High-throughput interrogation of programmed ribosomal frameshifting in human cells

Martin Mikl; Yitzhak Pilpel; Eran Segal

To not limit our analysis to endogenous frameshifting events, we designed completely synthetic 418 regions corresponding to a slippery site and the upstream and downstream region with different 419 sequence and structural properties. Some fully designed sequences were able to trigger frameshifting 420 and resulted in a -1 PRF signal of up to 11% GFP fluorescence, approximately corresponding to the 421 wild-type frameshifting rate of the most efficient event tested here (SARS coronavirus). We tested all 422 different downstream regions with synthetic slippery sequences resembling common types of -1 PRF 423 sites and found pronounced differences in combinatorial preferences (Fig 4G) , showing that also fully 424 synthetic frameshifting events exhibit the combinatorial preferences and context-dependent 425 peculiarities observed for native PRF events. whether they yielded a detectable frameshifting signal in our assay or not (Fig 5BC, Fig S10A; "-1 446 PRF events" vs. "all library variants"). 447