Reversible biotinylation phosphoramidite for 5'-end-labeling, phosphorylation, and affinity purification of synthetic...

A fluoride/amine-cleavable phosphoramidite designed for biotinylation, phosphorylation, and affinity purification of synthetic oligonucleotides was synthesized and coupled efficiently to the 5'-end of DNA on a solid-phase automatic synthesizer. The two hydroxyl groups of diethyl bis(hydroxymethyl)malonate were used to link biotin and the 5'-end of DNA together through a diisopropylsilyl acetal functionality and a phosphate ester group, respectively. The DNA was cleaved from solid support and fully deprotected by treating with a mixture of MeNH(2) ( approximately 40%) and NH(4)OH ( approximately 29%) (1:1, v/v, 65 degrees C, 30 min), and the linkage between biotin and DNA was found completely stable under these conditions. The biotinylated full-length DNA was efficiently attached to NeutrAvidin coated microspheres and failure sequences and other impurities were simply removed by washing with buffer and water. The microspheres were then treated with HF/pyridine/THF (rt, 1 h) and MeNH(2) ( approximately 40%, rt, 15 min) sequentially to yield high quality full-length 5'-end phosphorylated unmodified DNA as revealed by HPLC analysis. It is anticipated that this method will find applications in areas that require efficient isolation of 5'-end phosphorylated DNA from a complex mixture.