A Universal DNA Aptamer that Recognizes Spike Proteins of Diverse SARS‐CoV‐2 Variants of Concern

Abstract

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.

One Solution for All: Searching for Universal Aptamers for Constantly Mutating Spike Proteins of SARS‐CoV‐2

“…For example, the ranking of MSA52R1 increased from 46 th in the WH pool to 14 th in the CA pool, producing a ranking increase of 69.5 %. [38] The results once again confirmed that the members in this family competed well for the binding of all four variants used for selection. Taken together, the sequencing analysis identified MSA52 as a universal aptamer candidate for the S protein.…”

“…However, the ranking of MSA52 increased to 24 th , 19 th , 17 th , and 14 th after one‐round selection with the S protein from the UK (UKS), Brazil (BZS), South Africa (SAS), and California (CAS) variants, respectively, but remained largely unchanged (with a ranking of 46 th ) in the pool with the wildtype S protein (WHS). [38] These observations indicate that MSA52 competed favorably for binding all four S protein variants with the top‐ranking sequences established using WHS, pointing to the possibility of this aptamer being the universal aptamer that we were searching for.…”

“…This was the foundational hypothesis examined by us in a recent study. [38] To find these aptamers quickly, we carried out five parallel one‐round SELEX experiments with the G13 pool using five different S proteins as the binding targets (Figure 4 A). These variants were: the wildtype S protein plus the S proteins from B.1.1.7 (Alpha, United Kingdom, UK), B.1.351 (Beta, South Africa, SA), P.1 (Gamma, Brazil, BZ) and B.1.429 (Epsilon, California, CA) variants, all of which were commercially available at the time of the experiment.…”

“…The amplified DNA pools were then subjected to high‐throughput sequencing, followed by a comparative analysis of abundance changes of the aptamer sequences in these pools. [38] …”

“…The sequence and predicted secondary structure of MSA52 were provided in Figure 5 A. [38] MSA52 consists of 79 nucleotides and the overall structure contains 4 pairing arms (P1, P2, P3 and P4) and 7 unpaired elements (SS12, SS23, SS34, SS41, and the loops L2, L3, L4). P1 was the pairing arm that was pre‐designed in the structure of the library (Figure 3 A).…”