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Gene expression is the process in which information stored into a gene is used to create a functional gene product, for example a protein or a functional RNA. Gene expression is used by all living organism to control cell life, differentiation, regeneration and cancer genesis. Molecular beacons provide a useful way to detect gene expression inside living cells without influencing the cell behavior. Molecular beacons are a particular kind of nucleic acid based probes composed by an oligonucleotide chain which assumes a hairpin configuration, encoding the target sequence of interest (loop) flanked by two complementary strands (stem), conjugated respectively with a fluorophore and a quencher. Upon hybridization with complementary target, the stem bonds break, parting the fluorophore from the quencher and resulting in a detectable signal. This thesis is divided in two major sections. The first section gives initially an overview of the molecular beacon technology usability and working principle. Later on the mechanism of molecular beacon’s internalization by SLO treatment was theoretically and experimentally investigated to determine optimal transfection condition and actual cell loading. Finally the problem of signal generation by molecular beacons was tackled by developing two new methods. It was demonstrated that by carefully design of interacting beacons with a new geometrical configuration, it is possible to induce among them both a linear and an avalanche reaction which improve the signal emission greatly. The second section of the thesis investigates the possibility to remotely activate gold nanoparticles conjugated to molecular beacons through an external radio frequency field. The gold nanoparticle will serve both as quencher for the fluorescence emission and as local point source of heat. The interaction between gold nanoparticles and a RF field will be investigated both theoretically and experimentally. The new gold-conjugated molecular beacon, called Nanomachine, could potentially be used in living cells to track e.g. down regulation of gene expression.
|Published - 2013