Figure 3.

Main screening strategies for isolating light-up RNA aptamers. (a) Colony screening [31,62]. SELEX-enriched gene library is cloned and expressed in bacteria plated on a solid medium supplemented with the fluorogen. Upon incubation, colonies expressing light-up aptamers are identified by fluorescence emission (green shadow) when illuminated with fluorogen excitation wavelength. (b) FACS-based bacteria screening [31]. SELEX-enriched gene library is cloned and expressed in bacteria incubated in the presence of the fluorogen. Bacteria fluorescence is then analyzed on a FACS using a laser exciting the fluorogen. Fluorescence emitting bacteria (green shadow) are then deflected and sorted from the rest of the population. (c) Miniaturized in vitro screening using large integration scale microfluidic devices [64]. DNA coding for each variant is first spotted (red dots, Step 1) onto a surface prior to assembling the microfluidic chip. Then, each DNA cluster is transcribed in RNA later captured on a second spot of the chip (blue spot, Step 2). Finally, flowing the fluorogen into the microfluidic channels (Step 3) allows the detection of light-up aptamers (green spot). Quantifying the fluorescence emitted by each construct at various concentrations of fluorogen allows the determination of parameters such as the brightness and the dissociation constant (K_D). (d) Droplet-based microfluidic in vitro screening workflow [65]. A gene library is diluted into a PCR mixture (in dark blue) prior to individualizing the molecules into picoliter-sized water-in-oil droplets carried by an oil phase (in gray, Step 1). Upon off-chip PCR amplification, small and amplified DNA-containing droplets (dark blue) are synchronized and fused with larger droplets containing an in vitro transcription mixture supplemented with the fluorogen (light blue, Step 2). Upon an incubation step allowing for in vitro transcription to take place, the fluorescence of each droplet is analyzed and those droplets containing a light-up aptamer (in green) are sorted from the rest of the population (in blue). Both fusion and sorting events are triggered by the application of an electric field to built-in positive (pos, shown in red) and ground (gnd, shown in black) electrodes.