Complex nanostructures based on oligonucleotide optical switches and nanoparticles for intracellular mRNA sensing and silencing.

Summary Nanosensing by oligonucleotide optical switches coupled to nanoparticles is a highly promising and fascinating strategy for the intracellular detection of a plethora of targets, such as specific mRNA molecules, proteins and small molecules. The use of an antisense oligonucleotide which acts as molecular beacon and able to generate a fluorescent signal when it hybridizes with the target mRNA, may represent an innovative strategy that conjugates the ability of sensing specific mRNA with the pharmacological silencing activity preventing the overexpression of proteins associated to pathologic conditions. Motivation and results We describe here the design, implementation and characterization of complex nanostructures for intracellular mRNA monitoring constituted by a particular oligonucleotide optical switch [1], named molecular beacon (MB), immobilised on polymethylmethacrylate (PMMA) fluorescent nanoparticles (NPs). The MB molecule, fixed onto the nanoparticle for intracellular delivery, is capable to turn on the fluorescence emission of its fluorescent label, upon the interaction with the target mRNA. The mRNA specific for survivin has been chosen as target: survivin is a multifunctional protein that plays a role in cancer development and progression [2]. Survivin is overexpressed in cancer cells while it is undetectable in most healthy tissues. The MB for survivin mRNA was characterised in vitro and its functionality has been verified in solution and after immobilisation onto the PMMA NPs. The functionality and specificity of the MB were also examined in living cells by experiments performed on the human melanoma A375 cell line and using human monocytes as negative control. The MB was firstly transfected by the classical lipid agent, lipofectamine. Real-time RT-PCR and western blot were used to analyse survivin mRNA and protein expression, respectively. Our findings demonstrated a fluorescence increase in the cytoplasm 1 h after beginning the transfection without evidence of fluorescence signals in the extracellular environment. Moreover, no fluorescence was observed in transfected cells that did not express survivin. Real-time PCR data and Western blot analysis demonstrated that the MB significantly decreased survivin expression in A375 cells, thus confirming the pharmacological its silencing activity. After 48 and 72 h of MB treatment, A375 apoptotic cell death was observed. Bear and MB-coated PMMA NPs were then tested with adenocarcinomic human alveolar basal epithelial cells (A549) in terms of cell vitality and internalization. These experiments provided a clear evidence of the subcellular distribution of PMMA nanoparticles in living cells and of the ability of PMMA NPs to promote the MB internalization. We have shown that oligonucleotide optical switches, together with NPs, can play a fundamental role in achieving quantitative information on intracellular events. The conducted analytical characterisation has demonstrated that they can be used not only as simple on-off elements but also as real sensors and therapeutic agents. References [1] A. Giannetti, S. Tombelli and F. Baldini, Oligonucleotide optical switches for intracellular sensing, Anal. Bioanal. Chem., 405 (2013) 6181-6196. [2] J.A. McKenzie and D. Grossman, Role of the Apoptotic and Mitotic Regulator Survivin in Melanoma, Anticancer Res., 32 (2012), 397-404. Corresponding author: F. Baldini, Istituto di Fisica Applicata Nello Carrara - CNR, Via Madonna del Piano 10, 50019 Sesto Fiorentino (FI), Italy, Phone: +39 0555226323, f.baldini@ifac.cnr.i