DNA
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Living Materials

DNA nanotechnology has been extensively studied as a biomedical platform and as a biophysical probe. For both, the DNA nanostructure can intentionally, or unintentionally, measure and perturb the biological material it is interacting with. There are many important considerations when designing DNA nanostructures for use with cells in vitro or in vivo. If you intend to work in physiological fluids containing serum or free-floating proteins, these proteins can non-specifically interact with your nanostructure and alter its intended shape and function1. In addition, DNA nanostructures are highly negatively charged so they will interact electrostatically with cell surface glycocalyx, proteins, and lipid membranes2. For anchoring structures onto cell surfaces, researchers often functionalize their nanostructure with hydrophobic moieties like cholesterol which insert into lipid membranes3. Compared to proteins, DNA origami synthesized using the standard M13mp18 scaffold are significantly larger, on the order of 100× the size of a typical protein. For this reason, many applications of DNA nanotechnology for biophysics rely on small multi-strand DNA complexes, like DNA tetrahedrons, which are likely to perturb cells less and are on the same size scale as proteins.

For applications within cells, nanostructures must first be internalized. Given their hydrophilicity and highly negative charge this itself is often a challenge2. Improvements in uptake have been achieved by using transfection reagents4 or adding molecules that bind nanostructures to cell surfaces5. When uptaken, they are typically engulfed into cells via scavenger receptors, clathrin-mediated endocytosis, and caveolin-mediated endocytosis6. Nanostructure size, shape, aspect ratio, as well as other structural factors are known to play a role in uptake efficiency, however there is no clear consensus on the rules governing their uptake and it is likely cell-type specific7. Once inside the cell, nanostructures are in an environment with low levels of magnesium. The ionic concentrations in the cytoplasm are typically inconsistent with origami folding buffer and are often inadequate to maintain a stable structure.

Despite these challenges, biomedical and biophysical applications of DNA nanotechnology continue to be driven forward, and progress is being made in addressing the limitations.

References

  1. Smolková, B. et al. Protein Corona Inhibits Endosomal Escape of Functionalized DNA Nanostructures in Living Cells. ACS Applied Materials & Interfaces 13, 46375 (2021).
  2. Liu, Y. et al. The effects of overhang placement and multivalency on cell labeling by DNA origami. Nanoscale 13, 6819–6828 (2021).
  3. Wu, N. et al. Functional and Biomimetic DNA Nanostructures on Lipid Membranes. Langmuir 34, 14721–14730 (2018).
  4. Okholm, A. H. et al. Quantification of cellular uptake of DNA nanostructures by qPCR. Methods 67, 193–197 (2014).
  5. Mikkilä, J. et al. Virus-Encapsulated DNA Origami Nanostructures for Cellular Delivery. Nano Lett. 14, 2196–2200 (2014).
  6. Wang, W. et al. Cell-Surface Binding of DNA Nanostructures for Enhanced Intracellular and Intranuclear Delivery. ACS Appl. Mater. Interfaces 16, 15783–15797 (2024).
  7. Bastings, M. M. C. et al. Modulation of the Cellular Uptake of DNA Origami through Control over Mass and Shape. Nano Lett. 18, 3557–3564 (2018).
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