Synthesis and characterization of a new 5-thiol-protected deoxyuridine phosphoramidite for site-specific modification of DNA

A new nucleotide analogue was developed for site-specific incorporation of a reactive thiol group into DNA. This creates a unique site for the post-synthetic modification of that nucleotide with a variety of molecular tags, such as photo-cross-linkers and fluorescent or spin-label moieties. 5'-O-(4,4'-Dimethoxytrityl)-5-[S-(2,4-dinitrophenyl)thio]-2'-deoxyuridine 3'-O-(2-cyanoethyl N,N'-diisopropylphosphoramidite) was synthesized and incorporated at internal positions in several oligonucleotides using automated DNA synthesis and standard phosphoramidite chemistry. The coupling yield of the analogue was comparable to the coupling yield for a standard phosphoramidite, and no significant differences were observed in the overall yields of the dinitrophenyl-labeled oligonucleotides compared to the corresponding unmodified oligonucleotides. Characterization of the dinitrophenyl-modified oligonucleotides included enzymatic degradation, HPLC chromatography, and gel electrophoresis. Deprotection of the mercaptan group with beta-mercaptoethanol yielded an oligonucleotide containing 5-mercaptodeoxyuridine which was then selectively modified, without purification, by reaction with 5-(iodoacetamido)fluorescein. Incorporation of the dinitrophenyl-modified oligonucleotide into double-stranded DNA was achieved using the polymerase chain reaction. Characterization of the dinitrophenyl-labeled product by immunodetection with anti-dinitrophenyl antibodies confirmed the stability of the protecting group to the thermocycling and thus established the use of this thiol-protected mercaptodeoxyuridine phosphoramidite for preparation of site-specifically modified DNA.