The binding between the SARS-CoV-2 Spike protein and its cellular receptor ACE2 is essential for viral entry and infection and, therefore, represents a critical target for antiviral intervention. Single-molecule force spectroscopy (SMFS), conducted using atomic force microscopy (AFM), allows direct investigation of Spike–ACE2 interactions at the single-molecule level, providing insights into binding mechanisms not easily captured by conventional methods. This review discusses recent applications of SMFS to assess molecular inhibitors of Spike–ACE2 interactions, focusing specifically on soluble ACE2 and glycan-binding lectins (Clec4g, hCLEC4G, and engineered H84T-Banlec). These inhibitors demonstrated potent, concentration-dependent activity against multiple SARS-CoV-2 variants, including Delta and Omicron, achieving IC₅₀ values in the low nanomolar range (soluble ACE2: 0.25–0.38 nM; Clec4g/hCLEC4G: 36–58 nM; H84T-Banlec: 2.6–5.2 nM). Notably, single-molecule IC₅₀ measurements closely correlated with EC₅₀ values from cell-based assays, validating the physiological relevance and predictive capability of the SMFS method. Collectively, these findings underscore the utility of AFM-SMFS as a powerful approach for inhibitor evaluation and antiviral discovery, particularly for strategies targeting conserved structural features of the Spike protein to achieve broad-spectrum efficacy against current and emerging viral variants.

Conformational plasticity allows class I viral fusion proteins, including the SARS-CoV-2 spike, to undergo major structural rearrangements that support receptor binding and membrane fusion, making them key vaccine and antiviral targets. Using high-speed atomic force microscopy (AFM) and single-molecule force spectroscopy, we analyzed the ancestral and nine spike variants and found an evolutionary trend from increased flexibility in early strains to greater rigidification beginning with Delta, fluctuating plasticity in Omicron sublineages, and pronounced compaction in JN.1. Delta favored multivalent ACE2 engagement through its three receptor-binding domains, whereas later Omicron variants shifted toward predominantly single-site interactions. This change was compensated by stronger RBD-ACE2 bond stability. Overall, spike evolution appears to balance openness and compaction to regulate receptor accessibility, mobility, and immune escape while preserving or enhancing entry efficiency. These single-molecule measurements define functional constraints on viral adaptation and may inform prediction of future variants and design of improved countermeasures.

In this Comment, we direct attention to initial efforts to establish a high-quality databank of atomic force microscopy (AFM) data: bioAFM-DB. We outline the state of this endeavor, its challenges, and potential courses of action.

The dopamine transporter (DAT) is linked to neuropsychiatric disorders including ADHD, Parkinson's disease, and substance use disorders. Accordingly, DAT is the target of illicit drugs and clinically important medicines. However, the number and function of ligand binding sites in DAT is enigmatic due to conflicting data from available structures and molecular pharmacology. Herein, we design force sensors with DAT ligands and measure their interaction forces with wild-type and mutated DATs, from which two distinct populations of unbinding strengths and off-rates are detected. The high-force population is reduced by V152I and S422A mutations, or by substituting Na+ with K+ or NMDG+. In contrast, several modifications including mutation G386H, acetylation of K92 and K384, mutation K92A, mutation K384A, or protonation of H477 decrease the low-force population. The present data delineate the threshold of binding strength, which may account for certain ligand binding sites to be imperceptible in crystal or cryo-EM structures. Furthermore, the force spectra provide the information on the position and kinetic rates of a herein detected ligand binding site in DAT.

