In vitro photobleaching measurement

5S-F30-Mango I, II, III and IV as well as 5S-F30-Broccoli template regions were placed under the control of T7 RNA polymerase promoter. Genes were PCR-amplified, in vitro-transcribed, purified and quantified as before (see Enrichment measurement section). Then 1 volume of 3 µM RNA solution was added to 1 volume of 2-times concentrated buffer (280 mM KCl, 2 mM MgCl₂, 20 mM NaH₂PO₄ pH 7.5) supplemented with 3.6 µM of fluorogenic dye (DFHBI-1T for Broccoli aptamer, and TO1-B for Mango aptamers). The mixture was incubated for an hour at room temperature prior to being loaded into a length of PTFE tubing (Thermo Fisher) and infused into a droplet generator microfluidic device where it was dispersed into 100 pL droplets carried by HFE 7500 fluorinated oil (3 M) supplemented with 3% of a fluorosufactant as described previously³⁷. The resulting emulsion was then loaded into 5 µL capillary (Corning) and the montage was imaged on an epifluorescence microscope (TiE, Nikon). Depending on the dye, the emulsion was exposed to a constant illumination wavelength of 470 nm (DFHBI-1T) or 508 nm (TO1-B) at the maximum intensity of the light source (Spectra X, Lumencor), and the emitted fluorescence (respectively, 514 nm ± 24 and 540 nm ± 12) was collected by an Orca-Flash IV camera for 200 ms every 100 ms with ×20 objective (numerical aperture (NA) 0.45). Fluorescence intensity of each picture was then extracted using NiS software (Nikon) and the data were fit to an exponential decay equation to compute the fluorescence half-life.