Fig. 3. Unbiased PTM interference screen with RNA base editing.

a Various signaling cues related to essential biological processes represent attractive targets for doseable and transient manipulation by RNA base editing. b 70 + PTM sites (Y > C, K > R, S > G, T > A, etc.) on various endogenous signaling transcripts have been targeted in HeLa-PB-SA1Q cells using 2 pmol (per 96-well with 150 µl total volume) of the improved standard guide RNA design (5′−3 + 9 + C + 12 nt, 4 LNAs, BisBG) or variation thereof as indicated. c–g Analysis of effects of various parameters on RNA base editing efficiency. Mean editing yields (N = 2) of 70+ targets plotted against target codon (c), GC content of the guide RNA calculated with Oligo Calc⁴⁴ (d), minimum free energy of guide RNA hairpin folding secondary structure calculated with NUPACK⁴⁵ (e), relative gene expression level (mean TPM values of genes in Hela PB SA1Q cells with 48 h dox induced SA1Q expression (f). Half-life of the transcript, taken from⁴². In panels c-g, the color code indicates the nucleotide 5′ to the edited adenosine. Data in panel b is shown as the mean of N = 2 independent experiments in most cases and N = 3 or 4 in some cases, individual data points are given. In panel c, the box represents the interquartile range showing the middle 50% of the data points. The ends of upper or lower T-shaped whiskers extend to the maximum or minimum data point which is still within 1.5 times the interquartile range. Source data are provided as a Source Data file.