biology

DNA Origami Nanovaccine Demonstrates Full Protection Against SARS-CoV-2 in Mice

A study recently published in Communications Biology (Nature Portfolio) by Esra Oktay, Farhang Alem, Keziah Hernandez, Michael Girgis, Christopher Green, Divita Mathur, Igor L. Medintz, Aarthi Narayanan, and Rémí Veneziano introduces an innovative DNA origami–based nanovaccine platform targeting the receptor-binding domain (RBD) of SARS-CoV-2. Their findings highlight the potential of rationally engineered DNA nanoparticles to elicit strong and durable immune protection.

The researchers designed wireframe DNA origami nanoparticles (DNA-NPs) functionalized with ten trimerized RBD antigens and CpG oligonucleotides as adjuvants. Trimer assembly was achieved by peptide nucleic acid (PNA)–mediated conjugation of protein G to RBD-Fc, enabling precise orientation on DNA-NP overhangs. Biophysical characterization employed dynamic light scattering, Förster resonance energy transfer (FRET)–based stability assays, and quantitative fluorescence labeling. Structural confirmation was obtained via atomic force microscopy (AFM). For high-resolution AFM imaging, the team employed NanoWorld® USC-F0.3-k0.3 ultra-short cantilevers, chosen for their 300 kHz resonance frequency, ~0.3 N/m force constant, and <10 nm tip radius. These probes provided enhanced stability and resolution, enabling accurate nanoscale visualization of the vaccine constructs. In murine models, the fully assembled RBD-DNA-NP with CpG provided complete protection following viral challenge. Vaccinated mice exhibited no weight loss or mortality, in stark contrast to control groups that received unconjugated components, bare origami scaffolds, or RBD alone—several of which experienced significant morbidity or mortality. Immunological assays revealed sustained neutralizing antibody activity two months post-immunization, with robust titers of RBD-specific IgG, IgM, and IgA, underscoring the durability of the immune response. This work demonstrates that DNA origami nanostructures can serve as an adaptable and effective vaccine delivery platform. By integrating precise antigen display with adjuvant loading, the approach provides a promising blueprint for next-generation nanovaccines. The use of advanced AFM probes, such as the NanoWorld® USC-F0.3-k0.3, further underscores the importance of high-precision nanometrology tools in validating nanoscale biomedical designs.
a Hydrodynamic diameter measurement of DNA-NPs with DLS. b Atomic force microscopy imaging of DNA-NPs formation and conjugation with 3-mer RBDs (scale bar: 20 nm). c Fluorescence-intensity based determination of PG and RBD stoichiometry on DNA origami NP. i) Tryptophan fluorescence emission was used to determine the total number of PG loaded on the surface of the DNA-NPs. ii) Second, the stoichiometry of RBD on NP was quantified via measuring the emission of Cy5 dyes conjugated to the RBD antigens. The bar graph represents the total coverage percentage for the PG (green bar) and the 3-mer RBD (orange bar) on the DNA-NP surface normalized to the number of conjugation sites available. Data are shown as the mean ± SD (n = 3 independent experiments).

This article contains images reused from Jia et al., Nature Communications 14, 1394 (2023), licensed under CC BY 4.0.

Mechanism for Vipp 1 spiral formation, ring biogenesis, and membrane repair

The ESCRT-III-like protein Vipp1 couples filament polymerization with membrane remodeling. It assembles planar sheets as well as 3D rings and helical polymers, all implicated in mitigating plastid-associated membrane stress. The architecture of Vipp1 planar sheets and helical polymers remains unknown, as do the geometric changes required to transition between polymeric forms. *

In the article “Mechanism for Vipp1 spiral formation, ring biogenesis, and membrane repair” Souvik Naskar, Andrea Merino, Javier Espadas, Jayanti Singh, Aurelien Roux, Adai Colom and Harry H. Low show how cyanobacterial Vipp1 assembles into morphologically-related sheets and spirals on membranes in vitro.*

