Site-Specific DNA Labeling by Staudinger Ligation

Site-specific and chemoselective labeling of DNA is still a difficult task. The Staudinger ligation is a bioorthogonal reaction between azides and phosphines that requires no catalyst to proceed, allowing for mild reaction conditions. The reaction may be extended for site-specific labeling of DNA using azido-modified triphosphates, which can be incorporated site-specifically into DNA strands by DNA polymerases in a template-dependent manner. The azido-modified DNA, in turn, can be labeled by suitable phosphines. This protocol describes (1) the synthesis of an azido-TTP analogue; (2) the enzymatic synthesis of azido-modified DNA; (3) the synthesis of suitable phosphine labels; and (4) the labeling of azido-DNA with biotin–phosphine by Staudinger ligation with approximately 70% conversion.