journal article·2026-02Open access

Resolving DNA origami structural integrity and pharmacokinetics in vivo

Yang Wang, Iris Rocamonde-Lago, Janine Waldvogel, Boxuan Shen, Yi-Chia Wu, Junke Zhu, Shuya Zang, Yingbo Jia, Igor Baars, Alexander Kloosterman, Ian T. Hoffecker, Ming-Ru Wu, Qin He, Björn Högberg

Nature Nanotechnology· Volume 21· Issue 2

Abstract

Abstract DNA origami holds great potential for advancing therapeutics, but the lack of methods for the precise assessment of structural integrity in vivo prevents its translation. Here we introduce proximity ligation assay for structural tracking and integrity quantification (PLASTIQ) for resolving origami structural integrity with only 1 µl of blood sample and with a detection limit of 0.01 fM. Through PLASTIQ, we could observe and quantify the dynamics of DNA origami degradation during blood circulation and evaluate the effectiveness of PEGylation for slowing this process in a murine model. Additionally, by using a double-layered barrel-like origami structure, we found distinct degradation kinetics of DNA helices depending on their specific location, revealing the slower degradation of internal helices compared with the outer ones. Our results suggest that PLASTIQ offers a quantitative approach for assessing DNA origami integrity in vivo by longitudinal sampling, providing dynamic pharmaceutical-level insights for accelerating the development of DNA-nanostructure-based therapeutic molecules and drugs.
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Research context

OpenAlex topics: Advanced biosensing and bioanalysis techniques · Protein Degradation and Inhibitors · Advanced Materials and Mechanics

SciVX discovery tags are terminology-based. OpenAlex topics are source-provided classifications.

References (54)

