Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Designer DNA nanostructures for viral inhibition

Nature Protocols volume 17, pages 282–326 (2022)Cite this article

Subjects

Abstract

Emerging viral diseases can substantially threaten national and global public health. Central to our ability to successfully tackle these diseases is the need to quickly detect the causative virus and neutralize it efficiently. Here we present the rational design of DNA nanostructures to inhibit dengue virus infection. The designer DNA nanostructure (DDN) can bind to complementary epitopes on antigens dispersed across the surface of a viral particle. Since these antigens are arranged in a defined geometric pattern that is unique to each virus, the structure of the DDN is designed to mirror the spatial arrangement of antigens on the viral particle, providing very high viral binding avidity. We describe how available structural data can be used to identify unique spatial patterns of antigens on the surface of a viral particle. We then present a procedure for synthesizing DDNs using a combination of in silico design principles, self-assembly, and characterization using gel electrophoresis, atomic force microscopy and surface plasmon resonance spectroscopy. Finally, we evaluate the efficacy of a DDN in inhibiting dengue virus infection via plaque-forming assays. We expect this protocol to take 2–3 d to complete virus antigen pattern identification from existing cryogenic electron microscopy data, ~2 weeks for DDN design, synthesis, and virus binding characterization, and ~2 weeks for DDN cytotoxicity and antiviral efficacy assays.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Schematic of applications of the DNA star strategy.
Fig. 2: Schematic overview of the design, assembly, and characterization of DDNs for inhibition of virus infection.
Fig. 3: Schematic overview of DENV detection by DNA star sensor.
Fig. 4: Identification of DENV antigen spatial patterns.
Fig. 5: DNA star design.
Fig. 6: Process flow diagram of the DNA star design.
Fig. 7: Process flow schematic of the purification of DNA oligos using denaturing gel electrophoresis.
Fig. 8: Gel electrophoretic characterization of the DNA star nanostructure.
Fig. 9: Purification of the DNA star.
Fig. 10: AFM images of the DNA stars.
Fig. 11: SPR binding assays.
Fig. 12: Evaluation of the inhibitors.

Similar content being viewed by others

Data availability

All data supporting the findings of this work are available within this paper (figures and description) and the Supplementary Information. All the raw and source data have been deposited at figshare. They can be accessed at https://doi.org/10.6084/m9.figshare.c.5409411. These files include: DNA_Star_Viral_Protocol_Dengue_Model.pse. PyMol session file used for generating Fig. 3 (second panel), Fig. 4, Fig. 5a,b and Supplementary Figs. 1 and 2. Source data are included for Fig. 8 (DNA_Star_Viral_Protocol_Figure_8_Gel_Image.pdf), Fig. 11c (DNA_Star_Viral_Protocol_Figure_11c.xlsx), Fig. 12c (DNA_Star_Viral_Protocol_Figure_12c.xlsx), Supplementary Fig. 3 (DNA_Star_Viral_Protocol_Supplementary_Figure_3_Gel_Image.pdf) and Supplementary Fig. 4 (DNA_Star_Viral_Protocol_Supplementary_Figure_4_Gel_Image.pdf).

Code availability

The SEQUIN program package used in this study runs on a Windows 10 operating system. It is deposited at figshare and available for download without any access restrictions at https://doi.org/10.6084/m9.figshare.c.5409411. The files include SEQUIN_Program_Package.zip (software package) and SEQUIN_User_Instruction.pdf (software command instruction).

References

  1. Dawood, F. S. et al. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. Lancet Infect. Dis. 12, 687–695 (2012).

    Article  PubMed  Google Scholar 

  2. Shrestha, S. S. et al. Estimating the burden of 2009 pandemic influenza A (H1N1) in the United States (April 2009–April 2010). Clin. Infect. Dis. 52, S75–S82 (2011).

    Article  PubMed  Google Scholar 

  3. Ali, M. G. et al. Recent advances in therapeutic applications of neutralizing antibodies for virus infections: an overview. Immunol. Res. 68, 325–339 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Tirado, S. M. & Yoon, K. J. Antibody-dependent enhancement of virus infection and disease. Viral Immunol. 16, 69–86 (2003).

