SNAPpa: A Photoactivatable SNAP-tag for the Spatiotemporal Control of Protein Labeling.

SNAP-tag is one of the most commonly used self-labeling protein tags for cell imaging studies. To achieve selective spatiotemporal imaging of cells, we set out to engineer a photoactivatable SNAP-tag. For this, we incorporated the well-established and readily available photocaged unnatural amino acid -nitrobenzyl--tyrosine (ONBY) into all three tyrosine positions of SNAP. In-gel imaging analysis and fluorescence polarization measurements revealed that placing ONBY in position Y114 of the SNAP-tag facilitates the most effective and most efficient photoactivation of the irreversible self-labeling reaction with (sulfonated) benzyl guanine substrates, which is why we dubbed this photoactivatable SNPA-tag variant "SNAPpa". To demonstrated its potential for live-cell imaging, we further tested SNAPpa in HEK293 cells, either fused to a nuclear localization domain for intracellular imaging or fused to either a transmembrane region or the glucagon-like peptide 1 receptor for extracellular imaging. Each SNAPpa construct produced no fluorescence signal when ONBY remained in its photocaged state by keeping the cells in the dark. However, a clear fluorescence signal appeared after light-induced decaging of ONBY. Applying a localized light beam thereby highlighted the precise spatiotemporal control of cell imaging. In conclusion, SNAPpa can be used for the efficient light-induced activation of fluorescence labeling and can be easily established, readily implemented and effectively combined with the broad repertoire of substrates that is already available for SNAP.