Scientists have designed and synthesized chains of molecules with a precise sequence and length to efficiently protect 3-D DNA nanostructures from structural degradation under a variety of biomedically relevant conditions.
They demonstrated how these “peptoid-coated DNA origami” have the potential to be used for delivering anti-cancer drugs and proteins, imaging biological molecules, and targeting cell-surface receptors implicated in cancer.
Similar to the Japanese art of paper folding, DNA origami is the folding of long, flexible DNA chains into desired shapes at the nanoscale (billionths of a meter) by “stapling” different parts of the chain with the complementary base pairs of short DNA strands.
These programmable and precisely controlled nanoscale architectures could be beneficial for many biomedical applications, including the targeted delivery of drugs and genes to desired tissues or cells, imaging of biological processes inside the body, and biosensing for disease detection or health monitoring.
However, enabling such applications will require solutions for protecting DNA origami structures in complex biological fluids and enabling new functions that are not inherent to DNA.
In this research, we synthesized biocompatible molecules called peptoids with a well-defined molecular sequence composition and length. We coated octahedral-shaped DNA origami which has high mechanical stability and a large open space for carrying nanoscale cargo such as small-molecule anti-cancer drugs with these peptoids.
Our demonstrations showed that the peptoid coatings efficiently protected the DNA origami in various physiological conditions and supported the addition of different chemical functionalities for biomedical applications.
Peptoids resemble peptides, or short chains of amino acids. However, in peptoids, the side chains (chemical groups attached to the main chain or backbone of the molecule) are attached to nitrogen rather than to carbon.
Moreover, peptoids are more flexible, owing to the lack of hydrogen bonds in the backbone. This flexibility can be leveraged to control how the peptoids bind to the DNA origami.
To determine which type was better at providing protection, the scientists studied the binding between two-stranded (duplex) DNA and peptoids. Experiments with fluorescent dye (which binds to the DNA) showed that a specific brush-type architecture was most efficient at stabilizing duplex DNA coated with peptoids at high temperature.
A collaborator at RMIT University in Australia simulated the molecular-level DNA-peptoid interactions to understand why.
Guided by these studies, the team investigated the structural stability of the peptoid-coated DNA origami in several types of physiologically relevant conditions: in a solution containing a low concentration of positively charged magnesium (Mg) ions, in a solution containing a DNA-specific nuclease (type of enzyme), and incubated in cell culture media (containing both nucleases and Mg ions at low concentration).
Typically, a high Mg-ion concentration is required to stabilize DNA origami by reducing the repulsion of DNA-DNA negative charges, but physiological fluids contain much lower concentrations.
For their investigations, they used a combination of experimental techniques: agarose gel electrophoresis, a method for separating DNA fragments (or other macromolecules) on the basis of their charge and size; transmission electron microscopy imaging and dynamic light scattering at the CFN; and real-time small-angle x-ray scattering at the Life Science X-ray Scattering (LiX) beamline of Brookhaven’s National Synchrotron Light Source II (NSLS-II).
The results indicated that the structure of the origami had remained intact after it was coated with specifically designed peptoids and placed in the different physiological conditions.
Following these experiments, the scientists conducted a series of demonstrations in collaboration with the Bertozzi Group at Stanford University to explore how the peptoid-coated origami could be used in biomedical applications.
For example, they loaded the chemotherapy drug doxorubicin into the coated origami. Doxorubicin is one of the commonly administered drugs to patients with HER2-positive breast cancer, in which an overexpression of the HER2 protein (a receptor on breast cells) causes cells to divide and grow uncontrollably.
Over 48 hours, the coated origami released less of the doxorubicin than its noncoated counterpart, as measured through the intensity of the drug’s intrinsic fluorescence.
“The ultimate goal is to be able to modulate the release rate during the drug delivery process to control biological and toxic effects,” explained Wang.
In a second nanocargo demonstration, they investigated whether proteins could be delivered in a similar way.
They encapsulated a cow-derived protein (attached to fluorescent molecules for visualization) inside the coated origami in the presence of the protein-digesting enzyme trypsin. Digestion of this encapsulated protein by trypsin was reduced and slowed due to a combination of the DNA origami itself and the peptoid coating.
In follow-on experiments, Wang plans to explore the potential of combinatorial therapy, in which peptoid-coated DNA origami carrying doxorubicin and featuring a trastuzumab-functionalized surface targets HER2-positive breast cancer cells.