Recognition of Bungarus multicinctus Venom by a DNA Aptamer against β-Bungarotoxin

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.

RNA aptamers selected against yeast cells inhibit Candida albicans biofilm formation in vitro

“…Interestingly, the length of the randomized region of Ca‐apt‐1 [41 nucleotide (nt)] was longer than that of the original 40‐nt pool RNAs. Such changes may occur either during reverse transcription or PCR steps in multiple rounds of SELEX (Doudna, Cech, & Sullenger, ; Takemura et al., ; Ye et al., ).…”

Probing the Key Binding Sequence and Improvement of the Stability of a β‐Bungarotoxin‐binding Aptamer in Snake Venom

“…The Kd of the final modified aptamer βB-1.11LOT was determined by fluorescence spectroscopy assay as previously described. 20 As the anisotropy changes in binding buffer were small, the experiments were performed in 20 mM Tris, pH 7.4, 10 mM NaCl, and 0.5 mM MgCl 2 , and the Kd of βB-1 in this buffer was also tested as a control. Different concentrations of β-BuTx were titrated into 150 μL of 20 nM fluorescein labeled aptamer, and the fluorescence anisotropy was measured as mentioned above.…”

“…The aptamer, βB-1, has an equilibrium dissociation constant (Kd) of 66 nM, and can discriminate B. multicinctus venom from other species of snake venoms. 20 Aptamer βB-1 is very promising for the design and development of drugs and/or diagnostic tests for β-BuTx intoxication and B. multicinctus bites.…”

“…22,23 Our previous work has shown that the original aptamer βB-1 was completely degraded by the venom of Naja naja atra. 20 Hence, improving the stability of the aptamer βB-1 in snake venom is important for its diagnostic or therapeutic uses. The results would also help to find clues for stability improvement of other snake venom component aptamers.…”

Biotechnological innovations in farm and pet animal disease diagnosis

“…Aptamers, first reported in 1990, are attracting interest in the areas of diagnostics and offer themselves as ideal candidates for use as biocomponents in biosensors (aptasensors), possessing many advantages over state of the art affinity sensors (O'Sullivan, 2002). The aptamers have proved to be potential diagnostic assays, especially in the detection of toxins such as brevetoxin-2, potent marine neurotoxins (Shimaa et al, 2015), marine biotoxinpalytoxin (Shunxiang et al, 2017), β-bungarotoxin (β-BuTx), and a neurotoxin from the venom of Bungarus multicinctus (Ye et al, 2014a). Furthermore, aptamers have been used for the serological detection of Mycobacterium bovis (Fu et al, 2014), Cryptosporidium parvum (Iqbal et al, 2015), and prion disease (Saijin et al, 2012).…”