Determining the Cytosolic Stability of Small DNA Nanostructures In Cellula

Abstract

DNA nanostructures have proven potential in biomedicine. However, their intracellular interactionsespecially cytosolic stabilityremain mostly unknown and attempts to discern this are confounded by the complexities of endocytic uptake and entrapment. Here, we bypass the endocytic uptake and evaluate the DNA structural stability directly in live cells. Commonly used DNA structurescrosshairs and a tetrahedronwere labeled with a multistep Förster resonance energy transfer dye cascade and microinjected into the cytosol of transformed and primary cells. Energy transfer loss, as monitored by fluorescence microscopy, reported the structure’s direct time-resolved breakdown in cellula. The results showed rapid degradation of the DNA crosshair within 20 min, while the tetrahedron remained consistently intact for at least 1 h postinjection. Nuclease assays in conjunction with a current understanding of the tetrahedron’s torsional rigidity confirmed its higher stability. Such studies can inform design parameters for future DNA nanostructures where programmable degradation rates may be required.

Customized Scaffolds for Direct Assembly of Functionalized DNA Origami

“…Results, summarized in Figure e, show that PB NPs remained stable regardless of the scaffold used for their assembly. Previous studies on the cytosolic stability of polyhedral DNA-NPs as well as in the cell lysate agree with these results …”

Uptake and stability of DNA nanostructures in cells: a cross-sectional overview of the current state of the art

“…3 shows results from a recent report where dye-labeled DNA structures were directly microinjected into the cytosol of a variety of different primary and transformed cells and the stability of the structures evaluated by tracking the resulting FRET over time. 86 This format was exploited to bypass the complications of endocytic uptake so that the only variable became that of cytosolic stability. This study confirmed that the high-torsion DNA tetrahedron is more stable in the cellular cytosol than linear structures.…”

“…Furthermore, expanding cytosolic studies to other DNA NS types as well as integration of stabilizing techniques are needed. 86…”

“…Furthermore, expanding cytosolic studies to other DNA NS types as well as integration of stabilizing techniques are needed. 86 Feasibility of DNA NS based therapies at a scale where patient impact is significant are required as well. For example, to what extent are DNA NS based anti-cancer interventions advantageous from an economic standpoint is being evaluated.…”

Morphology Dictated Immune Activation with Framework Nucleic Acids

“…[6][7][8][9][10] This surge in popularity can be attributed to the programmability and precise control over the size, shape, function, and biocompatibility of these structures. FNAs have been shown to maintain their structural stability and integrity in biological fluids, [11][12] and can be effectively internalized by mammalian cells without the transfectants or transfection methods. [13] Much work has been done to explore the mechanisms of the cellular uptake of DNA nanostructures.…”