Super-Resolution Microscopy with DNA Molecules: Towards Localizomics

Abstract

Super-resolution fluorescence microscopy is a powerful tool for biological research. We use the transient binding of short fluorescently labeled oligonucleotides (DNA-PAINT) for simple and easy-to-implement multiplexed super-resolution imaging that technically achieves sub-5-nm spatial resolution. However, this does not translate to achievable image resolution in biological specimen, mainly due to the lack of readily available small and specific affinity reagents. We introduce Slow Off-rate Modified Aptamers (SOMAmers) for DNA-PAINT super-resolution microscopy as efficient and quantitative labeling reagents. We demonstrate the achievable image resolution and specificity by labeling and imaging of transmembrane as well as intracellular targets in fixed and live cell-specimen. Apart from ever increasing spatial resolution, efficient multiplexing strategies for the simultaneous detection of hundreds of molecular species are still elusive. We now introduce an entirely new approach to multiplexed super-resolution microscopy by designing the blinking behavior of targets with engineered binding frequency and duration in DNA-PAINT. We assay this kinetic barcoding approach in silico and in vitro using DNA origami structures, show the applicability for multiplexed RNA and protein detection in cells and finally experimentally demonstrate 124-plex super-resolution imaging within minutes.