Inosine induces context-dependent recoding and translational stalling

To test how inosine in a codon is translated, we synthesized short reporter-transcripts coding for an N-terminal Flag-tag and a test-peptide containing inosine in one defined codon. All codons containing inosines in at least one position were tested. Codons that would lead to ambiguous translation products were omitted. The transcripts were generated using in vitro transcription with ITP instead of GTP and subsequently in vitro translated using rabbit reticulocyte lysate and resulting peptides were analyzed by LC-MS/MS. The templates for in vitro transcription were generated using linearized DNA plasmids.