Show source-provided references
1. Afonin, K. A., Dobrovolskaia, M. A., Ke, W., Grodzinski, P. & Bathe, M. Critical review of nucleic acid nanotechnology to identify gaps and inform a strategy for accelerated clinical translation. Adv. Drug Deliv. Rev. 181, 114081 (2022).10.1016/j.addr.2021.114081 ↗
2. Rothemund, P. W. K. Folding DNA to create nanoscale shapes and patterns. Nature 440, 297–302 (2006).10.1038/nature04586 ↗
3. Zhang, Q. et al. DNA origami as an in vivo drug delivery vehicle for cancer therapy. ACS Nano 8, 6633–6643 (2014).10.1021/nn502058j ↗
4. Xu, T. et al. DNA origami frameworks enabled self-protective siRNA delivery for dual enhancement of chemo-photothermal combination therapy. Small 17, e2101780 (2021).10.1002/smll.202101780 ↗
5. Sigl, C. et al. Programmable icosahedral shell system for virus trapping. Nat. Mater. 20, 1281–1289 (2021).10.1038/s41563-021-01020-4 ↗
6. Li, L. et al. A DNA origami device spatially controls CD95 signalling to induce immune tolerance in rheumatoid arthritis. Nat. Mater. https://doi.org/10.1038/s41563-024-01865-5 (2024).10.1038/s41563-024-01865-5 ↗
7. Zhu, K. et al. Myeloid cell-specific topoisomerase 1 inhibition using DNA origami mitigates neuroinflammation. EMBO Rep. 23, e54499 (2022).10.15252/embr.202154499 ↗
8. Monferrer, A. et al. Broad-spectrum virus trapping with heparan sulfate-modified DNA origami shells. ACS Nano 16, 20002–20009 (2022).10.1021/acsnano.1c11328 ↗
9. Sun, Y. et al. DNA origami-based artificial antigen-presenting cells for adoptive T cell therapy. Sci. Adv. 8, eadd1106 (2022).10.1126/sciadv.add1106 ↗
10. Shaw, A. et al. Spatial control of membrane receptor function using ligand nanocalipers. Nat. Methods 11, 841–846 (2014).10.1038/nmeth.3025 ↗
11. Smyrlaki, I. et al. Soluble and multivalent Jag1 DNA origami nanopatterns activate notch without pulling force. Nat. Commun. 15, 465 (2024).10.1038/s41467-023-44059-4 ↗
12. Berger, R. M. L. et al. Nanoscale FasL organization on DNA origami to decipher apoptosis signal activation in cells. Small 17, e2101678 (2021).10.1002/smll.202101678 ↗
13. Spratt, J. et al. Multivalent insulin receptor activation using insulin-DNA origami nanostructures. Nat. Nanotechnol. 19, 237–245 (2024).10.1038/s41565-023-01507-y ↗
14. Dong, R. et al. DNA origami patterning of synthetic T cell receptors reveals spatial control of the sensitivity and kinetics of signal activation. Proc. Natl Acad. Sci. USA 118, e2109057118 (2021).10.1073/pnas.2109057118 ↗
15. Wang, Y., Baars, I., Fördös, F. & Högberg, B. Clustering of death receptor for apoptosis using nanoscale patterns of peptides. ACS Nano 15, 9614–9626 (2021).10.1021/acsnano.0c10104 ↗
16. Veneziano, R. et al. Role of nanoscale antigen organization on B-cell activation probed using DNA origami. Nat. Nanotechnol. 15, 716–723 (2020).10.1038/s41565-020-0719-0 ↗
17. Wagenbauer, K. F. et al. Programmable multispecific DNA-origami-based T-cell engagers. Nat. Nanotechnol. 18, 1319–1326 (2023).10.1038/s41565-023-01471-7 ↗
18. Kern, N., Dong, R., Douglas, S. M., Vale, R. D. & Morrissey, M. A. Tight nanoscale clustering of Fcγ receptors using DNA origami promotes phagocytosis. eLife 10, e68311 (2021).10.7554/eLife.68311 ↗
19. Zeng, Y. C. et al. Fine tuning of CpG spatial distribution with DNA origami for improved cancer vaccination. Nat. Nanotechnol. https://doi.org/10.1038/s41565-024-01615-3 (2024).10.1038/s41565-024-01615-3 ↗
20. Wamhoff, E.-C. et al. Enhancing antibody responses by multivalent antigen display on thymus-independent DNA origami scaffolds. Nat. Commun. 15, 795 (2024).
21. Perrault, S. D. & Shih, W. M. Virus-inspired membrane encapsulation of DNA nanostructures to achieve in vivo stability. ACS Nano 8, 5132–5140 (2014).10.1021/nn5011914 ↗
22. Benson, E. et al. DNA rendering of polyhedral meshes at the nanoscale. Nature 523, 441–444 (2015).10.1038/nature14586 ↗
23. Ponnuswamy, N. et al. Oligolysine-based coating protects DNA nanostructures from low-salt denaturation and nuclease degradation. Nat. Commun. 8, 15654 (2017).10.1038/ncomms15654 ↗
24. Chandrasekaran, A. R. Nuclease resistance of DNA nanostructures. Nat. Rev. Chem. 5, 225–239 (2021).10.1038/s41570-021-00251-y ↗
25. Anastassacos, F. M., Zhao, Z., Zeng, Y. & Shih, W. M. Glutaraldehyde cross-linking of oligolysines coating DNA origami greatly reduces susceptibility to nuclease degradation. J. Am. Chem. Soc. 142, 3311–3315 (2020).10.1021/jacs.9b11698 ↗
26. Gerling, T., Kube, M., Kick, B. & Dietz, H. Sequence-programmable covalent bonding of designed DNA assemblies. Sci. Adv. 4, eaau1157 (2018).10.1126/sciadv.aau1157 ↗
27. Wamhoff, E.-C. et al. Controlling nuclease degradation of wireframe DNA origami with minor groove binders. ACS Nano 16, 8954–8966 (2022).10.1021/acsnano.1c11575 ↗
