Impressive leap forwards for DNA origami: an elegant staple strand proximity ligation method for tracking DNA origami pharmacokinetics in vivo! This approach even allows analysis of stability of subregions within a DNA origami nanostructure. I think DNA origami has a lot of therapeutic potential, so it is exciting to see this solution to one of its translational barriers. Link: https://www.nature.com/articles/s41565-025-02091-z Paper title: “Resolving DNA origami structural integrity and pharmacokinetics in vivo”
Using origami samples in test tubes, we sequentially performed ligation, PCR and polyacrylamide gel electrophoresis (PAGE; Fig. 2a). For both Wrod and Lrod, amplification bands appeared only after ligation (Supplementary Fig. 3) and matched the sizes of single-LSP controls (Fig. 2d, g). When the origami was heat denatured before ligation, no LSP bands were detected (Fig. 2e, h), confirming that proximity ligation requires intact structures. By contrast, we showed that scaffold-targeted qPCR or origamiFISH assays37,38 still detected DNA regardless of the structural state (Fig. 2e, h), emphasizing their inability to distinguish intact origami from degraded origami.
Previous studies have shown that the coating of DNA nanostructures with the oligolysine-PEG polymer can protect them against nucleases and denaturation in low-salt environments, potentially increasing their stability in vivo23. Since PEGylation confers a physical barrier for the interaction of enzymes with DNA helices, we hypothesized that the ligase might also have decreased accessibility to PEGylated origamis. However, our in vitro experiments with PEGylated PEG-Lrod showed comparable ligation and amplification efficiencies to the bare Lrod (Supplementary Fig. 4). Another approach to enhance lattice-based origami stability in low-salt buffers and improved resistance to nucleases is sequence-specific covalent UV crosslinking26. We tested the application of the PLASTIQ protocol to a crosslinked version of the Lrod (UV-Lrod) with the same LSPs as Lrod. We observed a similar amplification pattern when compared to the non-crosslinked Lrod after PAGE electrophoresis of the pooled PCR-amplified LSPs (Extended Data Fig. 1).
Together, these results demonstrate that PLASTIQ reliably detects DNA origami integrity at the single-helix level for both wireframe and lattice designs, and that it is compatible with PEGylated or UV-crosslinked nanostructures.
