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

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

Abstract

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 (KD) 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.

Hiu, J.J., Tan, H.S. & Yap, M.K.K. Targeting cobra venom cytotoxin: a linear 40-mer ssDNA aptamer-based antivenom confers neutralisation potentials against cobra venom-induced cytotoxicity. Arch Toxicol (2025). https://doi.org/10.1007/s00204-025-04211-z