DNA Aptamers withChemically Locked Ends for VirusInfection Inhibition

Nucleic acid aptamers, known as chemical antibodies, demonstrate remarkable affinity and specificity for targets. Therefore, aptamers are proposed as an alternative to an antibody in extensive applications. However, nucleic acid aptamers exhibit poor tolerance to degradation by nucleases, which severely hampers their biological applications. Herein, we developed a biological regulation pattern for aptamers by utilizing small-molecule-mediated terminal manipulation, which could prevent the interaction of DNA aptamers with exonucleases and help aptamers persist in the desired conformation with high stability. Diagonal T-T bases were designed in the ends of aptamers and could be chemically cross-linked with trioxsalen via photocatalyzed cycloaddition. Aptamers with different patterns of terminal T-T cross-linking sites were synthesized. Experimental investigation and molecular dynamics simulations combinedly revealed that the cross-linking efficiency of ends depended on multiple factors: (i) the number of T-T cross-linking sites in the terminal sequences, (ii) the spatial conformation of aptamers, and (iii) the competitive binding ability of the T-T sites with trioxsalen compared to other base sites. The aptamers with locked ends exhibited superior exonuclease resistance, especially with both 3′- and 5′-cross-linked ends, thus demonstrating a great target binding capability. Notably, in the application exploration, the terminal locked aptamers, which bound to receptor-binding domains on SARS-CoV-2, showed superior performance in virus infection inhibition. This work puts forward a paradigm to develop a biological regulation pattern for aptamers based on chemical terminal manipulation of DNA, potentially promoting the clinical applications of nucleic acid drugs.