SNAP/CLIP-Tags and Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC)/Inverse Electron Demand Diels-Alder (IEDDA) for Intracellular Orthogonal/Bioorthogonal Labeling.

Abstract

Labeling a protein of interest (POI) with a fluorescent reporter is a powerful strategy for studying protein structures and dynamics in their native environments. Compared to fluorescent proteins, synthetic dyes provide more choices in photophysical or photochemical attributes to microscopic characterizations. The specificity of bioorthogonal reactions in conjunction with the fidelity of subcellular destinations of genetically encoded protein tags can be employed to label POIs in live and fixed cells in a two-step process. In the present study the orthogonality of the strain-promoted azide-alkyne cycloaddition (SPAAC) and the inverse electron demand Diels-Alder (IEDDA) reaction is corroborated in concurrent labeling of two different intracellular targets. An azido group and a strained alkene are first installed at specific subcellular locations via orthogonal enzymatic reactions of the genetically incorporated SNAP- and CLIP-tags. The subsequent bioorthogonal reactions with fluorophores carrying matching reactive functionalities result in simultaneous dual labeling. The two-step "orthogonal-bioorthogonal" labeling process would increase the utilities of SNAP/CLIP-tags and, as a consequence, would expand the capability of decorating biological specimens with functionalities beyond fluorophores to potentially include spin labels, radioactive tracers, or catalysts.