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Biomedical subjects

Christophe Escudé

Publications and source records attributed to Christophe Escudé.

8 recordsLinked to original sources
PubMed2005

Detection of single DNA molecules by multicolor quantum-dot end-labeling.

Observation of DNA-protein interactions by single molecule fluorescence microscopy is usually performed by using fluorescent DNA binding agents. However, such dyes have been shown to induce cleavage of the DNA molecule and perturb its interactions with proteins. A new method for the detection of surface-attached DNA molecules by fluorescence microscopy is introduced in this paper. Biotin- and/or digoxigenin-modified DNA fragments are covalently linked at both extremities of a DNA molecule via sequence-specific hybridization and ligation. After the modified DNA molecules have been stretched on a glass surface, their ends are visualized by multicolor fluorescence microscopy using conjugated quantum dots (QD). We demonstrate that under carefully selected conditions, the position and orientation of individual DNA molecules can be inferred with good efficiency from the QD fluorescence signals alone. This is achieved by selecting QD pairs that have the distance and direction expected for the combed DNA molecules. Direct observation of single DNA molecules in the absence of DNA staining agent opens new possibilities in the fundamental study of DNA-protein interactions. This work also documents new possibilities regarding the use of QD for nucleic acid detection and analysis.

Biotinylation↗
PubMed2005

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.

AT Rich Sequence↗
PubMed2004

Ligand-mediated transcription elongation control using triplex-based padlock oligonucleotides.

Triplex-forming oligonucleotides (TFOs) provide useful tools for the artificial regulation of gene expression at the transcriptional level. They can become topologically linked to their DNA target upon circularization, thereby forming very stable triple helical structures. These "padlock oligonucleotides" are able to interfere with transcription elongation when their target site is located in the transcribed region of a gene. In vitro transcription experiments showed that a bacterial RNA polymerase was stopped at the site of triple-helix formation, whereas expression of a reporter gene was inhibited in live cells. In both cases, the padlock oligonucleotide was more efficient at inhibiting transcription elongation than a linear TFO, and the inhibition was observed only in the presence of a triplex stabilizing agent. These results provide new insights into the ligand-modulated locking of padlock oligonucleotides around their DNA target.

Bacteria↗
PubMed2003

Sequence-specific fluorescent labeling of double-stranded DNA observed at the single molecule level.

Fluorescent labeling of a short sequence of double-stranded DNA (dsDNA) was achieved by ligating a labeled dsDNA fragment to a stem-loop triplex forming oligonucleotide (TFO). After the TFO has wound around the target sequence by ligand-induced triple helix formation, its extremities hybridize to each other, leaving a dangling single-stranded sequence, which is then ligated to a fluorescent dsDNA fragment using T4 DNA ligase. A non-repeated 15 bp sequence present on lambda DNA was labeled and visualized by fluorescence microscopy after DNA combing. The label was found to be attached at a specific position located at 4.2 +/- 0.5 kb from one end of the molecule, in agreement with the location of the target sequence for triple helix formation (4.4 kb from one end). In addition, an alternative combing process was noticed in which a DNA molecule becomes attached to the combing slide from the label rather than from one of its ends. The method described herein provides a new tool for the detection of very short sequences of dsDNA and offers various perspectives in the micromanipulation of single DNA molecules.

Bacteriophage lambda↗
PubMed2002

Padlock oligonucleotides as a tool for labeling superhelical DNA.

Labeling of a covalently closed circular double-stranded DNA was achieved using a so-called 'padlock oligonucleotide'. The oligonucleotide was targeted to a sequence which is present in the replication origin of phage f1 and thus in numerous commonly used plasmids. After winding around the double-stranded target DNA sequence by ligand-induced triple helix formation, a biotinylated oligonucleotide was circularized using T4 DNA ligase and in this way became catenated to the plasmid. A gel shift assay was developed to measure the extent of plasmid modification by the padlock oligonucleotide. A similar assay showed that a modified supercoiled plasmid was capable of binding one streptavidin molecule thanks to the biotinylated oligonucleotide and that this binding was quantitative. The catenated complex was visualized by electron and atomic force microscopies using streptavidin conjugates or single strand-binding proteins as protein tags for the padlock oligonucleotide. This method provides a versatile tool for plasmid functionalization which offers new perspectives in the physical study of supercoiled DNA and in the development of improved vectors for gene therapy.

Base Sequence↗
PubMedDate not supplied

Coupling of a targeting peptide to plasmid DNA using a new type of padlock oligonucleotide.

We have recently described a new method for attaching padlock oligonucleotides to supercoiled plasmid DNA at specific sequences. A variant of this method has been developed in order to allow the coupling of targeting moieties to plasmids using a convenient strategy. After sequence-specific winding around the double-stranded target DNA sequence by ligand-induced triple helix formation, the extremities of a triplex-forming oligonucleotide hybridize to each other, leaving a dangling single-stranded sequence, which is then ligated to a hairpin oligonucleotide using T4 DNA ligase. Any targeting moiety may be attached to the hairpin oligonucleotide. This strategy was used to attach an NLS peptide to a luciferase-expressing plasmid. Despite the presence of the padlock oligonucleotide, the reporter gene was efficiently expressed after transfection of the plasmid in HeLa or T24 cells, using either cationic lipids or cationic polymers as transfecting agents. However, no increase in gene expression could be observed as a result of peptide attachment. Nevertheless, the coupling strategy described in this paper may find applications as a tool for plasmid functionalization in other targeting experiments, and may lead to the development of improved vectors for gene therapy.

Amino Acid Sequence↗