Synthesis and Characterization of a [3-~(15)N]-Labeled Cis-Syn Thymine Dimer-Containing DNA Duplex

摘要

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 ~(15)N by two efficient routes from [3-~(15)N]-thymidine phosphoramidite.In the postsynthetic irradiation route,an ODN containing an adjacent pair of [3-~(15)N]-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-~(15)N]-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 ~(15)N-labeled thymine dimer-containing decamer duplex were assigned by 2D ~1H-~(15)N heterocorrelated HSQC NMR spectroscopy,and the ~(15)N-~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 H-bonding that is consistent with the more distorted nature of the 5'-base pair found in solution state NMR and crystallographic structures.