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 incorporatio n of a reactive thiol group into DNA. This creates a unique site for t he post-synthetic modification of that nucleotide with a variety of mo lecular tags, such as photo-cross-linkers and fluorescent or spin-labe l moieties. tyl)-5-[S-(2,4-dinitrophenyl)thio]-2'-deoxyuridine 3'-O-(2 -cyanoethyl N,N'-diisopropylphosphoramidite) was synthesized and incor porated at internal positions in several oligonucleotides using automa ted DNA synthesis and standard phosphoramidite chemistry. The coupling yield of the analogue was comparable to the coupling yield for a stan dard phosphoramidite, and no significant differences were observed in the overall yields of the dinitrophenyl-labeled oligonucleotides compa red to the corresponding unmodified oligonucleotides. Characterization of the dinitrophenyl-modified oligonucleotides included enzymatic deg radation, HPLC chromatography, and gel electrophoresis. Deprotection o f the mercaptan group with beta-mercaptoethanol yielded an oligonucleo tide containing 5-mercaptodeoxyuridine which was then selectively modi fied, without purification, by reaction with 5-(iodoacetamido)fluoresc ein. Incorporation of the dinitrophenyl-modified oligonucleotide into double-stranded DNA was achieved using the polymerase chain reaction. Characterization of the dinitrophenyl-labeled product by immunodetecti on with anti-dinitrophenyl antibodies confirmed the stability of the p rotecting group to the thermocycling and thus established the use of t his thiol-protected mercaptodeoxyuridine phosphoramidite for preparati on of site-specifically modified DNA.