Amplified Fluorescence in Situ Hybridization by Small and Bright Dye-Loaded Polymeric Nanoparticles
| Autor: | Egloff, Sylvie, Melnychuk, Nina, Cruz Da Silva, Elisabete, Reisch, Andreas, Martin, Sophie, Klymchenko, Andrey S. |
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| Přispěvatelé: | Laboratoire de Bioimagerie et Pathologies (LBP), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), European Project: 648528,H2020,ERC-2014-CoG,BrightSens(2015), European Project: ERC Proof of Concept,AmpliFISH |
| Rok vydání: | 2021 |
| Předmět: |
0303 health sciences
fluorescent nanoparticles mRNA RNA imaging General Engineering General Physics and Astronomy 010402 general chemistry fluorescence microscopy 01 natural sciences 0104 chemical sciences DNA-functionalized polymeric nanoparticles 03 medical and health sciences single-cell analysis [CHIM]Chemical Sciences [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology General Materials Science fluorescence in situ hybridization 030304 developmental biology |
| Zdroj: | ACS Nano ACS Nano, 2021, 16 (1), pp.1381-1394. ⟨10.1021/acsnano.1c09409⟩ |
| ISSN: | 1936-086X 1936-0851 |
| DOI: | 10.1021/acsnano.1c09409 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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