A Simple,High‐Yield Synthesis of DNA Duplexes Containing a Covalent,Thermally Cleavable Interstrand Cross‐Link at a Defined Location

Interstrand DNA–DNA cross‐links are highly toxic to cells because these lesions block the extraction of information from the genetic material. The pathways by which cells repair cross‐links are important, but not well understood. The preparation of chemically well‐defined cross‐linked DNA substrates represents a significant challenge in the study of cross‐link repair. Here a simple method is reported that employs “post‐synthetic” modifications of commercially available 2′‐deoxyoligonucleotides to install a single cross‐link in high yield at a specified location within a DNA duplex. The cross‐linking process exploits the formation of a hydrazone between a non‐natural N⁴‐amino‐2′‐deoxycytidine nucleobase and the aldehyde residue of an abasic site in duplex DNA. The resulting cross‐link is stable under physiological conditions, but can be readily dissociated and re‐formed through heating–cooling cycles.