Fig. 2. Schematic overview of the workflow in this study. A DNA library for DMS analysis was constructed by introducing mutations at FGF2 positions predicted to be involved in interactions with FGFR isoforms. mRNA display was then performed. The DNA library was transcribed into mRNA, and a puromycin linker was ligated to the 3′ end of each transcript. In vitro translation was carried out, resulting in the covalent linkage of the translated FGF2 mutants to their corresponding mRNA via puromycin. After reverse transcription, FGF2–mRNA : cDNA complexes were subjected to parallel affinity selection against individual FGFR isoforms. Recovered DNA was analyzed by NGS, yielding DMS-derived enrichment landscapes of FGF2 mutants toward each FGFR isoform. These data were subsequently used to design, evaluate, and analyze FGF2 variants with altered FGFR isoform specificity.