    Article  CAS  PubMed  Google Scholar 

  5. Whitehead, S. S., Blaney, J. E., Durbin, A. P. & Murphy, B. R. Prospects for a dengue virus vaccine. Nat. Rev. Microbiol. 5, 518–528 (2007).

    Article  CAS  PubMed  Google Scholar 

  6. Prasad, B. V. & Schmid, M. F. Principles of virus structural organization. Adv. Exp. Med. Biol. 726, 17–47 (2012).

    Article  CAS  PubMed  Google Scholar 

  7. Kwon, P. S. et al. Designer DNA architecture offers precise and multivalent spatial pattern-recognition for viral sensing and inhibition. Nat. Chem. 12, 26–35 (2020).

    Article  CAS  PubMed  Google Scholar 

  8. Fibriansah, G. et al. Structural changes in dengue virus when exposed to a temperature of 37 degrees C. J. Virol. 87, 7585–7592 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Kosuri, S. & Church, G. M. Large-scale de novo DNA synthesis: technologies and applications. Nat. Methods 11, 499–507 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Praetorius, F. et al. Biotechnological mass production of DNA origami. Nature 552, 84–87 (2017).

    Article  CAS  PubMed  Google Scholar 

  11. Ducani, C., Kaul, C., Moche, M., Shih, W. M. & Hogberg, B. Enzymatic production of ‘monoclonal stoichiometric’ single-stranded DNA oligonucleotides. Nat. Methods 10, 647–652 (2013).

    Article  CAS  PubMed  Google Scholar 

  12. Lin, C. et al. In vivo cloning of artificial DNA nanostructures. Proc. Natl Acad. Sci. USA 105, 17626–17631 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Seeman, N. C. Nucleic acid junctions and lattices. J. Theor. Biol. 99, 237–247 (1982).

    Article  CAS  PubMed  Google Scholar 

  14. Chandrasekaran, A. R. & Zhuo, R. A ‘tile’ tale: hierarchical self-assembly of DNA lattices. Appl. Mater. Today 2, 7–16 (2016).

    Article  Google Scholar 

  15. Duan, J., Wang, X. & Kizer, M. E. Biotechnological and therapeutic applications of natural nucleic acid structural motifs. Top. Curr. Chem. 378, 26 (2020).

    Article  CAS  Google Scholar 

  16. Chao, J. et al. Programming DNA origami assembly for shape-resolved nanomechanical imaging labels. Nat. Protoc. 13, 1569–1585 (2018).

    Article  CAS  PubMed  Google Scholar 

  17. Lanphere, C. et al. Design, assembly, and characterization of membrane-spanning DNA nanopores. Nat. Protoc. 16, 86–130 (2021).

    Article  CAS  PubMed  Google Scholar 

  18. Sigl, C. et al. Programmable icosahedral shell system for virus trapping. Nat. Mater. 20, 1281–1289 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Chauhan, N. & Wang, X. Nanocages for virus inhibition. Nat. Mater. 20, 1176–1177 (2021).

    Article  CAS  PubMed  Google Scholar 

  20. Kuzuya, A., Sakai, Y., Yamazaki, T., Xu, Y. & Komiyama, M. Nanomechanical DNA origami ‘single-molecule beacons’ directly imaged by atomic force microscopy. Nat. Commun. 2, 449 (2011).

    Article  PubMed  Google Scholar 

  21. Nikolovska-Coleska, Z. Studying protein–protein interactions using surface plasmon resonance. Methods Mol. Biol. 1278, 109–138 (2015).

    Article  CAS  PubMed  Google Scholar 

  22. Drescher, D. G., Selvakumar, D. & Drescher, M. J. Analysis of protein interactions by surface plasmon resonance. Adv. Protein Chem. Struct. Biol. 110, 1–30 (2018).

