Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused devastation to human society through its high virulence, infectivity, and genomic mutations, which reduced the efficacy of vaccines. Here, we report the development of aptamers that effectively interfere with SARS-CoV-2 infection by targeting its spike protein, which plays a pivotal role in host cell entry of the virus through interaction with the viral receptor angiotensin-converting enzyme 2 (ACE2). To develop highly effective aptamers and to understand their mechanism in inhibiting viral infection, we determined the three-dimensional (3D) structures of aptamer/receptor-binding domain (RBD) complexes using cryogenic electron microscopy (cryo-EM). Moreover, we developed bivalent aptamers targeting two distinct regions of the RBD in the spike protein that directly interact with ACE2. One aptamer interferes with the binding of ACE2 by blocking the ACE2-binding site in RBD, and the other aptamer allosterically inhibits ACE2 by binding to a distinct face of RBD. Using the 3D structures of aptamer–RBD complexes, we minimized and optimized these aptamers. By combining the optimized aptamers, we developed a bivalent aptamer that showed a stronger inhibitory effect on virus infection than the component aptamers. This study confirms that the structure-based aptamer-design approach has a high potential in developing antiviral drugs against SARS-CoV-2 and other viruses.

The sensing responses of SARS-CoV-2 spike protein using top-down-fabricated Si-based electrolyte-gated transistors (EGTs) have been investigated. An aptamer was employed as a receptor for the SARS-CoV-2 spike protein. The EGT demonstrated excellent intrinsic characteristics and higher sensitivity in the subthreshold regime compared to the linear regime. The limit of detection (LOD) was achieved as low as 0.94 pg/mL and 20 pg/mL for the current and voltage sensitivity, respectively. To analyze the sensing responses of EGT in detecting the aptamer–SARS-CoV-2 spike protein conjugate, a lumped-capacitive model with the presence of an effective dipole potential and an effective capacitance of the functionalized layer component was employed. The aptamer-functionalized EGT showed high sensitivity even in 10 mM phosphate-buffered saline (PBS) solution. These results suggest that Si-based EGTs are a highly promising method for detecting SARS-CoV-2 spike proteins.

The emergence of numerous SARS-CoV-2 variants, characterized by mutations in the viral RNA genome and target proteins, has presented challenges for accurate COVID-19 diagnosis. To address this, we developed universal aptamer probes capable of binding to the spike proteins of SARS-CoV-2 variants, including highly mutated strains like Omicron. These aptamers were identified through protein-based SELEX using spike proteins from three key variants (D614G-substituted Wuhan-Hu-1, Delta, and Omicron) and virus-based SELEX, known as viro-SELEX. Leveraging these universal aptamers, we created a highly sensitive lateral flow assay (LFA) and an ultra-sensitive molecular diagnostic platform that integrates a novel rapid PCR technique, enabling fast and reliable detection across all SARS-CoV-2 variants.