Site-specific labeling of supercoiled DNA at the A+T rich sequences

Vladimir N. Potaman, Alexander Y. Lushnikov, Richard R. Sinden, Yuri L. Lyubchenko

Research output: Contribution to journalArticle

21 Scopus citations

Abstract

Progress in structural biology studies of supercoiled DNA and its complexes with regulatory proteins depends on the availability of reliable and routine procedures for site-specific labeling of circular molecules. For this, we made use of oligonucleotide uptake by plasmid DNA under negative superhelical tension. Subsequent circularization of the oligonucleotide label facilitated by an oligonucleotide scaffold results in its threading between the two strands of duplex DNA. Several lines of evidence, including direct AFM mapping of the label, show that the circular oligonucleotide is stably localized at its target, an A+T rich region. The specific binding mode when the oligonucleotide threads the double helix results in a DNA kink that tends to occupy an apical position in a plectonemically wound supercoiled DNA, similar to the positioning of an A-tract bend. Site-specific labels may allow visualization techniques, such as electron and atomic force microscopies, to reliably map protein binding sites, identify local alternative structures in supercoiled DNA, and monitor structural dynamics of DNA molecules in real time. Site-specific oligonucleotide reactions with DNA may also have application in biotechnology and gene therapy.

Original languageEnglish (US)
Pages (from-to)13198-13206
Number of pages9
JournalBiochemistry
Volume41
Issue number44
DOIs
Publication statusPublished - Nov 5 2002

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ASJC Scopus subject areas

  • Biochemistry

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