An approach for the synthesis of duplexes containing N3T-butyl-N3T interstrand cross-links via a bisphosphoramidite strategy

Abstract

DNA duplexes containing an interstrand cross-link have been synthesized utilizing a bis-3′-O-phosphoramidite deoxythymidine dimer where the N3 atoms are bridged by a butyl linker. With this approach sufficient quantities of high purity cross-linked duplexes are obtained that will enable various biochemical and structural studies to aid in research directed towards understanding the mechanism of interstrand cross-linked DNA repair. This methodology has advantages over a previously reported method to synthesize cross-linked DNA duplexes involving a monophosphoramidite of the same cross-linked thymidine dimer including circumventing the use of costly 5′-O-deoxyphosphoramidites in the assembly of the cross-linked duplex by solid-phase synthesis. This strategy can be employed to produce cross-linked duplexes in which the lesions are engineered to have a directly opposed (1–1) or staggered (1–2 or 2–1) orientations. Biophysical studies of duplexes containing this N3T-butyl-N3T cross-link in staggered 1–2 and 2–1 orientations reveal that both duplexes have a higher Tm than a non-cross-linked duplex suggesting that these linkages do not result in the destabilization of duplex DNA. Circular dichroism spectra of the 1–2 and 2–1 cross-linked duplexes exhibit minor differences from B-form structure, which correlates with molecular modeling studies.Key words: chemically modified oligonucleotides, interstrand cross-link, DNA adduct, DNA repair.