The spirals converge to form a central ring similar to those described in membrane budding. Cryo-EM structures of helical filaments reveal a close geometric relationship between Vipp1 helical and planar lattices. Moreover, the helical structures reveal how filaments twist—a process required for Vipp1, and likely other ESCRT-III filaments, to transition between planar and 3D architectures. *

Overall, the authors’ results provide a molecular model for Vipp1 ring biogenesis and a mechanism for Vipp1 membrane stabilization and repair, with implications for other ESCRT-III systems. *

NanoWorld Ultra-Short Cantilevers USC-F0.3-k0.3 for High-Speed AFM (HS-AFM) with a typical spring constant of 0.3 N nm−1 and a typical resonance frequency of about 300 kHz were used for image acquisition with fast scanning atomic force microscopy.*

Fig. 2 from Souvik Naskar et al. 2024 “Mechanism for Vipp1 spiral formation, ring biogenesis, and membrane repair”:Vipp1 assembles dynamic networks of spirals, rings and sheets on membrane a, F-AFM phase timecourse showing Vipp1 recruitment to the highly curved edge of membrane patches. Scan rate, 70 Hz; 256 × 256 pixels. The area in the dashed box is enlarged in b. b, Spiral and ring formation localized to the membrane edge. Scan rate, 70 Hz; 256 × 256 pixels. c, Left, phase timecourse showcasing a dense network of sheets, spirals, and rings that ultimately cover the entire membrane plane. Right, average of six F-AFM height images. Scan rate, 120 Hz; 256 × 256 pixels. d, Average F-AFM height image showing Vipp1 sheet, spiral, and ring detail. Red arrows mark the sheet branching into filaments ~13 nm wide. Scan rate, 20 Hz; 256 × 256 pixels. e, Vipp1 sheet and spiral filament height offset from the membrane. f–i, Quantification of Vipp1 filament and spiral characteristics. n = 124, 13, 278, and 278 independent measurements for panels f, g, h, and i, respectively. Error bars show one s.d. of the mean. NanoWorld Ultra-Short Cantilevers USC-F0.3-k0.3-10 with a typical spring constant of 0.3 N nm−1 and a typical resonance frequency of about 300 kHz were used for image acquisition. — Fig. 2 from Souvik Naskar et al. 2024 “Mechanism for Vipp1 spiral formation, ring biogenesis, and membrane repair”:
Vipp1 assembles dynamic networks of spirals, rings and sheets on membrane
a, F-AFM phase timecourse showing Vipp1 recruitment to the highly curved edge of membrane patches. Scan rate, 70 Hz; 256 × 256 pixels. The area in the dashed box is enlarged in b. b, Spiral and ring formation localized to the membrane edge. Scan rate, 70 Hz; 256 × 256 pixels. c, Left, phase timecourse showcasing a dense network of sheets, spirals, and rings that ultimately cover the entire membrane plane. Right, average of six F-AFM height images. Scan rate, 120 Hz; 256 × 256 pixels. d, Average F-AFM height image showing Vipp1 sheet, spiral, and ring detail. Red arrows mark the sheet branching into filaments ~13 nm wide. Scan rate, 20 Hz; 256 × 256 pixels. e, Vipp1 sheet and spiral filament height offset from the membrane. f–i, Quantification of Vipp1 filament and spiral characteristics. n = 124, 13, 278, and 278 independent measurements for panels f, g, h, and i, respectively. Error bars show one s.d. of the mean.

