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.