Monoclonal antibodies and ligands targeting CD40 exhibit a wide range of agonistic activities and antitumor responses. Studies have shown that the flexibility and affinity of antibodies play a crucial role in their immunostimulatory activity. However, a systematic comparison with the natural ligand is yet missing and a detailed investigation with respect to molecular rigidity, binding kinetics, and bond lifetime has not been undertaken to date. Here, we study the dynamic binding features of clinically relevant anti-hCD40 antibody subclasses, ChiLob 7/4, and the trimeric human CD40L to hCD40 at the single-molecule level. We visualize resembling of hCD40 receptors into dimers and higher-order oligomers that are dynamically captured and released by both ChiLob 7/4 and hCD40L with their multiple binding sites. Thereby, ChiLob 7/4 acts as a nanomechanical calliper and rotates its Fab arms in a highly dynamic fashion to screen for hCD40 binding, while hCD40L undergoes significantly less conformational changes. Despite its minor molecular flexibility, hCD40L performs association, dissociation, and re-association of hCD40 ten times faster when compared to ChiLob 7/4. We uncover a distinct binding mechanism that may explain the enhanced cluster formation potential and agonistic activity of the natural ligand and will inspire the design of novel ligand formats.

This study explores the cross-fertilization of transgenic tobacco plants to produce dual-specific monoclonal antibodies (mAbs) targeting Ebola virus–like particles and HER2 proteins. We generated F1 plants by hybridizing individual transgenic lines expressing the anti-HER2 breast cancer VHH mAb (HV) and the H-13F6 human anti-Ebola large single chain mAb (EL). Hybridizing transgenic plants to express dual-antibodies between different structures VHH and LSCK indicate the potential of transgenic plants as a cost-effective and scalable production system for dual targeting mAbs. We performed polymerase chain reaction (PCR) analysis to confirm the integration of EL and HV genes in the F1 progeny. The reverse-transcription (RT)-PCR and immunoblotting were performed to confirm the expression of transgenes. Indirect enzyme-linked immunosorbent assay was conducted to confirm the functionality of purified EL and HV mAb. A PCR analysis confirmed the successful integration of both EL and HV mAb genes in the F1 progeny. Additionally, (RT)-PCR and immunoblotting validated the expression of these transgenes, with EL and HV mAbs purified from the F1 plants. Indirect enzyme-linked immunosorbent assay (ELISA) demonstrated that EL × HV mAb proteins maintained binding activity to Ebola virus–specific antigens, comparable to that of the EL mAb protein, while also exhibiting binding activity against HER2 proteins similar to that of the HV mAb. This study indicates the potential for transgenic plants to produce dually targeting mAbs, suggesting a promising application in enabling the co-expression of antibodies targeting two different diseases in a single plant.

Uniaxial tensile testing and atomic force microscopy (AFM) nanoindentation experiments are two valuable methods used to quantify changes in stiffness after corneal crosslinking (CXL). Here, we apply these methods by characterizing corneal stiffness ex vivo before and after CXL. Sixty-two fresh porcine corneas were divided into three groups: an untreated control group, a CXL3 group treated with the Dresden protocol, and a CXL9 group treated with the accelerated protocol. Biomechanical testing was then performed using either uniaxial tensile testing or AFM nanoindentation. Uniaxial tensile testing revealed a significant increase in corneal stiffness for the CXL3 group compared to the control group (p < 0.05). At 10% strain, the CXL3 and CXL9 groups exhibited increases in stiffness of 96% and 48%, respectively, compared to the control group. In contrast, AFM analysis revealed no significant differences in stiffness, showing 28% and 16% increases in the CXL3 and CXL9 groups, respectively, compared to the control group. The results suggest that uniaxial tensile testing provides a robust, sample-averaged measure of global stiffening. Interestingly, AFM nanoindentation enables mapping of localized biomechanical changes with high spatial resolution but is less sensitive to overall biomechanical changes induced by CXL.

The capability for varied functionalization and precise control at the nanoscale are significant advantages of DNA nanostructures. In the assembly of DNA nanostructure, the surface-assisted growth method utilizing double-crossover (DX) tile structures facilitates nucleation at relatively low concentrations on the surface based on electrostatic interactions, thereby enabling crystal growth over large areas. However, in surface-assisted growth, the geometrical hindrance of vertical structures on the DX tile structure surface makes it challenging to conjugate DNA nanostructures into fabricated surfaces. Here, the surface-assisted growth method was employed to extend the DX tile growth for forming vertical structure arrays on the substrate, providing attachment sites for functionalization on uniformly covered substrates at the macroscopic scale. Additionally, the spacing of the vertical structure arrays was demonstrated to be controllable through the strategic design of the repeating unit tiles that construct the DX crystals.