    Article  CAS  PubMed  Google Scholar 

  23. Douzi, B. Protein–protein interactions: surface plasmon resonance. Methods Mol. Biol. 1615, 257–275 (2017).

    Article  PubMed  Google Scholar 

  24. Rath, P. P., Anand, G. & Agarwal, S. Surface plasmon resonance analysis of the protein–protein binding specificity using Autolab ESPIRIT. Bio Protoc. 10, e3519 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Baer, A. & Kehn-Hall, K. Viral concentration determination through plaque assays: using traditional and novel overlay systems. J. Vis. Exp. e52065 (2014).

  26. Mendoza, E. J., Manguiat, K., Wood, H. & Drebot, M. Two detailed plaque assay protocols for the quantification of infectious SARS-CoV-2. Curr. Protoc. Microbiol. 57, ecpmc105 (2020).

    Article  PubMed  Google Scholar 

  27. Kwon, S. J. et al. Nanostructured glycan architecture is important in the inhibition of influenza A virus infection. Nat. Nanotechnol. 12, 48–54 (2017).

    Article  CAS  PubMed  Google Scholar 

  28. Nie, C. et al. Spiky nanostructures with geometry-matching topography for virus inhibition. Nano Lett. 20, 5367–5375 (2020).

    Article  CAS  PubMed  Google Scholar 

  29. Lauster, D. et al. Phage capsid nanoparticles with defined ligand arrangement block influenza virus entry. Nat. Nanotechnol. 15, 373–379 (2020).

    Article  CAS  PubMed  Google Scholar 

  30. King, D. J. & Noss, R. R. Toxicity of polyacrylamide and acrylamide monomer. Rev. Environ. Health 8, 3–16 (1989).

    Article  CAS  PubMed  Google Scholar 

  31. Malik, N. et al. Dendrimers: relationship between structure and biocompatibility in vitro, and preliminary studies on the biodistribution of 125I-labelled polyamidoamine dendrimers in vivo. J. Control Release 65, 133–148 (2000).

    Article  CAS  PubMed  Google Scholar 

  32. Ahmad, K. M., Xiao, Y. & Soh, H. T. Selection is more intelligent than design: improving the affinity of a bivalent ligand through directed evolution. Nucleic Acids Res 40, 11777–11783 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Strauch, E. M. et al. Computational design of trimeric influenza-neutralizing proteins targeting the hemagglutinin receptor binding site. Nat. Biotechnol. 35, 667–671 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Mei, Q. A. et al. Stability of DNA origami nanoarrays in cell lysate. Nano Lett. 11, 1477–1482 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Hahn, J., Wickham, S. F. J., Shih, W. M. & Perrault, S. D. Addressing the instability of DNA nanostructures in tissue culture. ACS Nano 8, 8765–8775 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Agarwal, N. P., Matthies, M., Gur, F. N., Osada, K. & Schmidt, T. L. Block copolymer micellization as a protection strategy for DNA origami. Angew. Chem. Int. Ed. Engl. 56, 5460–5464 (2017).

    Article  CAS  PubMed  Google Scholar 

  37. Perrault, S. D. & Shih, W. M. Virus-inspired membrane encapsulation of DNA nanostructures to achieve in vivo stability. ACS Nano 8, 5132–5140 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Li, S. et al. A DNA nanorobot functions as a cancer therapeutic in response to a molecular trigger in vivo. Nat. Biotechnol. 36, 258–264 (2018).

    Article  CAS  PubMed  Google Scholar 

  39. Kizer, M. E. et al. Hydroporator: a hydrodynamic cell membrane perforator for high-throughput vector-free nanomaterial intracellular delivery and DNA origami biostability evaluation. Lab Chip 19, 1747–1754 (2019).

    Article  CAS  PubMed  Google Scholar 

  40. Jiang, D. et al. DNA origami nanostructures can exhibit preferential renal uptake and alleviate acute kidney injury. Nat. Biomed. Eng. 2, 865–877 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Kim, Y. & Yin, P. Enhancing biocompatible stability of DNA nanostructures using dendritic oligonucleotides and brick motifs. Angew. Chem. Int. Ed. Engl. 59, 700–703 (2020).

