Uptake and Fate of Fluorescently Labeled DNA Nanostructures in Cellular Environments: A Cautionary Tale
| Autor: | Hanadi F. Sleiman, Empar Vengut-Climent, Aurélie Lacroix, Donatien de Rochambeau |
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| Rok vydání: | 2019 |
| Předmět: |
General Chemical Engineering
UNESCO::QUÍMICA 010402 general chemistry 01 natural sciences QUÍMICA [UNESCO] chemistry.chemical_compound Live cell imaging Cyanine QD1-999 Cellular localization Nuclease biology 010405 organic chemistry Oligonucleotide dna nanostructures General Chemistry Fluorescence 0104 chemical sciences Chemistry Förster resonance energy transfer chemistry biology.protein Biophysics cell uptake fluorescence DNA |
| Zdroj: | ACS Central Science, Vol 5, Iss 5, Pp 882-891 (2019) Lacroix, A.; Vengut-Climent, E.; de Rochambeau, D.; Sleiman, H. ACS Cent. Sci.2019, 5, 5, 882-891. ACS Publications RODERIC. Repositorio Institucional de la Universitat de Valéncia instname |
| ISSN: | 2374-7943 |
| Popis: | [Image: see text] Fluorescent dye labeling of DNA oligonucleotides and nanostructures is one of the most used techniques to track their fate and cellular localization inside cells. Here, we report that intracellular fluorescence, and even FRET signals, cannot be correlated with the cellular uptake of intact DNA structures. Live cell imaging revealed high colocalization of cyanine-labeled DNA oligos and nanostructures with phosphorylated small-molecule cyanine dyes, one of the degradation products from these DNA compounds. Nuclease degradation of the strands outside and inside the cell results in a misleading intracellular fluorescent signal. The signal is saturated by the fluorescence of the degradation product (phosphorylated dye). To test our hypothesis, we synthesized a range of DNA structures, including Cy3- and Cy5-labeled DNA cubes and DNA tetrahedra, and oligonucleotides with different stabilities toward nucleases. All give fluorescence signals within the mitochondria after cellular uptake and strongly colocalize with a free phosphorylated dye control. Kinetics experiments revealed that uptake of stable DNA structures is delayed. We also studied several parameters influencing fluorescent data: stability of the DNA strand, fixation methods that can wash away the signal, position of the dye on the DNA strand, and design of FRET experiments. DNA nanostructures hold tremendous potential for biomedical applications and biotechnology because of their biocompatibility, programmability, and easy synthesis. However, few examples of successful DNA machines in vivo have been reported. We believe this contribution can be used as a guide to design better cellular uptake experiments when using fluorescent dyes, in order to further propel the biological development, and application of DNA nanostructures. |
| Databáze: | OpenAIRE |
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