Uptake and Fate of Fluorescently Labeled DNA Nanostructuresin Cellular Environments: A Cautionary Tale

摘要

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 DNAstructures is delayed. We also studied several parameters influencingfluorescent data: stability of the DNA strand, fixation methods thatcan wash away the signal, position of the dye on the DNA strand, anddesign of FRET experiments. DNA nanostructures hold tremendous potentialfor biomedical applications and biotechnology because of their biocompatibility,programmability, and easy synthesis. However, few examples of successfulDNA machines in vivo have been reported. We believethis contribution can be used as a guide to design better cellularuptake experiments when using fluorescent dyes, in order to furtherpropel the biological development, and application of DNA nanostructures.