Synthesis and characterization of a [3-¹⁵N]-labeled cis-syn thymine dimer-containing DNA duplex

Cis-syn thymine dimers are the major photoproducts of DNA and are the principal cause of mutations induced by sunlight. To better understand the nature of base pairing with cis-syn thymine dimers, we have synthesized a decamer oligodeoxynucleotide (ODN) containing a cis-syn thymine dimer labeled at the N3 of both T's with ¹⁵N by two efficient routes from [3- ¹⁵N]-thymidine phosphoramidite. In the postsynthetic irradiation route, an ODN containing an adjacent pair of [3-¹⁵N]-labeled T's was irradiated and the cis-syn dimer-containing ODN isolated by HPLC. In the mixed building block route, a mixture of cis-syn and trans-syn dimer-containing ODNs was synthesized from a mixture of [3-¹⁵N]-labeled thymine dimer phosphoramidites after which the cis-syn dimer-containing ODN was isolated by HPLC. The N3-nitrogen and imino proton signals of an ¹⁵N-labeled thymine dimer-containing decamer duplex were assigned by 2D ¹H- ¹⁵N heterocorrelated HSQC NMR spectroscopy, and the ¹⁵N-¹H coupling constant was found to be 1.8 Hz greater for the 5′-T than for the 3′-T. The larger coupling constant is indicative of weaker Ç-bonding that is consistent with the more distorted nature of the 5′-base pair found in solution state NMR and crystallographic structures.