Robust Covalent Aptamer Strategy Enables Sensitive Detection and Enhanced Inhibition of SARS-CoV-2 Proteins

Abstract

Aptamer-based detection and therapy have made substantial progress with cost control and easy modification. However, the conformation lability of an aptamer typically causes the dissociation of aptamer−target complexes during harsh washes and other environmental stresses, resulting in only moderate detection sensitivity and a decreasing therapeutic effect. Herein, we report a robust covalent aptamer strategy to sensitively detect nucleocapsid protein and potently neutralize spike protein receptor binding domain (RBD), two of the most important proteins of SARS-CoV-2, after testing different crosslink electrophilic groups via integrating the specificity and efficiency. Covalent aptamers can specifically convert aptamer−protein complexes from the dynamic equilibrium state to stable and irreversible covalent complexes even in harsh environments. Covalent aptamer-based ELISA detection of nucleocapsid protein can surpass the gold standard, antibody-based sandwich ELISA. Further, covalent aptamer performs enhanced functional inhibition to RBD protein even in a blood vessel-mimicking flowing circulation system. The robust covalent aptamer-based strategy is expected to inspire more applications in accurate molecular modification, disease biomarker discovery, and other theranostic fields.

Aptamer‐based strategies against SARS‐CoV‐2 viruses

“…Multiple washing in ELISA experiments may affect the binding of aptamers to target molecules and thus affect the detection sensitivity. For this reason, Tan et al designed a covalent aptamer strategy [58] (Figure 4f, Table 2) and modified N protein specific aptamer with -11 of 23…”

“…In the development of covalent aptamers, important considerations are cross-link efficiency, specificity and kinetics of covalent aptamers binding on the target. Tan et al selected optimum NHS tag for a covalent RBD aptamer (NHS-RApt) for SARS-CoV-2 neutralization [58] (Figure 8b), based on the systematic evaluation of three types of covalent tags (sulfuryl fluoride (SF), NHS and acrylamide). NHS-RApt could bind RBD strongly, specifically, and rapidly, while NHS-RApt-virus mimics were highly stable in harsh environments (multiple centrifugations) compared with the original aptamer and antibody.…”

bioTCIs: Middle-to-Macro Biomolecular Targeted Covalent Inhibitors Possessing Both Semi-Permanent Drug Action and Stringent Target Specificity as Potential Antibody Replacements

“…In most cases, bioTCIs are created by a rational design through introduction of the warhead into the targeted middle/macro-biomolecules at a specific position (i.e., placement) [ 29 ] via chemical modification [ 31 ] including bio-conjugation [ 28 , 42 ] or unnatural amino acid (Uaa) incorporation via genetic code expansion [ 30 ]. Usually, the position is determined based on the three-dimensional structure of an inhibitor/target complex.…”

“…Aptamers have been touted as potential antibody replacements given their high specificity and affinity [ 83 , 85 ] but the limitation of a very short in vivo half-life has prevented their practical application [ 81 ]. In theory, these already identified aptamers can be rendered potential TCIs with a long drug half-life just by incorporating a warhead, as recently demonstrated for the thrombin binding aptamer [ 28 , 34 ] and SARS-CoV-2 spike protein binding aptamer [ 42 , 68 ]. Whether other warhead-introduced aptamers will conjugate with the target protein depends on the orientation of the aptamer to the target dictating the proper positioning of the warhead on the aptamer, and the availability of the interaction-capable amino acids on the target protein.…”

Highly catalytic and stable Au@AuPt nanoparticles for visual and quantitative detection of E. coli O157:H7

“…To further develop ELISA, the relatively fragile antibodies can be substituted with aptamers that possess comparable affinity and specificity but better stability towards environmental variance and are easily fabricated in ELISA. [13][14][15][16] Enhancing the catalytic activity and stability of enzymes is another critical step to further develop ELISA. Recent studies focus on nanozymes to mimic natural enzymes.…”