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

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.

Expanding the substrate selectivity of SNAP/CLIP-tagging of intracellular targets.

SNAP-tag belongs to a class of genetic tools of protein labeling that complements fluorescent proteins. This single-turnover enzyme is a mutant of human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hAGT). It accepts, in most cases, label-carrying O6-benzylguanines or benzyl-2-chloro-6-aminopyrimidines as suitable substrates. In this article, strategies and methods to expand the scope of the labels for intracellular proteins of live cells via the actions of SNAP-tag are presented. CLIP-tag is another mutant of the hAGT that was engineered to have mutually exclusive substrate specificity from SNAP-tag. The use of complementary bioorthogonal chemical reactions in conjunction with orthogonal enzymatic SNAP/CLIP-tags for the purpose of dual-color intracellular labeling is also described.