abberior dyes & labels
Cell‐Permeable Nanobodies Allow Dual‐Color Super‐Resolution Microscopy in Untransfected Living Cells
Schneider, A. F. L., Benz, L., Lehmann, M., & Hackenberger, C.
cell-penetrating peptides, nanobody, semi-synthesis
Super-resolution microscopy in living cells can be restricted by the availability of small molecule probes, which only exist against few targets and genetically encoded tags. Here, we expand the applicability of live-cell STED by engineering cell-permeable and highly fluorescent nanobodies as intracellular targeting agents. To ensure bright fluorescent signals at low concentrations we used the concept of intramolecular photostabilization by ligating a fluorophore along with the photostabilizer trolox to the nanobody using expressed protein ligation (EPL). Furthermore, these semi-synthetic nanobodies are equipped with a cleavable cell-penetrating peptide for efficient cellular entry, which enables super-resolution imaging of GFP and mCherry, as well as two endogenous targets, nuclear lamins and the DNA replication and repair protein PCNA. We monitored cell division and DNA replication via confocal and STED microscopy thus demonstrating the utility of these new intracellular tools for functional analysis.