Angew. Chem. Int. Ed. http://doi.org/f27zcg (2015)

Credit: © 2015 WILEY

Covalent crosslinking that can occur between the strands of a DNA duplex is of interest both in materials science, where it can impart stability to oligonucleotide-based assemblies, and in biochemistry, as it prevents replication and transcription processes and results in cytoxicity. Studies on crosslinking and the repair processes that naturally occur in cells have, however, been hindered by the fact that crosslinking agents typically promote the formation of a variety of linkages. Jacqueline Gamboa Varela and Kent Gates, from the University of Missouri, have now devised a simple, high-yield synthesis to crosslink complementary oligonucleotides at a specific location.

The researchers built on previous work that had identified the formation, within a duplex, of a linkage between the abasic site (Ap) of one oligonucleotide — generated by the loss or removal of a coding nucleobase — and a guanine or adenine residue of the opposite strand. Now, Gamboa Varela and Gates have instead crosslinked the Ap residue with a non-natural nucleobase, N4-amino-2′-deoxycytidine (dC*), installed for this purpose at a defined location. The Ap and dC* residues react through their respective aldehyde and hydrazine moieties to form a covalent hydrazone linkage. The complementary strands containing Ap and dC* were prepared through straightforward treatments of the corresponding deoxyuridine- and cytosine-containing oligonucleotides, using the enzyme uracil DNA glycosylase, and bisulfite and hydrazine in sodium phosphate, respectively. The Ap and dC* moieties are positioned in a manner that facilitates the formation of the hydrazone crosslink: the two residues are offset by one base in the hybridized duplex, and dC* is mispaired.

Characterization by gel electrophoresis analysis showed that the crosslink formation occurred in high yield at the predetermined location. The resulting dC*–Ap hydrazone linkage was stable under physiological conditions, increased the thermal stability of the duplex, and was found to be thermally reversible: repeated heating and cooling resulted in its cleavage and regeneration, respectively.