    Article  CAS  PubMed  Google Scholar 

  42. Gerling, T., Kube, M., Kick, B. & Dietz, H. Sequence-programmable covalent bonding of designed DNA assemblies. Sci. Adv. 4, eaau1157 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Rothemund, P. W. Folding DNA to create nanoscale shapes and patterns. Nature 440, 297–302 (2006).

    Article  CAS  PubMed  Google Scholar 

  44. Chandrasekaran, A. R., Anderson, N., Kizer, M., Halvorsen, K. & Wang, X. Beyond the fold: emerging biological applications of DNA origami. Chembiochem 17, 1081–1089 (2016).

    Article  CAS  PubMed  Google Scholar 

  45. Wilner, O. I. & Willner, I. Functionalized DNA nanostructures. Chem. Rev. 112, 2528–2556 (2012).

    Article  CAS  PubMed  Google Scholar 

  46. Shaw, A. et al. Spatial control of membrane receptor function using ligand nanocalipers. Nat. Methods 11, 841–846 (2014).

    Article  CAS  PubMed  Google Scholar 

  47. Shaw, A. et al. Binding to nanopatterned antigens is dominated by the spatial tolerance of antibodies. Nat. Nanotechnol. 14, 184–190 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Veneziano, R. et al. Role of nanoscale antigen organization on B-cell activation probed using DNA origami. Nat. Nanotechnol. 15, 716–723 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Lee, H. et al. Molecularly self-assembled nucleic acid nanoparticles for targeted in vivo siRNA delivery. Nat. Nanotechnol. 7, 389–393 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Klinman, D. M. Immunotherapeutic uses of CpG oligodeoxynucleotides. Nat. Rev. Immunol. 4, 249–258 (2004).

    Article  CAS  PubMed  Google Scholar 

  51. Liu, X. et al. A DNA nanostructure platform for directed assembly of synthetic vaccines. Nano Lett. 12, 4254–4259 (2012).

    Article  CAS  PubMed  Google Scholar 

  52. Auvinen, H. et al. Protein coating of DNA nanostructures for enhanced stability and immunocompatibility. Adv. Healthc. Mater. 6 (2017).

  53. Ponnuswamy, N. et al. Oligolysine-based coating protects DNA nanostructures from low-salt denaturation and nuclease degradation. Nat. Commun. 8, 15654 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. 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).

    Article  CAS  PubMed  Google Scholar 

  55. Ramakrishnan, S., Ijas, H., Linko, V. & Keller, A. Structural stability of DNA origami nanostructures under application-specific conditions. Comput. Struct. Biotechnol. J. 16, 342–349 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Bila, H., Kurisinkal, E. E. & Bastings, M. M. C. Engineering a stable future for DNA-origami as a biomaterial. Biomater. Sci. 7, 532–541 (2019).

    Article  CAS  PubMed  Google Scholar 

  57. Chandrasekaran, A. R. Nuclease resistance of DNA nanostructures. Nat. Rev. Chem. 5, 225–239 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Kick, B., Praetorius, F., Dietz, H. & Weuster-Botz, D. Efficient production of single-stranded phage DNA as scaffolds for DNA origami. Nano Lett. 15, 4672–4676 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Palluk, S. et al. De novo DNA synthesis using polymerase-nucleotide conjugates. Nat. Biotechnol. 36, 645–650 (2018).

    Article  CAS  PubMed  Google Scholar 

  60. Deng, Y. et al. Intracellular delivery of nanomaterials via an inertial microfluidic cell hydroporator. Nano Lett. 18, 2705–2710 (2018).

    Article  CAS  PubMed  Google Scholar 

  61. Ellington, A. D. & Szostak, J. W. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822 (1990).

    Article  CAS  PubMed  Google Scholar 

  62. Tuerk, C. & Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505–510 (1990).

    Article  CAS  PubMed  Google Scholar 

  63. Robertson, D. L. & Joyce, G. F. Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA. Nature 344, 467–468 (1990).

    Article  CAS  PubMed  Google Scholar 

  64. Kizer, M. E., Linhardt, R. J., Chandrasekaran, A. R. & Wang, X. A molecular hero suit for in vitro and in vivo DNA nanostructures. Small 15, e1805386 (2019).

