Directors

Summary

The State Key Laboratory of Precision Spectroscopy at East China Normal University and the Labo-ratoire de Physique at Ecole Normale Superieure de Lyon are joining forces in this collaborative research project to advance in luminescent nanocluster technologies and make it an operational experimental tool for the genome-wide analysis of DNA replication at the single DNA molecule level. If successful this pilot project will provide the proof of concept and pave the way for a novel marking strategy of DNA bers likely to improve the throughput of DNA combing analyses of DNA replication and make it amenable to genome-scale studies and, in turn, be used to clarify the replication pattern of the largest possible number of regions for which other replication mapping techniques have yielded controversial results.

The mapping of DNA replication origins and the regulation of replication fork progression and ter-mination in higher eukaryotic genomes are still challenging questions in molecular biology. Although genome-scale methods allow genome-wide pro ling of DNA replication, these methods only provide an average picture of genome replication in a cell population, while each cell uses a di erent cohort of origins to replicate its genome. Only single-molecule methods such as DNA combing can quantitate cell-to-cell heterogeneity in DNA replication patterns. However, the throughput of single-molecule methods is cur-rently too low to permit genome-wide analyses of the replication program. Recently, results have been gathered by the group of O. Hyrien (IBENS, Paris) showing that this bottleneck could likely be overcome by (i) de ning simple conditions for visualising replication progression on a DNA ber by the doubling of DNA uorescence intensity at replicated tracts and (ii) using uorescent labeling of nicking endonu-clease sites, allowing each and every molecule to be speci cally labeled and subsequently mapped on the genome based on this optical bar-coding. We believe that metal nanocluster marking of DNA bers could further improve the throughput of single- ber DNA replication experiments, allowing one marking step to combine both the replication progression and genomic localization information.

Metal nanoclusters (NCs) with size approaching the Fermi-wavelength of an electron (< 1 nm) and exhibiting molecular-like characteristics have been very actively investigated in a variety of elds including chemistry, medicine, and biology. Au and Ag NCs in particular have attracted tremendous interest because of their wide applications in single-molecule studies, sensing, biolabeling, catalysis, and biological uorescence imaging. Metal NCs can exhibit photoemission due to intrinsic states quantization e ects only when their size < 2 nm. However, photoemission has also been observed in NCs as large as 18 nm. This observation suggests that the photoemission mechanism of metal NCs cannot be simply attributed to the small size and quantization e ects. Recent studies have shown that in fact both the surface ligands and metal cores contribute to the photoemission properties of nanoclusters. In particular, the ECNU team have shown that Ag nanoclusters luminescence is due to (i) ligand to the metal - metal charge transfer (Ligand-to-Metal-Metal Charge Transfer) and (ii) the subsequent relaxation processes. DNA is an ideal template candidate to take advantage of the e ect of the surface ligand / metal cores interaction on the luminescent properties of NCs. \Art-like" one-, two-, and three- dimensional DNA structures can be designed to template the metal nanocluster cores in order to study the e ect of the geometry in a controlled manner. Moreover, the chemical nature of the surface ligands can also be controlled by the sequences of DNA templates. For example, placing DNA-templated Ag NCs in close proximity of a guanine rich DNA oligonucleotide resulted in a 500 fold enhancement of the red uorescence of these particles. It was also recently reported that the uorescence of double stranded DNA-hosted Cu nanoclusters is very sensitive to base type located in the DNA major groove, so that a single nucleotide mismatch in a speci c DNA sequence can be detected.

The project aims at developing novel strategies for synthesizing DNA-templated nanoclusters and gaining a better insight in their photophysics, with the speci c goal to apply nanocluster technology in genomics. The project focuses on NC properties when using double stranded DNA (simple 1D geometry) as a template in order to be applicable to \natural" DNA present in living organisms. The objective is to develop a synthesis method of DNA-templated NC that allows uorescent marking of DNA molecules in a base composition dependent manner. In the rst step, we will focus on short ( 50 bp) DNA molecules with diverse base composition: homopolymers with only A:T or G:C base pairs, alternating A:T and T:A base pairs, alternating G:C / C:G base pairs and mixed polymers with natural G+C content. These DNA templates will be used to de ne the most e cient synthesis protocol of Ag, Au or Cu DNA-templated NCs so that the uorescence properties (emission spectra, uorescence life time) depends on the sequence composition in a controlled manner. In the second step, the analysis will be extended to 200 bp DNA molecules of di erent shapes. Indeed, over the persistence length of DNA ( 150 bp) the local curvature at each DNA steps may results in global deformation of the axis of the double helix. We will focus on two extreme conditions where DNA molecule is either straight (I-shaped molecules) or present a global curvature (C-shaped molecules). The study will allow us to assess whether DNA curvature has an e ect on the ligand - metal interaction and, in turn, on the the uorescence properties of the synthesized NCs. Depending on the achievement of the two rst steps of the project, we will then progressively increase DNA molecule size to test whether homogeneous marking can be achieved.

The success of this pilot project will allow the collaboration to enter a new phase to test NC marking of long DNA molecules in order to address key questions about the applicability of this technology as a tool for DNA replication pro ling. Is this marking technique compatible with DNA combing protocols? Is the marking su ciently homogeneous to permit the visualization of replication progression on a DNA bre by the doubling of uorescence intensity at replicated tracts? Does the sensitivity of the uorescence properties to the DNA sequence provide su cient information to allow the identi cation of the original location of the DNA ber along the genome? Beyond application for DNA replication pro ling, NC marking of DNA molecules could also have a signi cant impact on genomic mapping protocols allowing one marking step to combine both the DNA molecule visualization and genomic localization information.