We report on a unique DNA aptamer, denoted MSA52, that displays universally high affinity for the spike proteins of wildtype SARS‐CoV‐2 as well as the Alpha, Beta, Gamma, Epsilon, Kappa, Delta and Omicron variants. Using an aptamer pool produced from round 13 of selection against the S1 domain of the wildtype spike protein, we carried out one‐round SELEX experiments using five different trimeric spike proteins from variants, followed by high‐throughput sequencing and sequence alignment analysis of aptamers that formed complexes with all proteins. A previously unidentified aptamer, MSA52, showed Kd values ranging from 2 to 10 nM for all variant spike proteins, and also bound similarly to variants not present in the reselection experiments. This aptamer also recognized pseudotyped lentiviruses (PL) expressing eight different spike proteins of SARS‐CoV‐2 with Kd values between 20 and 50 pM, and was integrated into a simple colorimetric assay for detection of multiple PL variants. This discovery provides evidence that aptamers can be generated with high affinity to multiple variants of a single protein, including emerging variants, making it well‐suited for molecular recognition of rapidly evolving targets such as those found in SARS‐CoV‐2.

Our previously discovered monomeric aptamer for SARS-CoV-2 (MSA52) possesses a universal affinity for COVID-19 spike protein variants but is ultimately limited by its ability to bind only one subunit of the spike protein. The symmetrical shape of the homotrimeric SARS-CoV-2 spike protein presents the opportunity to create a matching homotrimeric molecular recognition element that is perfectly complementary to its structural scaffold, causing enhanced binding affinity. Here, we describe a branched homotrimeric aptamer with three-fold rotational symmetry, named TMSA52, that not only possesses excellent binding affinity but is also capable of binding several SARS-CoV-2 spike protein variants with picomolar affinity, as well as pseudotyped lentiviruses expressing SARS-CoV-2 spike protein variants with femtomolar affinity. Using Pd−Ir nanocubes as nanozymes in an enzyme-linked aptamer binding assay (ELABA), TMSA52 was capable of sensitively detecting diverse pseudotyped lentiviruses in pooled human saliva with a limit of detection as low as 6.3 × 10 3 copies/mL. The ELABA was also used to test 50 SARS-CoV-2-positive and 60 SARS-CoV-2-negative patient saliva samples, providing sensitivity and specificity values of 84.0 and 98.3%, respectively, thus highlighting the potential of TMSA52 for the development of future rapid tests.

A unique DNA aptamer, denoted MSA52, displays universally high affinity for the spike proteins of the wild‐type SARS‐CoV‐2 as well as its Alpha, Beta, Gamma, Epsilon, Kappa, Delta and Omicron variants. This aptamer also recognizes pseudotyped lentiviruses expressing eight different spike proteins of SARS‐CoV‐2 with very high affinity, exhibiting dissociation constants (Kd) of 20–50 pM for these viruses. More information can be found in the Research Article by J. D. Brennan, Y. Li et al. (DOI: 10.1002/chem.202200078).

The cover feature image shows nucleic acid aptamers armed and ready for our battle against the monstrous SARS‐CoV‐2 virus. Often thought of as “chemical antibodies”, these molecular recognition elements are equipped with several unique benefits and have thus been a popular research subject worldwide. Many aptamers for recognizing the spike and nucleocapsid proteins of SARS‐CoV‐2 have been developed and examined as diagnostic and therapeutic weaponry for the war against COVID‐19 and future pandemics. More information can be found in the Review by J. D. Brennan, Y. Li, and co‐workers .

What is the most significant result of this study?This study reports on au nique DNA aptamer,d enoted MSA52, that displays auniversally high affinityfor the spike proteins of wildtype SARS-CoV-2 as well as the Alpha, Beta, Gamma, Epsilon, Kappa, Delta, and Omicron variants. This aptamer also recognizes pseudotyped lentiviruses (PL) expressing eight different spike proteins of SARS-CoV-2 with K d values between 20-50 pM. It was integrated into as imple colorimetric assay for the detection of multiple PL variants. This discovery provides evidence that aptamers can be generated with high affinity to multiple variants of asingle protein, including emerging variants, making them well-suited for molecular recognition of rapidly evolving targets such as those found in SARS-CoV-2.

The SARS‐CoV‐2 virus and COVID‐19 pandemic continue to demand effective diagnostic and therapeutic solutions. Finding these solutions requires effective molecular recognition elements. Nucleic acid aptamers represent a possible solution. Characterized by their high affinity and specificity, aptamers can be rapidly identified from random‐sequence nucleic acid libraries. Over the past two years, many labs around the world have rushed to create diverse aptamers that target two important structural proteins of SARS‐CoV‐2: the spike (S) protein and nucleocapsid (N) protein. These have led to the identification of many aptamers that show real promise for the development of diagnostic tests and therapeutic agents for SARS‐CoV‐2. Herein we review all these developments, with a special focus on the development of diverse aptasensors for detecting SARS‐CoV‐2. These include electrochemical and optical sensors, lateral flow devices, and aptamer‐linked immobilized sorbent assays.

Many DNA aptamers have been reported to target the spike protein (S‐protein) of SARS‐CoV‐2. These aptamers display different affinities or recognize different epitopes of the S‐protein. We conducted a comparative study of 9 DNA aptamers for binding to several variants of the S‐protein and pseudoviruses using the same testing methods, including dot‐blot assays and enzyme‐linked aptamer binding assays, to evaluate their affinity ranking and analytical utility. Moreover, the binding sites of these aptamers on the S‐protein were examined using aptamer competition assays to understand the effect of S‐protein mutations on aptamer affinity and the degree of overlapping of the binding sites by these aptamers.

Aptamers that can recognize the spike (S) protein of SARS-CoV-2 with high affinity and specificity are useful molecules towards the development of diagnostics and therapeutics to fight COVID-19. However, this S protein is constantly mutating, producing variants of concern (VoCs) that can significantly weaken the binding by aptamers initially engineered to recognize the S protein of the wildtype virus or a specific VoC. One strategy to overcome this problem is to develop universal aptamers that are insensitive to all or most of the naturally emerging mutations in the protein. We have recently demonstrated this concept by subjecting a pool of S protein-binding DNA aptamers for one-round parallel-SELEX experiments targeting 5 different S protein variants for binding-based sequence enrichment, followed by bioinformatic analysis of the enriched pools. This effort has led to the identification of a universal aptamer that recognizes 8 different variants of the spike protein with equally excellent affinity.