Abstract
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.
🔬 Techniques
🧬 Organisms
✨ Fluorophores
🧪 Sample Preparation
🔬 Cell Lines
🏭 Microscope Brands
🏛️ Research Organizations (ROR)
Affiliated research institutions:
📊 Figures
Figure 1
Cyanine-labelednDNA nanostructures. (A)Two wireframe DNA-minimalnnanostructures were used in the study: the DNA nanocube, composednof four 96-mers, and the DNA tetrahedron, composed of four 63-mers.n(...
Figure 2
Uptake of DNA oligonucleotides and DNA nanostructures.n(A) Experimentalnsetup: DNA structures are incubated with mammalian cells, directlynin the cellular media (FBS-supplemented). Fluorescent signal ...
Figure 3
Mitochondrial and lysosomal localizationnof DNA structures. Livenconfocal microscopy in HeLa cells revealed colocalization of DNA structures.nIn the image, we represent the Cy5-labeled DNA nanocube co...
Figure 4
Phosphorylated dyes colocalize with DNA structures. (A)nStructuresnof synthesized phosphorylated cyanine 3 (Cy3-P) and cyanine 5 (Cy5-P)ndyes. (B) Co-incubation of structures and model for degradation...
Figure 5
Changing thendye position within the structure. Representativenimages are shown in the figure. (A) Live confocal microscopy of HeLancells (24 h incubation) with the different clips and cubes at fixedn...
Figure 6
HEG-protection of the DNA tetrahedron. (A) Chemicalnstructure ofnHEG modification. (B) Gel electrophoresis from serum stability experiments.nThe component clip of the tetrahedron revealed higher stabi...
Figure 7
Lowering serum conditionsnand changing the dye to its sulfonatednversion. Representative images are shown in the figure. (A) Live confocalnmicroscopy in HeLa cells in low-serum conditions (0.1% FBS) r...
Figure 8
Fu00f6rsternresonance energy transfer (FRET) experiments. (A)nLive confocal microscopy after 24 h incubation in HeLa cells. Representativenimages are shown in the figure. (B) Quantification of the gra...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
💬 Discussion
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