SYNTHESIS AND CLEAVAGE OF OLIGODEOXYNUCLEOTIDES CONTAINING A 5-HYDROXYURACIL RESIDUE AT A DEFINED SITE

Oxidation and hydrolysis of a cytosine residue can lead to the formati on of 5-hydroxyuracil in DNA. The biological consequences of this modi fication are not fully understood. To facilitate biochemical and bioph ysical studies aimed at elucidating the effects of this modification i n DNA, we have developed a solid-phase synthetic method for the placem ent of 5-hydroxyuracil residues at defined sites in oligodeoxynucleoti des. This method is based upon the enhanced acidity of the g-hydroxyl proton which allows selective aqueous acetylation. Under standard aque ous ammonia deprotection conditions, however, we observed that 5-hydro xyuracil residues are lost substantially from synthetic oligonucleotid es. Substitution of aqueous ammonia with methanolic potassium carbonat e and the use of phosphoramidite derivatives with alternatively protec ted amino groups allow synthesis of oligonucleotides containing 5-hydr oxyuracil and all normal bases in high yield. The composition of the o ligodeoxynucleotides prepared by this method has been verified by enzy matic digestion followed by high-performance liquid chromatography (HP LC) analysis as well as acid hydrolysis followed by GC/MS analysis. Th e location of the 5-hydroxyuracil residue is demonstrated by selective permanganate oxidation of the 5-hydroxyuracil residue followed by p-e limination. We have also probed a synthetic oligonucleotide containing a unique 5-hydroxyuracil residue with uracil DNA N-glycosylase, previ ously reported to remove this lesion from DNA.