Cellular Imaging of Small RNAs using Fluorescent RNA-Mango Aptamers

Abstract

In recent years, there has been an explosion of fluorescent RNA aptamers that have been isolated using SELEX. Since their discovery, fluorogenic RNA aptamers, such as Spinach and Mango have held great potential to enable the visualisation of RNA molecules in cells. However, resolving complex RNA structures has been limited primarily to bacterial cells due to inefficiency in maintaining adequate aptamer stability and fluorescence. Evolving new RNA aptamers with improved physicochemical properties (i.e. thermal stability, fluorescence brightness and ligand affinity) should better their use in cellular imaging. Three new Mango-like aptamers have recently been evolved using microfluidic-assisted in vitro compartmentalization, mutagenesis and fluorescent selection, which improved fluorescence brightness, ligand binding affinity and thermal stability. We show that these aptamers are readily useable to image small non-coding RNAs (such as 5S rRNA and U6 snRNA) in both live and fixed human cells with improved sensitivity and resolution. The Mango tagged RNAs sub-cellular localisation pattern is conserved, as validated using immunofluoresence. Our data show that these new aptamers are significantly improved for cellular imaging over previous fluorogenic aptamers and can in principle be incorporated into a wide range of coding and non-coding RNAs. We anticipate that these new aptamers will drastically improve RNA imaging in cells.