Engineering Versatile Two-Dimensional Nanobody-Origami Architectures for Enhanced Antiviral Activity

Considering the serious global health burden posed by pathogenic viruses, the development of effective antiviral molecules and therapeutic strategies is critical for improving human health. Designing and synthesizing a versatile and biocompatible molecular platform offering neutralization of both coronaviruses and retroviruses, two virus families that are greatly problematic to the human population, remains a significant challenge. Here, we report a programmable and versatile platform built on a two-dimensional (2D) DNA origami that enables nanoscale spatial control of multivalent nanobody (Nb) patterns for a broad-spectrum antiviral application. By site-selectively conjugating a Nb to a DNA oligonucleotide and placing multiple of them to predefined sites on 2D DNA origami, we synthesized hybrid nano-architectures with tunable Nb patterns designed to approximately match the geometric presentation of viral surface proteins. We demonstrate that such specific Nb spatial configurations significantly enhance both viral binding affinity and neutralization potency. For SARS-CoV-2, a coronavirus, a triangular Nb pattern with matched spacing with the spike proteins achieved an IC ₅₀ of 1.52 nM, representing a 171-fold improvement over monomeric Nbs. Extending this strategy to a retroviral virus, Human Immunodeficiency Virus (HIV) by utilizing a gp120 spike-binding Nb, we observed a 233-fold increase in neutralization efficiency using a patterned 2D Nb nano-architecture. These findings suggest a generalizable and versatile platform strategy for engineering potent antiviral agents through spatially optimized Nb presentation for a corresponding viral pathogen, offering a promising avenue for future antibody and Nb-based drug development.