Synthesis and characterization of DNA duplexes containing an (NC)-C-4-ethyl-(NC)-C-4 interstrand cross-link

Short DNA duplexes containing an (NC)-C-4-ethyl-(NC)-C-4 interstrand cross- link, C-C, were synthesized on controlled pore glass supports. Duplexes hav ing two, three, or four A/T base pairs on either side of the C-C cross-link and terminating with a C-4 overhang at their 5'-ends were prepared. The cr oss-link was introduced using a convertible nucleoside approach. Thus, an o ligonucleotide terminating at its 5'-end with O-4-triazoyl-2'-deoxyuridine was first prepared on the support. The triazole group of support-bound olig omer was displaced by the aminoethyl group of 5'-dimethoxytrityl-3'-O-tert- butyldimethylsilyl-N-4-(2-aminoethyl)deoxycytidine to give the cross-link. The dimethoxytrityl group was removed, and the upper and lower strands of t he duplex were extended from two 5'-hydroxyl groups of the cross-link using protected nucleoside 3'-phosphoramidites. The rert-butyldimethylsilyl grou p of the resulting partial duplex was then removed, and the chain was exten ded in the 3'-direction from the resulting 3'-hydroxyl of the cross-link us ing protected nucleoside 5'-phosphoramidites. The cross-linked duplexes wer e purified by HPLC and characterized by enzymatic digestion and MALDI-TOF m ass spectrometry. Duplexes with three or four A/T base pairs on either side of the C-C cross-link gave sigmoidal shaped A(260) profiles when heated, a behavior consistent with cooperative denaturation of the A/T base pairs. E ach 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 fro m the site of ligation. The ability to synthesize duplexes with a defined i nterstrand cross-link and to incorporate these duplexes into longer pieces of DNA should enable preparation of substrates that can be used for a varie ty of biophysical and biochemical experiments, including studies of DNA rep air.