Fluorescence profiles of droplets obtained at each round of µIVC. Fusion efficiency can be assessed by the blue fluorescence of coumarin added in the droplets while the orange fluorescence informs on RNA function (Gemini-561 fluorescence activation). The population of droplets gated and sorted during the first (A) and the second (B) round of screening are dashed-boxed in black. Their DNA content was recovered and used to prime a new round of screening. During the third round (C), a relaxed (black dashed box, population R3A) and a stringent (red dashed box, population R3B) gating were used. (D) For each round of µIVC, the enriched DNA libraries were in vitro transcribed in the presence of 500 nM Gemini-561 and the fluorescence was monitored over time. The fluorescence apparition rate was computed for each library and normalized to that of the parental SRB-2 aptamer. The values are the mean of n = 3 independent experiments. The error bars correspond to ± 1 SD.
