AFM for DNA-based glyco-chip characterization on a molecular scale
Phaner-Goutorbe, Magali; Lallemand, David; Chevolot, Yann; Morvan, F; Meyer, A; Vidal, S; Jochum, A; Praly, J.P.; Cloarec, J. P.; Vasseur, J. J.; Souteyrand , Eliane France
Atomic Force Microscopy becomes an invaluable technique for the study of biological systems at a nanometric scale. During the past decade, it has been increasingly used to image and manipulate biomolecules and cell surfaces in air or in situ. Most of the experiments concerns the observation of adsorbed biological molecules, DNA or cells, few articles are dealing with AFM studies on real biosystems with direct applications such as DNA –chips or glyco-chips. The sensitivity of these objects depends on the molecular organization of the immobilized molecules on the substrate. Then, it appears necessary to characterize the chip on a molecular scale before recognition.
The present work reports an AFM characterization of a glyco-chip based on the use of DNA chip as an addressing tool for mixed DNA/Oligosaccharide molecules. Glycomic aims to understand the relationships between the structure of oligosaccharides and their function. Oligosaccharides are implicated in crucial events such as HIV virus infection, failure of xenotransplantation. There is a need to study the structural parameters governing the saccharides/protein recognition by using customized glycoarrays.
A model chip is fabricated with four different spots : one contains the 18-mer oligonucleotide covalently bonded to the silanized silicon substrate, the three others are composed with the first one hybridized to its complementary strand terminated by three saccharide residues to take advantage of the "cluster" effect. Saccharides are anchored to the complementary strand by a linker. The three spots differ from each other by the choice of different saccharides, Mannose and galactose and different linkers : cyclohexanedimethanol (L1), tetraethyleneglycol (L2).
Each spot is studied by AFM on a molecular scale in order to find the rlationship between the molecular organization of saccharides and their selectivity towards lectins. We show that the linker molecule has a larger effect than the saccharide molecule on the glyco-chip molecular organization. We obtain a homogeneous distribution of molecules with the (L1) linker whereas saccharides organize themselves in domains with the (L2) linker. AFM experiments are combined with fluorescent spectroscopy measurements.