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

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

Research output: Contribution to journalArticle

Abstract

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
Volume123
Issue number15
DOIs
StatePublished - 2001

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Nucleosides
Base Pairing
DNA
Hydroxyl Radical
DNA Ligases
Ligation
Deoxyuridine
Deoxycytidine
Denaturation
Triazoles
Oligomers
Oligonucleotides
Mass spectrometry
Repair
DNA Repair
Glass
Digestion
Mass Spectrometry
High Pressure Liquid Chromatography
Substrates

ASJC Scopus subject areas

  • Chemistry(all)

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Synthesis and characterization of DNA duplexes containing an N4C-ethyl-N4C interstrand cross-link. / Noll, D. M.; Noronha, A. M.; Miller, P. S.

In: Journal of the American Chemical Society, Vol. 123, No. 15, 2001, p. 3405-3411.

Research output: Contribution to journalArticle

Noll, D. M. ; Noronha, A. M. ; Miller, P. S. / Synthesis and characterization of DNA duplexes containing an N4C-ethyl-N4C interstrand cross-link. In: Journal of the American Chemical Society. 2001 ; Vol. 123, No. 15. pp. 3405-3411.
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