A highly specific aptamer for the SARS-CoV-2 spike protein from the authentic strain.

DNA aptamers are oligonucleotides that specifically bind to target molecules, similar to how antibodies bind to antigens. We identified an aptamer named MEZ that is highly specific to the receptor-binding domain, RBD, of the SARS-CoV-2 spike protein from the Wuhan-Hu-1 strain. The SELEX procedure was utilized to enrich the initial 31-mer oligonucleotide library with the target aptamer. The aptamer identification was performed using the novel protocol based on nanopore sequencing developed in this study. The MEZ aptamer was chemically synthesized and tested for binding with the SARS-CoV-2 RBD of the spike protein from different strains. The Kd is 6.5 nM for the complex with the RBD from the Wuhan-Hu-1 strain, which is comparable with known aptamers; the advantage is that the MEZ aptamer is smaller than known analogs. The proposed aptamer is highly selective for the RBD protein from the Wuhan-Hu-1 strain and does not form complexes with the RBD from Beta, Delta and Omicron strains. Experimental and theoretical studies together revealed the molecular mechanism of aptamer binding. The aptamer occupies the same binding site as ACE2 when bound to the RBD. The 3'-end of the MEZ aptamer is important for complex formation and is responsible for the discrimination of the RBD protein from a specific strain. The 5'-end is responsible for the formation of a loop in the 3D structure of the aptamer, which is important for proper binding.

Nanoisland SERS-Substrates for Specific Detection and Quantification of Influenza A Virus.

Surface-enhanced Raman spectroscopy (SERS)-based aptasensors for virus determination have attracted a lot of interest recently. This approach provides both specificity due to an aptamer component and a low limit of detection due to signal enhancement by a SERS substrate. The most successful SERS-based aptasensors have a limit of detection (LoD) of 10-100 viral particles per mL (VP/mL) that is advantageous compared to polymerase chain reactions. These characteristics of the sensors require the use of complex substrates. Previously, we described silver nanoisland SERS-substrate with a reproducible and uniform surface, demonstrating high potency for industrial production and a suboptimal LoD of 4 × 105 VP/mL of influenza A virus. Here we describe a study of the sensor morphology, revealing an unexpected mechanism of signal enhancement through the distortion of the nanoisland layer. A novel modification of the aptasensor was proposed with chromium-enhanced adhesion of silver nanoparticles to the surface as well as elimination of the buffer-dependent distortion-triggering steps. As a result, the LoD of the Influenza A virus was decreased to 190 VP/mL, placing the nanoisland SERS-based aptasensors in the rank of the most powerful sensors for viral detection.