Preparation and Analysis of Oligonucleotides Containing Lesions Resulting from C5‘-Oxidation

Hydrogen atom abstraction from the C5‘-position of nucleotides in DNA results in direct strand scission. The newly formed 5‘-termini of the cleaved DNA consists of alkali-labile fragments of the oxidized nucleotide. One terminus contains a 5‘-aldehyde as part of an otherwise undamaged nucleotide (T-al). A second more structurally distinct product that is produced in lower yields results from cleavage of the C4‘−C5‘ carbon−carbon bond. The 1,4-dioxo-2-phosphorylbutane (DOB) is a precursor of the alkylating agent but-2-ene-1,4-dial. To facilitate studies on these lesions, methods for synthesizing oligodeoxynucleotides containing DOB or T-al at their 5‘-termini were developed. The effects of these lesions on the UV-melting temperatures of duplex DNA, and their cleavage labilities were determined. T-al cleaves very slowly (t1/2 = 100.7 h), whereas DOB has a half-life at 37 °C (pH 7.2) of less than 11 h. In addition, DOB forms a stable adduct very efficiently with Tris, which protects the abasic site against cleavage.