Current cancer treatments include surgery, chemotherapy, radiation therapy, hormone therapy, and targeted therapy. However, challenges such as recurrence, metastasis, drug side effects, and resistance were still remained. Immunotherapy, particularly antibody therapy, offers new potential in cancer treatment. The biopharmaceutical sector, which includes antibody-based therapeutics, is one of the fastest-growing areas of the pharmaceutical market, with annual sales already exceeding 100 billion. Mammalian cells are commonly used to produce monoclonal antibodies (mAbs), but plant-based expression systems offer several advantages. Although the capacity to target and manipulate plant-expressed proteins is still in its early stages, the use of leucine-rich repeats (LRRs) shows promising potential. LRRs, which mediate protein–protein interactions, provide binding specificity to variable lymphocyte receptor (VLR) antibodies in jawless vertebrates, functioning similarly to immunoglobulin-based antibodies in jawed vertebrates. In the future, VLRs could be used in plants to bind specific proteins or carbohydrates, inhibit their functions, or create chimeric proteins with novel specificities by replacing endogenous LRR domains with those from VLRs. This paper discusses recent advances in plant expression systems for the production of therapeutic recombinant proteins, such as antibodies and vaccines.

Epithelial cell adhesion molecule (EpCAM) fused to IgG, IgA and IgM Fc domains was expressed to create IgG, IgA and IgM-like structures as anti-cancer vaccines in Nicotiana tabacum. High-mannose glycan structures were generated by adding a C-terminal endoplasmic reticulum (ER) retention motif (KDEL) to the Fc domain (FcK) to produce EpCAM-Fc and EpCAM-FcK proteins in transgenic plants via Agrobacterium-mediated transformation. Cross-fertilization of EpCAM-Fc (FcK) transgenic plants with Joining chain (J-chain, J and JK) transgenic plants led to stable expression of large quaternary EpCAM-IgA Fc (EpCAM-A) and IgM-like (EpCAM-M) proteins. Immunoblotting, SDS-PAGE and ELISA analyses demonstrated that proteins with KDEL had higher expression levels and binding activity to anti-EpCAM IgGs. IgM showed the strongest binding among the fusion proteins, followed by IgA and IgG. Sera from BALB/c mice immunized with these vaccines produced anti-EpCAM IgGs. Flow cytometry indicated that the EpCAM-Fc fusion proteins significantly activated CD8+ cytotoxic T cells, CD4+ helper T cells and B cells, particularly with EpCAM-FcKP and EpCAM-FcP (FcKP) × JP (JKP). The induced anti-EpCAM IgGs captured human prostate cancer PC-3 and colorectal cancer SW620 cells. Sera from immunized mice inhibited cancer cell proliferation, migration and invasion; down-regulated proliferation markers (PCNA, Ki-67) and epithelial-mesenchymal transition markers (Vimentin); and up-regulated E-cadherin. These findings suggest that N. tabacum can produce effective vaccine candidates to induce anti-cancer immune responses.

Ultraviolet-C (UV-C) radiation and ozone gas are potential mechanisms employed to inactivate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), each exhibiting distinct molecular-level modalities of action. To elucidate these disparities and deepen our understanding, we delve into the intricacies of SARS-CoV-2 inactivation via UV-C and ozone gas treatments, exploring their distinct molecular-level impacts utilizing a suite of advanced techniques, including biological atomic force microscopy (Bio-AFM) and single virus force spectroscopy (SVFS). Whereas UV-C exhibited no perceivable alterations in virus size or surface topography, ozone gas treatment elucidated pronounced changes in both parameters, intensifying with prolonged exposure. Furthermore, a nuanced difference was observed in virus-host cell binding post-treatment: ozone gas distinctly reduced SARS-CoV-2 binding to host cells, while UV-C maintained the status quo. The results derived from these methodical explorations underscore the pivotal role of advanced Bio-AFM techniques and SVFS in enhancing our understanding of virus inactivation mechanisms, offering invaluable insights for future research and applications in viral contamination mitigation.

