title: Site-specific DNA labeling by Staudinger ligation.
abstract: 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.
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Weisbrod
authors.forename: Anna
authors.initials: A
authors.lastname: Baccaro
authors.forename: Andreas
authors.initials: A
authors.lastname: Marx
mesh_terms: D001386:Azides / Q000737:chemistry; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003857:Deoxyuridine / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D010720:Phosphines / Q000737:chemistry; D013194:Staining and Labeling / Q000379:methods*; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000737:chemistry
chemical_list: D001386:Azides; D010710:Phosphates; D010720:Phosphines; D013942:Thymine Nucleotides; D001710:Biotin; D004247:DNA; D004259:DNA-Directed DNA Polymerase; C024157:thymidine 5'-triphosphate; D003857:Deoxyuridine