Selection and Identification of a DNA Aptamer That Mimics Saxitoxin in Antibody Binding

Hu, Pan; Liu, Zengshan; Tian, Rui-Yun; Ren, Hong-Lin; Wang, Xiaoxu; Lin, Clement; Gong, Sheng; Meng, Xianmei; Wang, Guangming; Zhou, Yu; Lu, Shiying

doi:10.1021/jf400880r

Cited by 18 publications

(9 citation statements)

References 43 publications

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“…Following washes by washing buffer and ddH 2 O to remove unbound ssDNA, the β-BuTx-bound aptamers were eluted by heat shock at 95°C with ddH 2 O. High concentration of guanidinium isothiocyanate buffer was often used in elution of aptamers from the plate, and a phenol-chloroform extraction and an alcohol precipitation procedure was performed to purify the ssDNA [44], [45]. We found that a heat shock with ddH 2 O was sufficient to elute bound ssDNA from the plate, and it could be directly served as the template in the following PCR reactions without any purification steps.…”

Section: Resultsmentioning

confidence: 99%

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

Zheng²,

Wang³

et al. 2014

PLoS ONE

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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.

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Section: Resultsmentioning

confidence: 99%

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

Zheng²,

Wang³

et al. 2014

PLoS ONE

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“…Compared with antibodies, aptamers possess some distinct advantages: in vitro screening of aptamers avoids ethical problems associated with animal experiments arising in antibody preparation. This further excludes the negative influences of immunogenicity and toxicity, and extends the range of targets to include metal ions, small molecules, biomacromolecules, , and even cells. , The obtained aptamer sequence can be rapidly produced by chemical synthesis with high purity, at low cost, and with minor batch-to-batch variations. , They can also be easily modified to enhance their chemical and thermal stability for long-term storage, and are labeled with various signal molecules to facilitate detection. − The antibody-based immunosensors have already been made commercially available, such as enzyme-linked immunosorbent assay (ELISA) kits and immunochromatographic strips. , Because of the numerous advantages of aptamers, they have been promising alternative molecular recognition elements to antibodies in analytics, medical diagnosis, and food safety inspection. − So it is of great significance to screen aptamers with high affinity and specificity.…”

Section: Introductionmentioning

confidence: 99%

Magnetic Separation-Based Multiple SELEX for Effectively Selecting Aptamers against Saxitoxin, Domoic Acid, and Tetrodotoxin

Duan

Xia

et al. 2018

J. Agric. Food Chem.

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In this study, a novel magnetic separation-based multiple systematic evolution of ligands by exponential enrichment (SELEX) was applied to select aptamers simultaneously against three kinds of marine biotoxins, including domoic acid (DA), saxitoxin (STX), and tetrodotoxin (TTX). Magnetic reduced graphene oxide (MRGO) was prepared to adsorb unbound ssDNAs and simplify the separation step. In the multiple SELEX, after the initial twelve rounds of selection against mixed targets and the subsequent four respective rounds of selection against each single target, the three resulting ssDNA pools were cloned, sequenced, and analyzed. Several aptamer candidates were selected and subjected to the binding affinity and specificity test. Finally, DA-06 ( K = 62.07 ± 19.97 nM), TTX-07 ( K = 44.12 ± 15.38 nM), and STX-41 ( K = 61.44 ± 23.18 nM) showed high affinity and good specificity for DA, TTX, and STX, respectively. They were also applied to detect and quantify DA, TTX, and STX successfully. The other two multitarget aptamers, DA-01 and TTX-27, were also obtained, which can bind with either DA or TTX. These aptamers provide alternative recognition molecules to antibodies for biosensor applications.

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“…Hu et al [ 106 ] have selected a very specific ssDNA aptamer that mimicked STX in antibody binding, with a dissociation constant (K d ) in the nM range. Its high binding affinity and specificity were confirmed by indirect ELISA, indirect competitive ELISA, and equilibrium filtration method.…”

Section: Aptasensors For Aquatic Phycotoxins and Cyanotoxins Detecmentioning

confidence: 99%

Aptamer-Based Biosensors to Detect Aquatic Phycotoxins and Cyanotoxins

Cunha

Biltes

Sales

et al. 2018

Sensors

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Aptasensors have a great potential for environmental monitoring, particularly for real-time on-site detection of aquatic toxins produced by marine and freshwater microorganisms (cyanobacteria, dinoflagellates, and diatoms), with several advantages over other biosensors that are worth considering. Freshwater monitoring is of vital importance for public health, in numerous human activities, and animal welfare, since these toxins may cause fatal intoxications. Similarly, in marine waters, very effective monitoring programs have been put in place in many countries to detect when toxins exceed established regulatory levels and accordingly enforce shellfish harvesting closures. Recent advances in the fields of aptamer selection, nanomaterials and communication technologies, offer a vast array of possibilities to develop new imaginative strategies to create improved, ultrasensitive, reliable and real-time devices, featuring unique characteristics to produce and amplify the signal. So far, not many strategies have been used to detect aquatic toxins, mostly limited to the optic and electrochemical sensors, the majority applied to detect microcystin-LR using a target-induced switching mode. The limits of detection of these aptasensors have been decreasing from the nM to the fM order of magnitude in the past 20 years. Aspects related to sensor components, performance, aptamers sequences, matrices analyzed and future perspectives, are considered and discussed.

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Selection and Identification of a DNA Aptamer That Mimics Saxitoxin in Antibody Binding

Cited by 18 publications

References 43 publications

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

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

Magnetic Separation-Based Multiple SELEX for Effectively Selecting Aptamers against Saxitoxin, Domoic Acid, and Tetrodotoxin

Aptamer-Based Biosensors to Detect Aquatic Phycotoxins and Cyanotoxins

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