FastMiR: An RNA-based Sensor for in vitro Detection of Small RNA

The direct detection of small RNAs could revolutionize analyses of small RNAs. These 21- to 24-nucleotide molecules, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), play a variety of important regulatory roles in plants and animals. Here we describe an RNA-based, fluorescence sensor for small RNA detection that utilizes the fluorophore-binding sequence of “Spinach”, a GFP-like RNA aptamer for which the RNA-fluorophore complex exhibits strong and consistent fluorescence under excitation wavelength when combined with the sxRNA switch for detection of miRNA-mRNA interactions. Our sensors, FastMiR171 and FastMiR122, can rapidly detect and quantify the levels of miR171 and miR122 in vitro with similar sensitivity to small RNA sequencing. We demonstrate that live cell imaging using a Spinach-based RNA aptamer is possible, but presently lacks the sensitivity for in vivo studies. However, with advances in fluorescent RNA aptamer detection in vivo, our sensor would have applications in localizing and tracking small RNAs and RNAs in live cells.