Synthesis and characterization of DNA duplexes containing an N4C-ethyl-N4C interstrand cross-link

Article abstract of DOI:10.1021/ja003340t

Short DNA duplexes containing an N⁴C-ethyl-N⁴C interstrand cross-link, C-C, were synthesized on controlled pore glass supports. Duplexes having two, three, or four A/T base pairs on either side of the C-C cross-link and terminating with a C₄ overhang at their 5′-ends were prepared. The cross-link was introduced using a convertible nucleoside approach. Thus, an oligonucleotide terminating at its 5′-end with O⁴-triazoyl-2′-deoxyuridine was first prepared on the support. The triazole group of support-bound oligomer was displaced by the aminoethyl group of 5′-dimethoxytrityl-3′-O-tert-butyldimethylsilyl-N⁴- (2-aminoethyl)deoxycytidine to give the cross-link. The dimethoxytrityl group was removed, and the upper and lower strands of the duplex were extended from two 5′-hydroxyl groups of the cross-link using protected nucleoside 3′-phosphoramidites. The tert-butyldimethylsilyl group of the resulting partial duplex was then removed, and the chain was extended in the 3′-direction from the resulting 3′-hydroxyl of the cross-link using protected nucleoside 5′-phosphoramidites. The cross-linked duplexes were purified by HPLC and characterized by enzymatic digestion and MALDITOF mass spectrometry. Duplexes with three or four A/T base pairs on either side of the C-C cross-link gave sigmoidal shaped A₂₆₀ profiles when heated, a behavior consistent with cooperative denaturation of the A/T base pairs. Each cross-linked duplex could be ligated to an acceptor duplex using T4 DNA ligase, a result that suggests that the C-C cross-link does not interfere with the ligation reaction, even when it is located only two base pairs from the site of ligation. The ability to synthesize duplexes with a defined interstrand cross-link and to incorporate these duplexes into longer pieces of DNA should enable preparation of substrates that can be used for a variety of biophysical and biochemical experiments, including studies of DNA repair.