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 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.
Géron-Landre
Bénédicte
B
Laboratoire Régulation et Dynamique des Génomes, Département Régulations, Développement et Diversité Moléculaire, Muséum National d'Histoire Naturelle, Paris Cedex, France.
Roulon
Thibaut
T
Escudé
Christophe
C
D020856:AT Rich Sequence / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D004270:DNA, Circular / Q000737:chemistry / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D020862:GC Rich Sequence / Q000235:genetics; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000737:chemistry* / Q000235:genetics; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D013194:Staining and Labeling / Q000379:methods*
D004270:DNA, Circular; D015345:Oligonucleotide Probes; C051802:triplex DNA; D004247:DNA