*Souvik Naskar, Andrea Merino, Javier Espadas, Jayanti Singh, Aurelien Roux, Adai Colom and Harry H. Low
Mechanism for Vipp1 spiral formation, ring biogenesis, and membrane repair
Nature Structural & Molecular Biology (2024)
DOI: https://doi.org/10.1038/s41594-024-01401-8

Open Access The article “Mechanism for Vipp1 spiral formation, ring biogenesis, and membrane repair” by Souvik Naskar, Andrea Merino, Javier Espadas, Jayanti Singh, Aurelien Roux, Adai Colom and Harry H. Low is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Real-time multistep asymmetrical disassembly of nucleosomes and chromatosomes visualized by high-speed atomic force microscopy

During replication, expression, and repair of the eukaryotic genome, cellular machinery must access the DNA wrapped around histone proteins forming nucleosomes. These octameric protein·DNA complexes are modular, dynamic, and flexible and unwrap or disassemble either spontaneously or by the action of molecular motors. Thus, the mechanism of formation and regulation of subnucleosomal intermediates has gained attention genome-wide because it controls DNA accessibility.*

In the article “Real-Time Multistep Asymmetrical Disassembly of Nucleosomes and Chromatosomes Visualized by High-Speed Atomic Force Microscopy” Bibiana Onoa, César Díaz-Celis, Cristhian Cañari-Chumpitaz, Antony Lee and Carlos Bustamante describe how they imaged nucleosomes and their more compacted structure with the linker histone H1 (chromatosomes) using high-speed atomic force microscopy to visualize simultaneously the changes in the DNA and the histone core during their disassembly when deposited on mica.*

Furthermore, Bibiana Onoa et al. trained a neural network and developed an automatic algorithm to track molecular structural changes in real time. *

The authors’ results show that nucleosome disassembly is a sequential process involving asymmetrical stepwise dimer ejection events. The presence of H1 restricts DNA unwrapping, significantly increases the nucleosomal lifetime, and affects the pathway in which heterodimer asymmetrical dissociation occurs. *

Bibiana Onoa et al. observe that tetrasomes are resilient to disassembly and that the tetramer core (H3·H4)2 can diffuse along the nucleosome positioning sequence. Tetrasome mobility might be critical to the proper assembly of nucleosomes and can be relevant during nucleosomal transcription, as tetrasomes survive RNA polymerase passage. These findings are relevant to understanding nucleosome intrinsic dynamics and their modification by DNA-processing enzymes. *

To characterize the nucleosomes dynamics in 2D, individual molecules were observed in buffer using an Ando-type high speed atomic force microscope together with NanoWorld Ultra-Short Cantilevers for HS-AFM of the USC-F1.2-K0.15 AFM probe type ( typical spring constant 0.15 N/m, typical resonance frequency in air 1200 kHz, resonance frequency 500–600 kHz in liquid). *

The AFM data presented in the article allow the authors to directly visualize the dynamics of DNA and histones during nucleosome and chromatosome disassembly, providing a simultaneous observation of DNA unwrapping and histone dissociation. *

The experimental and analytical strategy presented shows that real-time HS-AFM is a robust and powerful tool for studying single nucleosomes and chromatin dynamics. *

graphical abstract from Bibiana Onoa et al 2024 "Real-Time Multistep Asymmetrical Disassembly of Nucleosomes and Chromatosomes Visualized by High-Speed Atomic Force Microscopy" - NanoWorld Ultra-Short Cantilevers of the USC-F1.2-k0.15 AFM probe type were used for the high-speed atomic force microscopy — graphical abstract from Bibiana Onoa et al 2024 “Real-Time Multistep Asymmetrical Disassembly of Nucleosomes and Chromatosomes Visualized by High-Speed Atomic Force Microscopy”

*Bibiana Onoa, César Díaz-Celis, Cristhian Cañari-Chumpitaz, Antony Lee and Carlos Bustamante
Real-Time Multistep Asymmetrical Disassembly of Nucleosomes and Chromatosomes Visualized by High-Speed Atomic Force Microscopy
ACS Central Science 2024, 10, 1, 122–137
DOI: https://doi.org/10.1021/acscentsci.3c00735

Open Access The article “Real-Time Multistep Asymmetrical Disassembly of Nucleosomes and Chromatosomes Visualized by High-Speed Atomic Force Microscopy” by Bibiana Onoa, César Díaz-Celis, Cristhian Cañari-Chumpitaz, Antony Lee and Carlos Bustamante is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.