TAILIEUCHUNG - Báo cáo khoa học: Stem–loop oligonucleotides as tools for labelling double-stranded DNA

We report on a sequence-specific double-stranded DNA labelling strategy in which a stem–loop triplex forming oligonucleotide (TFO) is able to encircle its DNA target. Ligation of this TFO to either a short hairpin oligonucleotide or a long double-stranded DNA fragment leads to the for-mation of a topological complex. This process requires the hybridization of both extremities of the TFO to each other on a few base pairs. The effects of different factors on the formation of these complexes have been investi-gated. . | ềFEBS Journal Stem-loop oligonucleotides as tools for labelling double-stranded DNA Bénédicte Geron-Landre Thibaut Roulon and Christophe Escude Laboratoire Regulation et Dynamique des Genomes Departement Regulations Developpement et Diversite Moleculaire Museum Nationald Histoire Naturelle Paris Keywords triple helix DNA labeling stem-loop oligonucleotide sequence specificity padlock oligonucleotide Correspondence C. Escude Laboratoire Regulation et Dynamique des Genomes Departement Regulations Developpement et Diversite Moleculaire USM 0503 Museum National d Histoire Naturelle CNRS UMR5153 INSERM U565 Case Postale 26 43 rue Cuvier F-75231 Paris Cedex 05 France Fax 33 14079 3705 Tel 33 14079 3774 E-mail escude@ Received 23 June 2005 revised 17 August 2005 accepted 23 August 2005 We report on a sequence-specific double-stranded DNA labelling strategy in which a stem-loop triplex forming oligonucleotide TFO is able to encircle its DNA target. Ligation of this TFO to either a short hairpin oligonucleotide or a long double-stranded DNA fragment leads to the formation of a topological complex. This process requires the hybridization of both extremities of the TFO to each other on a few base pairs. The effects of different factors on the formation of these complexes have been investigated. Efficient complex formation was observed using both GT or TC TFOs. The stem-loop structure enhances the specificity of the complex. The topologically linked TFO remains associated with its target even under conditions that do not favour triple-helix formation. This approach is sufficiently sensitive for detection of a 20-bp target sequence at the subfemtomolar level. This study provides new insights into the mechanics and properties of stem-loop TFOs and their complexes with double-stranded DNA targets. It emphasizes the interest of such molecules in the development of new tools for the specific labelling of short DNA sequences. doi Triple-helix

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