Laboratoire sur puce pour étudier la réplication cellulaire – Replichip

We wish to develop micro and nano fluidic systems aimed at studying chromatin, and biological processes involving chromatin at the single molecule or at the single cell level. Chromatin is the nucleo-protein structure inside the nucleus of eucaryotes, and it is essential for genome maintenance and regulation. In this project, we are particularly interested in studying replication in the DNA and in the chromatin context using nanofabrication approaches. Replication is a key step of the cell cycle during which one mother cell duplicates its genetic material to provide two daughter cells with the same genetic material. Replication insures genetic stability over the cell cycles, and it is thus a central issue in modern biology. Alterations of the replication program occur in cancer cells, and there is a crucial need of high-throughput methods to characterize replication in cancer diagnosis. However, the current techniques are not efficient to perform such large scale analysis. The overall aim of our project is hence to implement an integrated system for high-through replication analysis. Replichip lies at the frontiers of technology, biology and physics. On the technological side, we propose to design square nano-capillaries ~100 nm in width and ~100 µm in length. These nano-capillaries will be filled with buffers and connected to classical microfluidic channels in order to convey DNA molecules by electrophoresis. DNA molecules are highly confined inside nano-capillaries, and this confinement induces an entropic spreading along the nano-capillary axis. Spreaded molecules in the process of replication can be characterized using fluorescence techniques. Indeed, these DNAs contain regions with two duplicated DNA strands, and other unreplicated regions in which there is only one DNA strand, and this difference in DNA content is associated to two-fold variations in fluorescence intensity. Parallel nanofluidic channels can be fabricated, and we intend to measure fluorescence intensities in several nanochannels in order to perform high-throughput replication analysis over a population of molecules. Different technological strategies, which are either based on quartz / Silicium e-beam lithography, or on soft lithography technologies (PDMS), are proposed to set up this integrated system. These technologies are already established at LAAS. In addition, we wish to develop nanoimprint to fabricate nano-channels, and this technology is under development in the coordinating laboratory. The biophysical side of the project, and in particular the manipulation and visualization of single molecules under electric fields, will be carried out by A. Bancaud. The biological work will be performed by the team 'Genetic Instabilities and Cancer' at IPBS, which is specialized on DNA replication, and which will provide either calibrated replication material or samples relevant for cancer diagnosis. Because the nano-capillary technology is versatile, we also wish to implement multi-color read-outs in order to investigate replication in the context of specific regions of the genome, and in particular fragile sites, which are regions known to be sensitive to replication stress, and to be associated to cancer development. In addition, we intend to adapt nano-capillaries to studying chromatin fibers instead of mere DNA, and we are particularly interested in investigating chromatin large scale structure, and its interplay with replication. To conclude, Replichip is a multidisciplinary project through which we will gain fundamental knowledge on replication, and which is adapted to cancer diagnosis. It reinforces the scientific orientations of LAAS towards nano-biotechnology, and it fits into the scope of the emerging Cancer-Bio-Santé pole. Finally, because nanobiotechnology are an emerging field of research, this project gives the opportunity to Aurélien Bancaud to gain international exposure, and to become an independent researcher at the frontiers of biology, technology and physics.