer than the encoded tail lengths. These observations were consistent with transcriptional slippage of T7 RNAP taking place within a poly A sequence. The type of RNAP did not alter the observed tail distribution, and comparison of T3, T7, and SP6 showed all three RNAPs produced equivalent tail length distributions. The addition of a sequence at the 3′ end of the poly A tail did, however, produce narrower tail length distributions which supports a previously described model of slippage where the 3′ end can be locked in place by having a G or C after the poly nucleotide region.
KeywordsmRNA Poly A Tail length Mass spectrometry Slippage In vitro transcription
Support for this work was provided by DARPA grant HR-0011-13-3-0003.
Compliance with ethical standards
Conflict of interest
The authors declare they have no conflict of interest.
The views, opinions, and/or finding expressed are those of the authors and should not be interpreted as representing the official views or policies of the Department of Defense or the US Government.
- 17.Muddiman DC, Null AP, Hannis JC. Precise mass measurement of a double-stranded 500 base-pair (309 kDa) polymerase chain reaction product by negative ion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Rapid Commun Mass Spectrom. 1999;13(12):1201–4.CrossRefGoogle Scholar
- 25.Hail ME, Elliott B, Anderson K. High-throughput analysis of oligonucleotides using automated electrospray ionization mass spectrometry. Am Biotechnol Lab. 2004;22:12–3.Google Scholar
- 26.Haukanes BI, Kvam C. Application of magnetic beads in bioassays. Biotechnology (N Y). 1993;11(1):60–3.Google Scholar
- 39.Anikin M, Molodtsov V, Temiakov D, McAllister WT. Transcript slippage and recoding. In: Atkins JF, Gesteland RF, editors. Recoding: expansion of decoding rules enrishes gene expression, Nucleic acid and molecular biology: Springer; 2010. p. 409–32.Google Scholar