This study explored the application of tobacco plant expression system for production of dual target monoclonal (mAb) proteins and computational analysis to evaluate their biochemical characteristics. The murine anti-colorectal cancer large single-chain antibody (CL) and H-13F6 human anti-Ebola virus full-size monoclonal antibody (EF) or anti-Ebola large single-chain antibody (EL) co-expressed in F1 plant through crossbreeding transgenic plants expressing each specific antibody. The binding activities of these antibodies to their respective target antigens were then examined. PCR analysis confirmed the presence of the CL gene and the heavy (HC) and light chain (LC) genes of EF in F1 plants resulting from the crossbreeding. Additionally, both CL and EL genes were identified in F1 plants produced through the crossbreeding of transgenic plants expressing each CL and EL. RT-PCR and immunoblot analyses confirmed the mRNA and protein expression of all transgenes in F1 plants. CL, EF, and EL were successfully purified from F1 plants. Indirect ELISA analysis revealed that CL × EF proteins exhibited binding activity to Ebola virus-specific antigenic protein, whereas CL × EL lost this binding activity. Conversely, both CL × EF and CL × EL proteins exhibited enhanced binding activity to the colorectal cancer-specific antigenic protein GA733. In addition, computational analyses [Molecular Dynamics (MD) and Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA) approach] were performed for EBOV antigen-antibody complexes and possible three combinations of Fc consistent with experimental results, supporting our assumption and interpretation.

The production of recombinant glycoproteins is pivotal for numerous therapies targeting human diseases, forming a cornerstone of the medical industry. The baculovirus-insect cell system has emerged as a leading method for producing high-value therapeutic glycoproteins. This system has several advantages, including rapid cultivation of insect cells, high production yields, and relatively straightforward post-translational processing. Insect cells, being eukaryotic, have functional glycosylation pathways, offering a significant advantage over prokaryotic cells which lack these capabilities. These advantages are particularly promising for the expression and testing of post-translational processing in de novo proteins, which are newly developed and extensively researched through AI-based computational methods. However, challenges remain, particularly with expression levels and glycosylation modifications, which need to be addressed to optimize protein production. This review aims to provide a comprehensive overview of recent advancements in the baculovirus-insect cell expression system, highlighting progress in insect cell utilization, expression optimization, glycosylation modifications, and the production of therapeutic proteins.

Traditional monoclonal antibodies such as Trastuzumab encounter limitations when treating Human Epidermal Growth Factor Receptor 2 (HER2)-positive breast cancer, particularly in cases that develop resistance. This study introduces plant-derived anti-HER2 variable fragments of camelid heavy chain domain (VHH) fragment crystallizable region (Fc) KEDL(K) antibody as a potent alternative for overcoming these limitations. A variety of biophysical techniques, in vitro assays, and in vivo experiments uncover the antibody's nanoscale binding dynamics with transmembrane HER2 on living cells. Single-molecule force spectroscopy reveals the rapid formation of two robust bonds, exhibiting approximately 50 pN force resistance and bond lifetimes in the second range. The antibody demonstrates a specific affinity for HER2-positive breast cancer cells, including those that are Trastuzumab-resistant. Moreover, in immune-deficient mice, the plant-derived anti-HER2 VHH-FcK antibody exhibits superior antitumor activity, especially against tumors that are resistant to Trastuzumab. These findings underscore the plant-derived antibody's potential as an impactful immunotherapeutic strategy for treating Trastuzumab-resistant HER2-positive breast cancer.

