Unità di analisi di interazioni molecolari

Human diseases are usually the outcome of the contribution of many processes, as for viral infection and neovascularization that start and sustain tumor growth and metastatization. At a molecular level, these processes are governed by an intricate network of interactions among viral proteins, cytokines, growth factors, extracellular matrix components and cellular receptors (the so called “interactome”). Understanding how macromolecules interact under physiologic and pathological conditions is an essential step in research and diagnostic laboratories. Unfortunately, the most common procedures for interaction analysis are rather lengthy, complex and demanding, allowing only qualitative results (i.e. immunofluorescence or immunoprecipitation), being affected by limitations related to the use of large amounts of purified proteins (i.e microcalorimetry), involving the labeling of the interactants (i.e radioisotope or fluorochrome tags), or using secondary antibodies or enzymes to generate a signal (i.e ELISA assay). Surface plasmon resonance (SPR) is a handy-user, reliable and high-throughput optical technique to evaluate biomolecular interactions. Launched less than 20 years ago, its use has seen tremendous growth and this trend is predicted to continue as the technology becomes more accessible and its applications more diverse. Briefly, a polarized beam of visible monochromatic light passes through a prism fitted with a glass slide coated with a gold film. Once reflected off the gold surface, the intensity of the beam is detected at the specular angle. When the light hits the glass, an electric field (evanescent wave) is generated and absorbed by the free electron clouds in the gold layer, causing a decrease of the intensity of the reflected beam that depends on the refractive index (RI) of the material present within 300 nm from the gold surface. In a SPR assay, a molecule (ligand) is immobilized onto the gold film and exposed to a sample containing a binder (analyte). The interaction of the analyte with the ligand causes a change of the RI at the gold surface resulting in the shift of the resonance angle and thus in the label-free transduction of the binding reaction that is presented as a real-time graph of the response units (RU) against time (sensorgram). The Macromolecular Interaction Analysis Unit is available for scientific collaborations or for fee-for-service by the university community and by biotechnological or pharmaceutical companies: 1) SPR analyses of interactions with measurements of kinetic parameters: association and dissociation rate (Kon and Koff), kinetic and steady state dissociation constant (Kd). 2) SPR analyses of drug/target interactions: direct interaction with measurement of kinetic parameters or competition experiments with calculation of the inhibitory potency (ID50) of the drug on the target ligand/receptors interaction. 3) Characterization of the biological consequences of the interactions analyzed:in vitro cell culture assays, ex vivo assays for endothelial physio-pathology, oncology and cardiovascular diseases.