A general strategy to inhibiting viral -1 frameshifting based on upstream attenuation duplex formation

Hu, Hao-Teng; Cho, Che-Pei; Lin, Ya-Hui; Chang, Kung-Yao

A stable structure could act as a roadblock to cause ribosomal drop-off in addition to stimulating −1 PRF. Accordingly, a frameshifting pseudoknot can prohibit a significant fraction of frameshifted ribosomes that it stimulated from reaching the −1 frame stop codon. Eventually, it leads to the compromise of observed −1 PRF efficiency (49) . By contrast, the trans-formed −1 PRF attenuator duplex identified in this work is upstream of the slippery site and the potential ribosomal drop-off effect mediated by the duplex should occur before the ribosome reaches the slippery site. While the drop-off effect could lead to under-estimation Figure S6) . The frameshifting assays were conducted in human 293T cell lysate using capped mRNA obtained by invitro transcription. The dual-luciferase activity was measured to calculate the in vitro frameshifting efficiency with the frameshifting efficiency of the reporter without antisense addition being treated as 1. Value for each bar is the mean of three independent experiments with standard error of the mean. P-values were determined by a student's t-test with P-value < 0.0001 designated by an '*' and referring to the comparison with the construct without the addition of a 2 OMe-modified RNA. (B) Summary of unpaired two-sample t-test for the effects of MERS 5 as-2 OMe-RNA on MERSex −1 PRF frameshifting activity. Calculated frameshifting activity for MERSex against a read-through control (Supplementary Figure S6 ) with or without 10 M of 2 OMe-modified RNA antisense in the same condition as those in (A). The data was statistically analyzed by a procedure suggested for bicistronic reporter assay (36) with results summarized in the right panel.