DNA breakage associated with targeted gene alteration directed by DNA oligonucleotides

Understanding the mechanism by which single-stranded oligonucleotides (ODNs) elicit targeted nucleotide exchange (TNE) is imperative to achieving optimal correction efficiencies and medical applicability. It has been previously shown that introduction of an ODN into cells results in the activation of DNA damage response pathways, but there has been no evaluation of the damage created at the level of the DNA. The activation of H2AX, a hallmark protein of DNA breakage, suggests that a double-strand break (DSB) could be occurring during the targeted gene alteration (TGA) reaction. Using the human HCT116 cell line with a single integrated mutant eGFP gene as our model system, we demonstrate that the DNA strand breakage occurs when a specific ODN, designed to direct TGA, is transfected into the cells. Both single- and double-stranded DNA cleavage is observed dependent on the level of ODN added to the reaction. Possible mechanisms of ODN-dependent DSB formation, as a function of TGA, are discussed herein.