Targeting cobra venom cytotoxin: a linear 40-mer ssDNA aptamer-based antivenom confers neutralisation potentials against cobra venom-induced cytotoxicity

Cytotoxin (CTX) is one of the major cobra venom components that contributes to dermonecrosis due to its cytotoxicity. However, current antibody-based antivenoms exert limited neutralisation effects against CTX-induced dermonecrosis. This study focused on discovering aptamer-based antivenom that specifically targets CTX, using repetitive centrifugation-based Systematic Evolution of Ligands by EXponential enrichment (SELEX) selection approach and Illumina amplicon next-generation sequencing. A total of 12 repetitive centrifugation-based selection rounds including a negative selection between rounds 7 and 8 were performed. This was followed by amplicon next-generation sequencing and sequencing bioinformatics workflow to analyse the abundance and persistence of the CTX-binding candidates. Sequences with log₁₀ read counts of 2-3 with a round representation of 3-4 were selected as the final candidates. A linear and single-stranded DNA, 40T, was discovered and it exhibited high binding affinity and specificity to CTX with dissociation constant (K_D) of 0.33-0.41 µM, as demonstrated by direct and competitive enzyme-linked aptamer assay (ELAA). 40T acquired a 'sandwich' configuration binding to CTX at the functional epitopes. It exhibited neutralisation potency against the CTX-induced cytotoxicity with EC₅₀ of 0.47 µM. To mimic the real envenomation situation, venoms from Naja sputatrix, Naja siamensis, and Naja sumatrana were used to induce experimentally envenomed model for treatment with 40T. 40T demonstrated notable cell viability-restoring effects against these venoms at low micromolar ratios. These findings suggested a modified selection and sequencing workflow to discover the potential of 40T as aptamer-based antivenom to mitigate venom-induced dermonecrosis.