Photochemically and Photoenzymatically Cleavable DNA

Methodology for the synthesis of site-specifically modified DNA has facilitated the physical, chemical, and enzymatic manipulation of DNA, as well as the study of important biological processes and the development of new classes of reagents and drugs. Herein we report the design, synthesis, and characterization of a DNA synthesis building block for the site-specific introduction of a photochemically and photoenzymatically cleavable linkage that is based on the photochemistry and photoenzymology of the cis-syn thymidine dimer. The phosphoramidite building block was synthesized in six steps from 5′-O-DMT-thymidine and 3′-O-TBDMS-thymidine and used to prepare a 22-mer containing a central dimer by standard automated DNA synthesis. Direct 254-nm photolysis of the 22-mer led to formation of the expected cleavage products in quantitative yield, as did visible light photolysis in the presence of Escherichia coli photolyase. An appreciable amount of cleavage (20%) by photolyase could also be induced by a single camera flash. The ability to photochemically and photoenzymatically cleave DNA site-specifically might find useful applications in the study of nucleic acid conformational transitions and enzymatic reactions, as well as the activation of prodrug forms of antisense oligonucleotides and ribozymes.