28. Manuguri, S., Nguyen, M.-K., Loo, J., Natarajan, A. K. & Kuzyk, A. Advancing the utility of DNA origami technique through enhanced stability of DNA-origami-based assemblies. Bioconjug. Chem. 34, 6–17 (2023).10.1021/acs.bioconjchem.2c00311 ↗
29. Youssef, S., Tsang, E., Samanta, A., Kumar, V. & Gothelf, K. V. Reversible protection and targeted delivery of DNA origami with a disulfide-containing cationic polymer. Small 20, e2301058 (2024).10.1002/smll.202301058 ↗
30. Wang, S.-T. et al. DNA origami protection and molecular interfacing through engineered sequence-defined peptoids. Proc. Natl Acad. Sci. USA 117, 6339–6348 (2020).10.1073/pnas.1919749117 ↗
31. Auvinen, H. et al. Protein coating of DNA nanostructures for enhanced stability and immunocompatibility. Adv. Health. Mater. 6, 1700692 (2017).10.1002/adhm.201700692 ↗
32. Krishnamurthy, K., Rajendran, A., Nakata, E. & Morii, T. Near quantitative ligation results in resistance of DNA origami against nuclease and cell lysate. Small Methods 8, e2300999 (2024).10.1002/smtd.202300999 ↗
33. Rajendran, A., Krishnamurthy, K., Giridasappa, A., Nakata, E. & Morii, T. Stabilization and structural changes of 2D DNA origami by enzymatic ligation. Nucleic Acids Res 49, 7884–7900 (2021).10.1093/nar/gkab611 ↗
34. Agarwal, N. P., Matthies, M., Gür, F. N., Osada, K. & Schmidt, T. L. Block copolymer micellization as a protection strategy for DNA origami. Angew. Chem. Int. Ed. 56, 5460–5464 (2017).10.1002/anie.201608873 ↗
35. Xu, S. et al. Multifunctional self-delivery micelles targeting the invasion-metastasis cascade for enhanced chemotherapy against melanoma and the lung metastasis. Asian J. Pharm. Sci. 16, 794–805 (2021).
36. Wamhoff, E.-C. et al. Evaluation of nonmodified wireframe DNA origami for acute toxicity and biodistribution in mice. ACS Appl. Bio Mater. 6, 1960–1969 (2023).10.1021/acsabm.3c00155 ↗
37. Okholm, A. H. et al. Quantification of cellular uptake of DNA nanostructures by qPCR. Methods 67, 193–197 (2014).10.1016/j.ymeth.2014.01.013 ↗
38. Wang, W. X. et al. Universal, label-free, single-molecule visualization of DNA origami nanodevices across biological samples using origamiFISH. Nat. Nanotechnol. 19, 58–69 (2024).10.1038/s41565-023-01449-5 ↗
39. Söderberg, O. et al. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat. Methods 3, 995–1000 (2006).10.1038/nmeth947 ↗
40. Gullberg, M. et al. Cytokine detection by antibody-based proximity ligation. Proc. Natl Acad. Sci. USA 101, 8420–8424 (2004).10.1073/pnas.0400552101 ↗
41. Fredriksson, S. et al. Protein detection using proximity-dependent DNA ligation assays. Nat. Biotechnol. 20, 473–477 (2002).10.1038/nbt0502-473 ↗
42. Cedervall, T. et al. Understanding the nanoparticle–protein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles. Proc. Natl Acad. Sci. USA 104, 2050–2055 (2007).10.1073/pnas.0608582104 ↗
43. Riches, A. C., Sharp, J. G., Thomas, D. B. & Smith, S. V. Blood volume determination in the mouse. J. Physiol. 228, 279–284 (1973).10.1113/jphysiol.1973.sp010086 ↗
44. Guo, C. et al. The interplay between PEGylated nanoparticles and blood immune system. Adv. Drug Deliv. Rev. 200, 115044 (2023).10.1016/j.addr.2023.115044 ↗
45. Zalba, S., ten Hagen, T. L. M., Burgui, C. & Garrido, M. J. Stealth nanoparticles in oncology: facing the PEG dilemma. J. Control. Release 351, 22–36 (2022).10.1016/j.jconrel.2022.09.002 ↗
46. Partikel, K. et al. Effect of nanoparticle size and PEGylation on the protein corona of PLGA nanoparticles. Eur. J. Pharm. Biopharm. 141, 70–80 (2019).10.1016/j.ejpb.2019.05.006 ↗
47. Bertosin, E. et al. Cryo-electron microscopy and mass analysis of oligolysine-coated DNA nanostructures. ACS Nano 15, 9391–9403 (2021).10.1021/acsnano.0c10137 ↗
48. Wang, X. et al. Albumin-hitchhiking drug delivery to tumor-draining lymph nodes precisely boosts tumor-specific immunity through autophagy modulation of immune cells. Adv. Mater. 35, e2211055 (2023).10.1002/adma.202211055 ↗
49. Chen, D. et al. Autophagy-activating aluminum hydroxide nanovaccine for enhanced antigen presentation and anti-tumor immunity. J. Control. Release 377, 223–235 (2025).10.1016/j.jconrel.2024.11.018 ↗
50. Jepsen, M. D. E. et al. Single molecule analysis of structural fluctuations in DNA nanostructures. Nanoscale 11, 18475–18482 (2019).10.1039/C9NR03826D ↗
51. 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).10.1021/acs.nanolett.8b00660 ↗
52. Comberlato, A., Koga, M. M., Nüssing, S., Parish, I. A. & Bastings, M. M. C. Spatially controlled activation of toll-like receptor 9 with DNA-based nanomaterials. Nano Lett. 22, 2506–2513 (2022).10.1021/acs.nanolett.2c00275 ↗
53. Du, R. R. et al. Innate immune stimulation using 3D wireframe DNA origami. ACS Nano 16, 20340–20352 (2022).10.1021/acsnano.2c06275 ↗
54. Douglas, S. M. et al. Self-assembly of DNA into nanoscale three-dimensional shapes. Nature 459, 414–418 (2009).10.1038/nature08016 ↗