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

D. M. Noll, A. M. Noronha, P. S. Miller

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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.

Original languageEnglish (US)
Pages (from-to)3405-3411
Number of pages7
JournalJournal of the American Chemical Society
Issue number15
StatePublished - 2001


ASJC Scopus subject areas

  • Chemistry(all)

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