    Article  PubMed  Google Scholar 

  65. Ke, Y., Castro, C. & Choi, J. H. Structural DNA nanotechnology: artificial nanostructures for biomedical research. Annu. Rev. Biomed. Eng. 20, 375–401 (2018).

    Article  CAS  PubMed  Google Scholar 

  66. Bujold, K. E., Lacroix, A. & Sleiman, H. F. DNA nanostructures at the interface with biology. Chem 4, 495–521 (2018).

    Article  CAS  Google Scholar 

  67. Jorge, A. F. & Eritja, R. Overview of DNA self-assembling: progresses in biomedical applications. Pharmaceutics 10, 268 (2018).

    Article  CAS  PubMed Central  Google Scholar 

  68. Udomprasert, A. & Kangsamaksin, T. DNA origami applications in cancer therapy. Cancer Sci. 108, 1535–1543 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Xu, W. et al. Functional nucleic acid nanomaterials: development, properties, and applications. Angew. Chem. Int. Ed. Engl. (2019).

  70. Weng, Y. et al. Improved nucleic acid therapy with advanced nanoscale biotechnology. Mol. Ther. Nucleic Acids 19, 581–601 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  71. Kaur, H., Bruno, J. G., Kumar, A. & Sharma, T. K. Aptamers in the therapeutics and diagnostics pipelines. Theranostics 8, 4016–4032 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Shum, K. T., Zhou, J. & Rossi, J. J. Aptamer-based therapeutics: new approaches to combat human viral diseases. Pharmaceuticals 6, 1507–1542 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  73. Keller, A. & Linko, V. Challenges and perspectives of DNA nanostructures in biomedicine. Angew. Chem. Int. Ed. Engl. 59, 15818–15833 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Jiang, S., Ge, Z., Mou, S., Yan, H. & Fan, C. Designer DNA nanostructures for therapeutics. Chem 7, 1156–1179 (2021).

    Article  CAS  Google Scholar 

  75. Zeng, Y., Nixon, R. L., Liu, W. & Wang, R. The applications of functionalized DNA nanostructures in bioimaging and cancer therapy. Biomaterials 268, 120560 (2021).

    Article  CAS  PubMed  Google Scholar 

  76. Wang, H., Luo, D., Wang, H., Wang, F. & Liu, X. Construction of smart stimuli-responsive DNA nanostructures for biomedical applications. Chemistry 27, 3929–3943 (2021).

    Article  CAS  PubMed  Google Scholar 

  77. He, L., Mu, J., Gang, O. & Chen, X. Rationally programming nanomaterials with DNA for biomedical applications. Adv. Sci. 8, 2003775 (2021).

    Article  CAS  Google Scholar 

  78. Huang, Z., Qiu, L., Zhang, T. & Tan, W. Integrating DNA nanotechnology with aptamers for biological and biomedical applications. Matter 4, 461–489 (2021).

    Article  CAS  Google Scholar 

  79. Smith, D. M. & Keller, A. DNA nanostructures in the fight against infectious diseases. Adv. Nanobiomed. Res. 2000049 (2021).

  80. Lippe, R. Flow virometry: a powerful tool to functionally characterize viruses. J. Virol. 92, e01765–17 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Zamora, J. L. R. & Aguilar, H. C. Flow virometry as a tool to study viruses. Methods 134–135, 87–97 (2018).

    Article  PubMed  Google Scholar 

  82. Xia, S. et al. Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion. Cell Res 30, 343–355 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Nie, J. et al. Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2. Emerg. Microbes Infect. 9, 680–686 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Shang, J. et al. Cell entry mechanisms of SARS-CoV-2. Proc. Natl Acad. Sci. USA 117, 11727–11734 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Koczula, K. M. & Gallotta, A. Lateral flow assays. Essays Biochem 60, 111–120 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  86. Li, N. et al. Photonic resonator interferometric scattering microscopy. Nat. Commun. 12, 1744 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Tang, Z. et al. Aptamer switch probe based on intramolecular displacement. J. Am. Chem. Soc. 130, 11268–11269 (2008).

