Abstract
Antibody-based technology is the main method for diagnosis and treatment of snake bite envenoming currently. However, the development of an antibody, polyclonal or monoclonal, is a complicated and costly procedure. Aptamers are single stranded oligonucleotides that recognize specific targets such as proteins and have shown great potential over the years as diagnostic and therapeutic agents. In contrast to antibodies, aptamers can be selected in vitro without immunization of animals, and synthesized chemically with extreme accuracy, low cost and high degree of purity. In this study we firstly report on the identification of DNA aptamers that bind to β-bungarotoxin (β-BuTx), a neurotoxin from the venom of Bungarus multicinctus. A plate-SELEX method was used for the selection of β-BuTx specific aptamers. After 10 rounds of selection, four aptamer candidates were obtained, with the dissociation constant ranged from 65.9 nM to 995 nM measured by fluorescence spectroscopy. Competitive binding assays using both the fluorescently labeled and unlabeled aptamers revealed that the four aptamers bound to the same binding site of β-BuTx. The best binder, βB-1, bound specifically to β-BuTx, but not to BSA, casein or α-Bungarotoxin. Moreover, electrophoretic mobility shift assay and enzyme-linked aptamer assay demonstrated that βB-1 could discriminate B. multicinctus venom from other snake venoms tested. The results suggest that aptamer βB-1 can serve as a useful tool for the design and development of drugs and diagnostic tests for β-BuTx poisoning and B. multicinctus bites.
Highlights
Snakebite is a major public health issue that affects residents of rural communities living in tropical and subtropical countries [1]
systematic evolution of ligands by exponential enrichment (SELEX) Selection Starting from a library of,1014 different sequences of oligodeoxyribonucleotides, we selected aptamers binding to bBuTx, using plate SELEX technique
The critical step in SELEX is the partitioning of the target-bound oligonucleotides from the unbound ones
Summary
Snakebite is a major public health issue that affects residents of rural communities living in tropical and subtropical countries [1]. The anisotropy of 2 nM of fluorescein labeled aptamer bB-1 in 2 mL of binding buffer was measured.
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