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International audience; Detection and imaging of RNA at the single-cell level is of outmost importance for fundamental research and clinical diagnostics. Current techniques of RNA analysis, including fluorescence in situ hybridization (FISH), are long, complex and expensive. Here, we report a methodology of amplified FISH (AmpliFISH) that enables simpler and faster RNA imaging using small and ultrabright dyeloaded polymeric nanoparticles (NPs) functionalized with DNA. We found that the small size of NPs (below 20 nm) was essential for their access to the intracellular mRNA targets in fixed permeabilized cells. Moreover, proper selection of the polymer matrix of DNA-NPs minimized nonspecific intracellular interactions. Optimized DNA-NPs enabled sequence-specific imaging of different mRNA targets (survivin, actin and polyA tails), using a simple 1h staining protocol. Encapsulation of cyanine and rhodamine dyes with bulky counterions yielded green, red and far-red emitting NPs that were 2-100-fold brighter than corresponding quantum dots. These NPs enabled multiplexed detection of three mRNA targets simultaneously, showing distinctive mRNA expression profiles in three cancer cell lines. Image analysis confirmed the single-particle nature of the intracellular signal, suggesting single-molecule sensitivity of the method. AmpliFISH was found to be semi-quantitative, correlating with RT-qPCR. In comparison with the commercial LNA-based FISH technique, AmpliFISH provides 8-200-fold stronger signal (dependent on the NP color) and requires only 3 steps vs. ~20 steps together with much shorter time. Thus, combination of bright fluorescent polymeric NPs with FISH yields a fast and sensitive single-cell transcriptomic analysis method for RNA research and clinical diagnostics.

The detection of ultralow concentrations of biomolecules is challenging because of limited brightness of fluorescent molecular probes. The aim of my PhD project is to functionalize ultrabright dye-loaded polymeric nanoparticles (NPs) with oligonucleotides and obtain nanoprobes for nucleic acids. We found that NPs with efficiently exposed azide group can be readily functionalized with DNA through a Cu-free “click” reaction. DNA-functionalized NPs were then converted into biosensors based on energy transfer (FRET), providing amplified fluorescence ratiometric response to the target DNA/RNA in solution and at the single-particle level. Finally, we made drastically improved nanoprobes that enabled detecting a single hybridization event by switching on/off FRET from thousands of dyes in the nanoantenna particle to a single acceptor. The obtained nanoprobes constitute a new powerful platform for detection of cancer biomarkers., La détection à de très faibles concentrations de biomolécules est un défi en raison de la luminosité limitée des sondes moléculaires fluorescentes. Le but de mon projet de thèse est de fonctionnaliser des nanoparticules polymères (NP) ultra-brillantes dopées de fluorophores avec des oligonucléotides et d’obtenir des nano-sondes pour les acides nucléiques. Nous avons constaté que les NP avec un groupement azide bien orienté peuvent être facilement fonctionnalisées avec l'ADN par le biais d'une réaction de type «click» sans cuivre. Les NP fonctionnalisées par l'ADN ont été ensuite converties en biocapteurs grâce au transfert d’énergie de résonance de type Förster (FRET), fournissant une réponse ratiométrique de fluorescence amplifiée vers la cible ADN/ARN en solution et au niveau particulaire. Enfin, nous avons mis au point des nano-sondes radicalement améliorées permettant de détecter un seul événement d'hybridation en activant / désactivant le FRET de milliers de colorants de la particule de nanoantenne jusqu’à un accepteur unique. Les nano-sondes obtenues constituent une nouvelle plate-forme puissante pour la détection de biomarqueurs du cancer.