    Article  CAS  PubMed  Google Scholar 

  88. Yip, K. M., Fischer, N., Paknia, E., Chari, A. & Stark, H. Atomic-resolution protein structure determination by cryo-EM. Nature 587, 157–161 (2020).

    Article  CAS  PubMed  Google Scholar 

  89. Nakane, T. et al. Single-particle cryo-EM at atomic resolution. Nature 587, 152–156 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Zhang, X. et al. Cryo-EM structure of the mature dengue virus at 3.5-A resolution. Nat. Struct. Mol. Biol. 20, 105–110 (2013).

    Article  PubMed  Google Scholar 

  91. Wadood, A. et al. Epitopes based drug design for dengue virus envelope protein: a computational approach. Comput. Bio. Chem. 71, 152–160 (2017).

    Article  CAS  Google Scholar 

  92. Lin, B., Parrish, C. R., Murray, J. M. & Wright, P. J. Localization of a neutralizing epitope on the envelope protein of dengue virus type 2. Virology 202, 885–890 (1994).

    Article  CAS  PubMed  Google Scholar 

  93. Crill, W. D. & Roehrig, J. T. Monoclonal antibodies that bind to domain III of dengue virus E glycoprotein are the most efficient blockers of virus adsorption to Vero cells. J. Virol. 75, 7769–7773 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Sukupolvi-Petty, S. et al. Type- and subcomplex-specific neutralizing antibodies against domain III of dengue virus type 2 envelope protein recognize adjacent epitopes. J. Virol. 81, 12816–12826 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Gromowski, G. D. & Barrett, A. D. Characterization of an antigenic site that contains a dominant, type-specific neutralization determinant on the envelope protein domain III (ED3) of dengue 2 virus. Virology 366, 349–360 (2007).

    Article  CAS  PubMed  Google Scholar 

  96. Lok, S. M. et al. Binding of a neutralizing antibody to dengue virus alters the arrangement of surface glycoproteins. Nat. Struct. Mol. Biol. 15, 312–317 (2008).

    Article  CAS  PubMed  Google Scholar 

  97. Williams, K. L., Wahala, W. M., Orozco, S., de Silva, A. M. & Harris, E. Antibodies targeting dengue virus envelope domain III are not required for serotype-specific protection or prevention of enhancement in vivo. Virology 429, 12–20 (2012).

    Article  CAS  PubMed  Google Scholar 

  98. Dong, Y. et al. DNA functional materials assembled from branched DNA: design, synthesis, and applications. Chem. Rev. 120, 9420–9481 (2020).

    Article  CAS  PubMed  Google Scholar 

  99. Wang, Y. L., Mueller, J. E., Kemper, B. & Seeman, N. C. Assembly and characterization of five-arm and six-arm DNA branched junctions. Biochemistry 30, 5667–5674 (1991).

    Article  CAS  PubMed  Google Scholar 

  100. Fu, T. J. & Seeman, N. C. DNA double-crossover molecules. Biochemistry 32, 3211–3220 (1993).

    Article  CAS  PubMed  Google Scholar 

  101. Wang, X. & Seeman, N. C. Assembly and characterization of 8-arm and 12-arm DNA branched junctions. J. Am. Chem. Soc. 129, 8169–8176 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Wang, X. et al. Paranemic crossover DNA: there and back again. Chem. Rev. 119, 6273–6289 (2019).

    Article  CAS  PubMed  Google Scholar 

  103. Seeman, N. C. De novo design of sequences for nucleic acid structural engineering. J. Biomol. Struct. Dyn. 8, 573–581 (1990).

    Article  CAS  PubMed  Google Scholar 

  104. He, Y. et al. Hierarchical self-assembly of DNA into symmetric supramolecular polyhedra. Nature 452, 198–201 (2008).

