Exosome nanovesicles displaying G protein-coupled receptors for drug discovery.

Exosomes are naturally occurring nanovesicles that can be tailored to display a broad range of drug targets, including G protein-coupled receptors. Such vesicles provide a new source of complex membrane proteins that are maintained in their native conformation. Given the difficulties to isolate receptors for drug target validation and discovery, receptor presentation on exosome emerges as a promising new tool for drug screening.

Multiple topological labeling for imaging single plasmids.

Sequence-specific labeling methods for double-stranded DNA are required for mapping protein binding sites or specific DNA structures on circular DNA molecules by high-resolution imaging techniques such as electron and atomic force microscopies. Site-specific labeling can be achieved by ligating a DNA fragment to a stem-loop-triplex-forming oligonucleotide, thereby forming a topologically linked complex. The superhelicity of the plasmid is not altered and the process can be applied to two different target sites simultaneously, using DNA fragments of different sizes.

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.

Exosome Display technology: applications to the development of new diagnostics and therapeutics.

Exosome Display is a novel methodology enabling the manipulation of exosome protein content. This technology stems from the identification of addressing domains that mediate the specific distribution of proteins on exosomes. More particularly, Lactadherin expressed in non-mammary gland tissue has been found to localize to exosomes via binding of its C1C2 domain to exosome lipids.

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.

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.

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.

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 Ligand-Modulated Padlock Oligonucleotide for Supercoiled Plasmids.

Catenation of a circular oligonucleotide to a supercoiled plasmid can be achieved in high yields by means of ligand-induced triple-helix formation. The noncovalent interactions in this supramolecular structure can be modulated by a triplex-stabilizing agent (TSA; see picture). The circular oligonucleotide represents a noncovalent anchor for plasmid functionalization.

Purification and characterization of human DNA topoisomerase IIIalpha.

Human topoisomerase IIIalpha (hTopo IIIalpha), the recently identified first member of the topoisomerase IA subfamily in humans, has a central domain which is highly homologous to the yeast topoisomerase III, but an overall organization closer to that of Escherichia coli DNA topoisomerase I. In order to determine the properties of hTopo IIIalpha, compared to those of other topoisomerase IA subfamily members, we purified this enzyme to near homogeneity, together with an active site-mutant Y337F. We show that hTopo IIIalpha is able to relax negatively supercoiled DNA in a distributive manner, leading to the total disappearance of the initial substrate and the appearance of intermediate topoisomers.