Solid phase synthesis and restriction endonuclease cleavage of oligodeoxynucleotides containing 5-(hydroxymethyl)-cytosine

Emerging data suggest an important role for cytosine methylation in tumorigenesis. Simultaneously, recent studies indicate a significant contribution of endogenous oxidative DNA damage to the development of human disease. Oxidation of the 5-methyl group of 5-methylcytosine (5(m)C) residues in DNA results in the formation of 5-(hydroxymethyl)cytosine (C-hm). The biological consequences of C-hm residues in vertebrate DNA are as yet unknown; however, conversion of the hydrophobic methyl group to the hydrophilic hydroxymethyl group may substantially alter the interaction of sequence-specific binding proteins with DNA, Central to both biophysical and biochemical studies on the potential consequences of specific DNA damage products such as C-hm are efficient methods for the synthesis of oligodeoxynucleotides containing such modified bases at selected positions, In this paper, we describe a method for the placement of C-hm residues in oligodeoxynucleotides using established phosphoramidite chemistry. In addition, we have examined the influence of specific C-hm residues on enzymatic cleavage of oligodeoxynucleotides by the methylation-sensitive restriction endonucleases Mspl and Hpall.