Fig. 3: Polysome profiling MPRA on fully randomized 5’UTR libraries.
From: Optimizing 5’UTRs for mRNA-delivered gene editing using deep learning
New libraries contain a 25nt- or 50nt-long randomized region in the 5’UTR, preceded only by the G triplet appended by IVT. A Schematic of mRNA library preparation based on template switching (TS). (i) Reverse transcription (RT) proceeds from the EGFP CDS into the mRNA 5’end. (ii) The reverse transcriptase (RTase) adds three deoxycytosines (CCC) to the 3’ of the cDNA, to which a TS primer ending in three riboguanines (rGrGrG) binds. (iii) the RTase switches templates, thereby adding the reverse complement of the TS primer to the 3’ end of the cDNA. (iv) Illumina adapters are incorporated using PCR via primers that bind to the flanking constant regions. B-C Median MRL of all sequences containing a uAUG (B) or a 5nt-long oligopyrimidine (C or U) tract (C) at the indicated position from the start of the transcript, for the 25nt- (yellow) or 50nt-long (orange) randomized 5’UTR libraries, as well as our previous fixed-end 50nt library (green). MRL was normalized to the median of each library. D Architecture of Optimus 5-Prime(25), trained on data from the random-end 25nt MPRA library. E Performance of Optimus 5-Prime(25) on a set of 2,000 5’UTRs from the random-end 25nt library held out from training. Source data are provided as a Source Data file.