    Article  CAS  PubMed  Google Scholar 

  105. Wang, P. et al. Retrosynthetic analysis-guided breaking tile symmetry for the assembly of complex DNA nanostructures. J. Am. Chem. Soc. 138, 13579–13585 (2016).

    Article  CAS  PubMed  Google Scholar 

  106. Caruthers, M. H. A brief review of DNA and RNA chemical synthesis. Biochem. Soc. Trans. 39, 575–580 (2011).

    Article  CAS  PubMed  Google Scholar 

  107. Roy, S. & Caruthers, M. Synthesis of DNA/RNA and their analogs via phosphoramidite and H-phosphonate chemistries. Molecules 18, 14268–14284 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  108. Hughes, R. A. & Ellington, A. D. Synthetic DNA synthesis and assembly: putting the synthetic in synthetic biology. Cold Spring Harb. Perspect. Biol. 9, a023812 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  109. Shlyakhtenko, L. S., Gall, A. A. & Lyubchenko, Y. L. Mica functionalization for imaging of DNA and protein–DNA complexes with atomic force microscopy. Methods Mol. Biol. 931, 295–312 (2013).

    Article  CAS  PubMed  Google Scholar 

  110. Fosmire, J. A., Hwang, K. & Makino, S. Identification and characterization of a coronavirus packaging signal. J. Virol. 66, 3522–3530 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  111. Kuo, L., Koetzner, C. A. & Masters, P. S. A key role for the carboxy-terminal tail of the murine coronavirus nucleocapsid protein in coordination of genome packaging. Virology 494, 100–107 (2016).

    Article  CAS  PubMed  Google Scholar 

  112. Biosafety in Microbiological and Biomedical Laboratories 6th edn (US Department of Health and Human Services, Centers for Disease Control and Prevention, National Institutes of Health, 2020).

  113. Jonsson, U. et al. Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology. Biotechniques 11, 620–627 (1991).

    CAS  PubMed  Google Scholar 

  114. van de Loosdrecht, A. A., Beelen, R. H., Ossenkoppele, G. J., Broekhoven, M. G. & Langenhuijsen, M. M. A tetrazolium-based colorimetric MTT assay to quantitate human monocyte mediated cytotoxicity against leukemic cells from cell lines and patients with acute myeloid leukemia. J. Immunol. Methods 174, 311–320 (1994).

    Article  PubMed  Google Scholar 

  115. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63 (1983).

    Article  CAS  PubMed  Google Scholar 

  116. Chacon, E., Acosta, D. & Lemasters, J. J. Primary cultures of cardiac myocytes as in vitro models for pharmacological and toxicological assessments. in In Vitro Methods in Pharmaceutical Research 209–223 (Elsevier, 1997).

  117. Patravale, V., Dandekar, P. & Jain, R. Nanoparticulate Drug Delivery: Perspectives on the Transition from Laboratory to Market 123–155 (Elsevier, 2012).

  118. Calisher, C. H., Monath, T. P., Karabatsos, N. & Trent, D. W. Arbovirus subtyping: applications to epidemiologic studies, availability of reagents, and testing services. Am. J. Epidemiol. 114, 619–631 (1981).

    Article  CAS  PubMed  Google Scholar 

  119. Lindsey, H. S., Calisher, C. H. & Mathews, J. H. Serum dilution neutralization test for California group virus identification and serology. J. Clin. Microbiol. 4, 503–510 (1976).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  120. Russell, P. K., Nisalak, A., Sukhavachana, P. & Vivona, S. A plaque reduction test for dengue virus neutralizing antibodies. J. Immunol. 99, 285–290 (1967).

    CAS  PubMed  Google Scholar 

  121. Abou-Karam, M. & Shier, W. T. A simplified plaque reduction assay for antiviral agents from plants. Demonstration of frequent occurrence of antiviral activity in higher plants. J. Nat. Prod. 53, 340–344 (1990).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Preparation of this paper was partially supported by NIH NIAAA (UO1 AA029348) and NIH NIAID (RO1 AI159454) to X.W. The authors thank A. F. Payne for helping with the list of equipment and supplies used in assays to measure DNA star cytotoxicity and plaque reduction test.

