Minimal genetically encoded tags for fluorescent protein labeling in living neurons

Modern light microscopy, including super-resolution techniques, brought about a demand for small labeling tags that bring the fluorophore closer to the target. This challenge can be addressed by labeling unnatural amino acids (UAAs) with click chemistry. UAAs are site-specifically incorporated into a protein of interest by genetic code expansion. If the UAA carries a strained alkene or alkyne moiety it can be conjugated to a tetrazine-bearing fluorophore via a strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC), a variant of bioorthogonal click chemistry. The minimal size of the incorporated tag and the possibility to couple the fluorophores directly to the protein of interest with single-residue precision make SPIEDAC live-cell labeling unique. However, until now, this type of labeling has not been used in complex, non-dividing cells, such as neurons. Using neurofilament light chain as a target protein, we established SPIEDAC labeling in living primary neurons and applied it for fixed-cell, live-cell, dual-color pulse--chase and super-resolution microscopy. We also show that SPIEDAC labeling can be combined with CRISPR/Cas9 genome engineering for tagging endogenous NFL. Due to its versatile nature and compatibility with advanced microscopy techniques, we anticipate that SPIEDAC labeling will contribute to novel discoveries in neurobiology.