Reverse transcription of RNA templates containing modified ribonucleosides transfers modification-related information as misincorporations, arrest or nucleotide skipping events to the newly synthesized cDNA strand. The frequency and proportion of these events, merged from all sequenced cDNAs, yield a so-called RT signature, characteristic for the respective RNA modification and reverse transcriptase (RT). While known for DNA polymerases in so-called error-prone PCR, testing of four different RTs by replacing Mg 2+ with Mn 2+ in reaction buffer revealed the immense influence of manganese chloride on derived RT signatures, with arrest rates on m 1 A positions dropping from 82% down to 24%. Additionally, we observed a vast increase in nucleotide skipping events, with single positions rising from 4% to 49%, thus implying an enhanced read-through capability as an effect of Mn 2+ on the reverse transcriptase, by promoting nucleotide skipping over synthesis abortion. While modifications such as m 1 A, m 2 2G, m 1 G and m 3 C showed a clear influence of manganese ions on their RT signature, this effect was individual to the polymerase used. In summary, the results imply a supporting effect of Mn 2+ on reverse transcription, thus overcoming blockades in the Watson-Crick face of modified ribonucleosides and improving both read-through rate and signal intensity in RT signature analysis.
Keywords: RNA modifications; RT signature; m1A; manganese chloride; reverse transcription.
The authors declare no conflict of interest.