Author information

Author notes

  1. These authors contributed equally: Shaokang Ren, Keith Fraser, Lili Kuo.

Authors and Affiliations

  1. Nick Holonyak Jr. Micro and Nanotechnology Laboratory (HMNTL), University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Shaokang Ren, Neha Chauhan, Addison T. Adrian, Paul S. Kwon & Xing Wang

  2. Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Shaokang Ren, Neha Chauhan, Addison T. Adrian & Xing Wang

  3. Department of Biological Science, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA

    Keith Fraser & Robert J. Linhardt

  4. Wadsworth Center, New York State Department of Health, Albany, NY, USA

    Lili Kuo

  5. Centre for Pathogen Diagnostics, DREMES at the University of Illinois at Urbana-Champaign and the Zhejiang University–University of Illinois at Urbana-Champaign Institute, Urbana, IL, USA

    Neha Chauhan, Addison T. Adrian & Xing Wang

  6. Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA

    Fuming Zhang & Robert J. Linhardt

  7. Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA

    Robert J. Linhardt

  8. Carl R. Woese Institute for Genomic Biology (IGB), University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Xing Wang

Authors
  1. Shaokang Ren
    View author publications

    Search author on:PubMed Google Scholar

  2. Keith Fraser
    View author publications

    Search author on:PubMed Google Scholar

  3. Lili Kuo
    View author publications

    Search author on:PubMed Google Scholar

  4. Neha Chauhan
    View author publications

    Search author on:PubMed Google Scholar

  5. Addison T. Adrian
    View author publications

    Search author on:PubMed Google Scholar

  6. Fuming Zhang
    View author publications

    Search author on:PubMed Google Scholar

  7. Robert J. Linhardt
    View author publications

    Search author on:PubMed Google Scholar

  8. Paul S. Kwon
    View author publications

    Search author on:PubMed Google Scholar

  9. Xing Wang
    View author publications

    Search author on:PubMed Google Scholar

Contributions

X.W. conceived and supervised the study and the entire manuscript preparation. K.F. contributed the protocol preparation for the configuration of virus antigen spatial patterns. S.R., and X.W. contributed the protocol preparation for the DNA star design and characterization. N.C., P.S.K. and X.W. contributed the protocol for the DNA oligos purification using denaturing gel electrophoresis. S.R., N.C., A.A. and X.W. contributed the protocol for the purification of a DDN complex. L.K. (with an assist from X.W.) contributed the protocol preparation for purification of DENV. F.Z. and R.J.L. (with an assist from X.W.) contributed the protocol preparation for SPR analysis on the interaction between viral particles and the DNA star complexes. L.K. (with an assist from X.W.) contributed the protocol preparation for the assays of DNA star cytotoxicity and antiviral efficacy. K.F. and X.W. wrote the introduction of the manuscript. X.W. (with assists from S.R. and L.K) wrote the troubleshooting section. R.J.L. and X.W. edited the entire manuscript with the inputs from other authors. S.R., K.F. and L.K. contributed equally to this protocol development.

Corresponding author

Correspondence to Xing Wang.

Ethics declarations

Competing interests

A Patent Cooperation Treaty application (PCT/US2020/033398) has been filed on DNA stars by X.W., R.J.L. and P.S.K.

Additional information

Peer review information Nature Protocols thanks Veikko Linko, Chuanxiong Nie and Wei Tao for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Kwon, P. S. et al. Nat. Chem. 12, 26–35 (2020): https://doi.org/10.1038/s41557-019-0369-8

Kwon, S. J. et al. Nat. Nanotechnol. 12, 48–54 (2017): https://doi.org/10.1038/nnano.2016.181

Supplementary information

Supplementary Information

Supplementary Tables 1–3 and Supplementary Figs. 1–4.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ren, S., Fraser, K., Kuo, L. et al. Designer DNA nanostructures for viral inhibition. Nat Protoc 17, 282–326 (2022). https://doi.org/10.1038/s41596-021-00641-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41596-021-00641-y

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing