Abstract:

Live cellular RNA imaging has become an indispensable tool in cellular biology as vital roles of biologically significant RNAs are discovered. Fluorogenic RNA aptamers are single-stranded oligonucleotides that exhibit strong fluorescence upon target-binding and showcase great potential in real-time RNA visualization. Therefore, we have developed a droplet-based microfluidic platform that combines the generation, injection, and sorting of droplets, for high-throughput screening of fluorogenic RNA aptamers. The sorting is programmed to select sequences that specifically bind to a new green fluorescent protein (GFP) fluorophore-mimic from a large DNA library by generating millions of droplets, each containing no more than one DNA sequence, and sorting the top 0.5%-2% of the fluorescent droplets at about 175 Hz. We have completed three rounds of sequence enrichment with our microfluidic platform, with droplets exhibiting strong fluorescence following each selection round.