Epitope-based discovery of aptamer targeting cobra venom cytotoxin

Cytotoxin (CTX) is a three-finger toxin found predominantly in cobra venoms, responsible for dermonecrosis. CTX is generally low in immunogenicity, resulting in the ineffectiveness of existing antibody-based antivenoms in attenuating the local symptoms of dermonecrosis. Aptamers emerge as promising new antivenoms for affordability, manufacturing feasibility and low immunogenicity. We found four epitopes within its functional loops: 'KLVPLFYK', 'AGKNL', 'MFMVSTPKVPV', and 'DVCPKNSLL'. This study aimed to discover aptamers that can specifically recognise these functional epitopes. The binding affinity, neutralisation efficacy, and mechanisms were determined for the aptamer candidates. Our results show that four different aptamer candidates exhibited different degrees of binding to functional epitopes 1, 2, 3, and 4. These aptamers were Apt15, Apt33, Apt26, and Apt31, respectively. Both functional epitopes 1 and 4 demonstrated the highest synergistic cytotoxicity in human skin keratinocytes, with elevated levels of receptor-interacting protein kinase-3 (RIPK3), implying that necroptosis associated with the cytotoxicity. Based on the enzyme-linked aptamer assay (ELAA), Apt31 had the strongest binding affinity towards functional epitope 4, with a K_D value of 187.7 nM. In the in vitro neutralisation assay, Apt31 conferred neutralisation potency with an ED₅₀ of 0.03 nM. However, Apt31 did not rescue human skin keratinocytes in experimental post-envenomed conditions, although increasing concentrations of Apt31 reduced the RIPK3 levels in the post-envenomed keratinocytes. The results conclude that Apt31 potently neutralised the in vitro cytotoxicity caused by functional epitopes but not the post-envenomed conditions.