Transgenic tobacco plant expressed EpCAM-Fc fusion proteins to induce in vivo immune responses producing anti-EpCAM antibodies inhibiting human colorectal cancer cell invasion and migration. Plant is emerging as a promising alternative to produce valuable immunotherapeutic vaccines. In this study, we examined the in vivo anti-cancer efficacy of epidermal cell adhesion molecule (EpCAM)-Fc and EpCAM-FcK fusion proteins produced in transgenic plants as colorectal cancer vaccine candidates. Mice were injected with plant-derived EpCAM-Fc (EpCAM-FcP) and EpCAM-FcP tagged with KDEL (ER retention signal) (EpCAM-FcKP), using mammalian-derived EpCAM-Fc (EpCAM-FcM) as positive control. Total IgGs from the immunized mice were used to assess immune responses. ELISA tests revealed that IgGs from mice immunized with EpCAM-FcKP (EpCAM-FcKP IgG) exhibited the highest absorbance value for binding affinity to recombinant EpCAM-FcM compared to IgGs from mice immunized with EpCAM-FcP (EpCAM-FcP IgG) and EpCAM-FcM (EpCAM-FcM IgG). Bio-layer interferometry revealed that EpCAM-FcKP IgG had a higher affinity value than EpCAM-FcM IgG and EpCAM-FcP IgG. Cell ELISA revealed that EpCAM-FcKP IgG exhibited the highest binding activity to EpCAM-positive cells SW480 and SW620 compared to EpCAM-FcP IgG, EpCAM-FcM IgG, and anti-EpCAM mAb. In the transwell invasion assay, EpCAM-FcKP IgG significantly decreased the numbers of invaded SW480 and SW620 cells compared to EpCAM-FcP IgG, whereas EpCAM-FcM IgG had similar numbers. In the wound healing assay, EpCAM-FcKP IgG showed higher migration inhibition compared to EpCAM-FcP IgG in both cell types, with similar results to EpCAM-FcM IgG in SW620 cells. These results confirm the applicability of plant systems to produce EpCAM-Fc vaccine candidates, inducing the production of anti-EpCAM IgGs against colorectal cancer cells.

Production of therapeutic monoclonal antibody (mAb) in transgenic plants has several advantages such as large-scale production and the absence of pathogenic animal contaminants. However, mAb with high mannose (HM) type glycans has shown a faster clearance compared to antibodies produced in animal cells. The neonatal Fc receptor (FcRn) regulates the persistence of immunoglobulin G (IgG) by the FcRn-mediated recycling pathway, which salvages IgG from lysosomal degradation within cells. In this study, Fc-engineering of antirabies virus therapeutic mAb SO57 with the endoplasmic reticulum (ER)-retention peptide signal (Lys-Asp-Glu-Leu; KDEL) (mAbpK SO57) in plant cell was conducted to enhance its binding activity to human neonatal Fc receptor (hFcRn), consequently improve its serum half-life. Enzyme-linked immunosorbent assay (ELISA) and Surface plasmon resonance assay showed altered binding affinity of the Fc region of three different mAbpK SO57 variants [M252Y/S254T/T256E (MST), M428L/N434S (MN), H433K/N434F (HN)] to hFcRn compared to wild type (WT) of mAbpK SO57. Molecular modeling data visualized the structural alterations in these mAbpK SO57. All of the mAbpK SO57 variants had HM type glycan structures similar to the WT mAbpK SO57. In addition, the neutralizing activity of the three variants against the rabies virus CVS-11 was effective as the WT mAbpK SO57. These results indicate that the binding affinity of mAbpK SO57 variants to hFcRn can be modified without alteration of N-glycan structure and neutralization activity. Taken together, this study suggests that Fc-engineering of antirabies virus mAb can be applied to enhance the efficacy of therapeutic mAbs in plant expression systems.

The binding of ligands to receptors within a nanoscale small space is relevant in biology, biosensing, and affinity filtration. Binding in confinement can be studied with biological systems but under the limitation that essential parameters cannot be easily controlled including receptor type and position within the confinement and its dimensions. Here we study molecular recognition with a synthetic confined nanopore with controllable pore dimension and molecular DNA receptors at different depth positions within the channel. Binding of a complementary DNA strand is studied at the single-molecule level with atomic force microscopy. Following the analysis, kinetic association rates are lower for receptors positioned deeper inside the pore lumen while dissociation is faster and requires less force. The phenomena are explained by the steric constraints on molecular interactions in confinement. Our study is the first to explore recognition in DNA nanostructures with atomic force microscopy and lays out new tools to further quantify the effect of nanoconfinement on molecular interactions.