Generation and application of novel DNA aptamers targeted to the nucleocapsid protein of the SARS-CoV-2

Coronavirus disease 2019 (COVID-19) is a respiratory disease caused by SARS-CoV-2, with common symptoms of fever, cough, and dyspnea. SARS-CoV-2 transmission with high prevalence includes aerosols, respiratory droplets, and fecal-oral routes. To date, COVID-19 has infected more than 7.7 million people, seriously threatening people around the world. Therefore, rapid diagnosis of COVID-19 is crucial for epidemic prevention and control. In this study, two novel DNA aptamers (apt1 and apt2) targeting the nucleocapsid protein of the SARS-CoV-2 (CoV2-N) were screened through selective evolution of ligands by exponential enrichment (SELEX). Subsequently, we devised a simple and sensitive lateral flow biosensor (LFB) for quick detection of the CoV2-N. Through secondary structure and minimum free energy predictions, we found that both apt1 and apt2 form stable stem-loop structures, with free energies of -16.01 and -14.04 kJ/mol, respectively. Moreover, they showed high binding affinities to CoV2-N, with calculated binding affinities (K_d) of 18.9 nM for apt1 and 55.7 nM for apt2. The aptamer-based LFB features simple operation, with a detection limit of 500 ng/mL for proteins and visually interpretable results within 5 min. Together, we obtained aptamers that specifically recognized CoV2-N and established a new detection method with high specificity and sensitivity.