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Research - Commercialisation

Biosensors and the dynamics of macromolecular interactions

Malcolm Buckle, research director

Malcolm Buckle research directorMembers
Malcolm BUCKLE (team leader) , Hervé LEH (research engineer), Claude NOGUES (post doctoral fellow), Andrew TRAVERS (visiting professor) and Evangeline BARBIER (M2 student)

Collaborations
Kevin Gaston (UK) , Sheela Jayaraman (UK), Andrew Travers (UK) , Georgi Muskhelishvili (D), Ashley Buckle (Aus), Than Chi Nguyen (F), Bruno Le Pioufle (F), Shoje Takesushi (J), Anne-Françoise Obaton (F), Dimitra Markovitsa (F), Chong Yen (F), Karel van Wely (Esp), Ed Nice (Aus), Yaneck Gottesmann (F), Hsin-Chih Lai (Taiwan), Hermann Heumann (D).

This group investigates the role of macromolecular interactions in the regulation of gene expression notably extending an accumulated expertise in kinetic analysis of nucleoprotein complexes into more complex situations. Since the unique and original methodological base established by this team has lead to the establishment of a technological platform a large part of the basic research undertaken in this team involves the development of novel technologies and has thus recently expanded into surface chemistry and new biosensor devices (micro-resonator cavities and surface plasmon imagery). The team has essentially concentrated on consolidating the methodologies of real time laser photocharacterisation of nucleoprotein complexes, identification of protein partners and protein interfaces in macromolecular complexes, quantification using surface Plasmon resonance imagery and coupling of SPR and SPRi to downstream identification devices such as mass spectrometers. Ongoing projects involve the developments of dedicated surface chemistries that provide extremely high signal to noise ratio's for SPR analysis. This has enabled novel collaborations with a number of groups working on macromolecular assemblies.
The main thrust of the group at the moment coincides with the arrival of Pr. Andrew Travers in the context of a Chaire d'excellence attributed by the ANR and coordinated by the Fondation Pierre Gilles de Gennes and is centred on a project entitled Chromatin Organisation: a Regulatory Dynamic Analysis.  The packaging of genomic DNA in both the bacterial nucleoid and the eukaryotic nucleus is constrained by apparently opposing requirements: on the one hand, the maintenance of DNA compaction, and, on the other, the provision of accessibility for enzymatic manipulation necessary for, for example, transcription. These considerations imply that chromatin structure is both dynamic and highly organised. While immense progress has been made in the understanding of the nature of the signals involved in maintaining and altering chromatin structure, much less is known about the details of chromatin structure itself, especially the transitions required for turning genes on and off. Without knowledge of the structure the detailed functioning of the chromatin machine remains an enigma. The major goal of this project is to investigate the nature of these structural transitions both at the local level of promoter structure and at the more global level of the folding and unfolding of the chromatin fibre.  We are looking at the structure of chromatin at the level of  1) mono and dinucleosomes, 2) nucleosome arrays 3) 30nm fibre and above.  Specific aims of the project  are thus to examine reconstitution of monocleosomes, to attempt to visualise in vitro the higher-order folding of the 30 nm fibre, to determine whether different linker histones could modulate this structure. and finally to characterise the nature of the structural transitions, if any, that accompany the formation of more highly ordered structures beyond the 30 nm fibre. technically the approach adopted consists of:
1.Reconstitution by SPRi. Using the unique proprietary protocols developed in the group and the novel reconstitution protocol introduced by A. Travers, we will characterise reconstitution using a large range of histones. Surfaces with multiple spots containing different immobilised DNA  sequences will be used to look at the kinetics of histone binding.
2.Analysis by AFM: We will first be looking at 30nm fibres using the selected nucleosome arrays from SPRi analysis.  This will be followed by studies on higher order structures beyond the 30nm fibre. Expected transitions between  toroidal or plectoneme structures should be discernible by AFM.
3.Analysis by AFM:  We will orient nucleosomal fragments using various combing techniques.4.Use of footprinting. DNase I and laser photofootprinting to determine the orientation of minor and major groves in specific structures
5.Chemical probing of local distortions in DNA using singlet oxygen a technique originally developed in the lab for use with NdYAG lasers.

A final aim will be to test the idea that abundant chromosomal proteins interact under different regimes to stabilise specific states of compaction of eukaryotic chromatin essentially through a topological process involving individual nucleosomes.

A second project in strong collaboration with other groups within the Institute d'Alembert (notably LPQM), involves the design, elaboration, characterization, and validation of a polymer based, label-free optofluidic sensor for chemical and biological applications, focused toward the real-time investigation of the kinetics of specific macromolecular interactions. Thus we are looking at the design and construction of organic micro-resonators as label-free chemio- and bio-sensors in a micro-fluidic environment.