D. Mathur, K. Rogers, S.A. Díaz, M. Muroski, W.P. Klein, L. Field, J.B. Delehanty, I.L. Medintz
George Mason University,
Keywords: DNA nanotechnology, DNA origami, cell cytosol, microinjections, confocal microscopy, FRET, fluorescence, drug delivery, biomaterials, therapeutics, mammalian cells, cytoplasm
Summary:DNA nanostructures have proven as minimally cytotoxic therapeutic carriers. While DNA-based drug carriers can be taken up by mammalian cells via endocytosis, it is largely unclear how these DNA nanostructures can reach the cytosol and, once there, can controllably maintain stability. With the enabling science explaining their behavior and mechanisms of controlling their stability in the cell cytosol it will be possible to advance the engineering of DNA-based mRNA and other therapeutic delivery systems. To that end, the current work identifies time-resolved breakdown of small DNA nanostructures inside a mammalian cell cytosol. Two DNA structures commonly applied in biomedical research – DNA crosshairs and tetrahedron – were directly introduced into monkey kidney cell cytosol via cell microinjections and tracked for loss of structural integrity. Tracking was performed by embedding the nanostructures with a multistep Förster resonance energy transfer cascade such that loss of energy transfer between the constituent dyes was a function of DNA nanostructure cytosolic degradation. Results indicate rapid degradation of the DNA crosshairs, with 65% loss of structural integrity and ejection from the cytosol within 25 minutes post injection. On the other hand, the DNA tetrahedron performs significantly better showing only 3% loss of structure 1 hour post injection. Comparison of the two structures in the presence of common nucleases as well as their physical properties indicate a general robustness against digestion in the DNA tetrahedron due to its compact shape and concomitant inaccessibility to being attacked by the enzymes. With the enabling technology of cell microinjections it would be possible to advance our understanding of the cell-specific cytosolic behavior of various DNA structures.