Special CPM seminar

A new high resolution optical detection scheme to study the interaction of enzymes along a replication fork

Vincent Croquette

École Supérieure de Physique et de Chimie Industrielles

We have developed a single molecule micromanipulation technique where double-stranded DNA molecules in the form of ‘hairpins’ are tethered at one end to a glass substrate and the other to small magnetic beads. These hairpins can be denatured (i.e. ‘unzipped’) by applying a magnetic force of > 15 pN. If the force is subsequently reduced to below this force, the hairpins will re-form. As this reformation takes place, the DNA fork is highly sensitive to any obstacle present on either of the remaining single strands of the molecule. These obstacles can include DNA secondary structures such as G4 quadruplexes or simple hybridized oligonucleotides. Upon colliding with such obstacle, the hairpin remains transiently blocked with an extension related to the obstacle position. In addition, proteins that recognize special features on DNA or a molecular motor moving along the DNA template can be tracked in real time using this system.

We have developed a new tracking technique allowing to measure the position of the magnetic beads using a dark field interferometry methods which enhance the accuracy and does not require anymore a look-up table.

Tracking in real-time the fork position we study the kinetics properties of hybridized oligonucleotides, DNA secondary structures or the activity of helicases and polymerases on single DNA. Using a set of short oligonucleotides, we shown that we can recognize a single molecule sequence by measuring accurately its specific hybridization pattern (fingerprint). Finally, we have demonstrated that we can detect epigenetic modifications on DNA molecules by analyzing the blockages observed when modification-specific antibodies bind to their antigens