The teams of Eric Le Cam, from the "Molecular Interactions and Cancer" lab (UMR 8126 - Institut Gustave Roussy/CNRS/Universite Paris Sud) and David Pastre, from the "Structure and Activity of Normal and Pathological Biomolecules - SABNP" lab (Inserm/Universite d'Evry-Val-d'Essonne, U829, Genopole Evry) have developed a new method for observing DNA using atomic force microscopy (AFM) under intracellular physico-chemical conditions.
These findings, published in the Journal "Biomacromolecules" on November 19, 2007, were validated by the observation of various degrees of DNA compaction -- a key factor in replication and transcription. This method will facilitate research into DNA/protein interactions and their impacts on DNA activity, DNA chips and nanobiotechnology.
The first phase of these investigations was to find a method for absorbing DNA on a mica surface (a clay mineral traditionally used in biomolecular imaging), in the presence of monovalent salts.
These findings, themselves representing a major advance in current techniques, make it possible to observe DNA molecules using atomic force microscopy (AFM) under intracellular physico-chemical conditions (molecular crowding, monovalent salts). Until recently, it was in fact impossible to obtain nanometric 3D high resolution images of DNA molecules using AFM under such lifelike conditions.
To validate this method and demonstrate the relevance of these novel imaging conditions, researchers chose to examine how macromolecular crowding affected the compaction of DNA molecules.
The presence of very large molecules in the intracellular environment impacts directly the degree of DNA compaction, and thus its activity. This phenomenon, which is well-known among biologists, but poorly studied has a critical influence on replication, transcription and, consequently, on gene expression.
This method enables the observation of DNA molecules in various physiological conditions, in particular in interaction with proteins; it can also be used in the fields of DNA chips or nanobiotechnology.