Detection of Methylation on dsDNA at Single-Molecule Level using Solid-State Nanopores

Abstract

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.