Molecular beacon as oligonucleotide nanosensors for intracellular mRNA

Molecular beacons, molecules capable to turn on or to modify their light emission upon the interaction with well- defined molecular targets, are among the most promising optical intracellular nanosensors proposed in the recent years. They are oligonucleotidic sequences labeled at the two side-ends, with a fluorophore (F1) at one side and a quencher Q (or a second fluorophore ) at the other side, characterized by an absorption band overlapping with the F1 emission band. In the absence of the target, these structures have a closed conformation with F1 and Q so closed that in the presence of F1 excitation fluorescence resonance energy transfer (FRET) occurs and no emission is observed. In the presence of the target, these structures open and fluorescence emission is observed since F1 and Q are sufficiently distant that FRET does not take place. Their intracellular internalization must be performed using suitable carriers capable to allow these nanosensors to penetrate the cell membrane and enter the cell. We describe here the design, implementation and characterization of structured polymethylmethacrylate (PMMA) nanoparticles (NPs) and carbon nanotubes (CNTs) for intracellular mRNA monitoring. PMMA NPs and CNTs were characterized as potential intracellular nanocarriers of the molecular beacon (MB) for the detection of mRNA encoding surviving, a protein member of the inhibitor of apoptosis family, highly expressed in most types of cancer. Atto647N and Blackberry 650 are the MB fluorophore/quencher pair. Hybridization studies with a target sequence analogous to survivin specific mRNA were conducted in vitro, and the MB functionalities, in solution and once anchored to NPs and CNTs, were successfully demonstrated. An increase of fluorescence signal was observed with the increase of the target concentration from 10 to 500 nM, confirming the dependence of the MB fluorescence on the presence of the target also after its immobilization onto the NPs. MB functionality and specificity were also tested in living cells by transfection with a classical lipid agent, lipofectamine, and by confocal microscopy imaging. A fluorescence increase was observed in the cytoplasm after 1 h from the transfection without fluorescent aggregates or fluorescence in the extracellular environment. On the contrary, no fluorescence was observed in transfected cells not expressing surviving.