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2017 | 64 | 3 | 401-406
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Selection and analysis of a DNA aptamer binding α-amanitin from Amanita phalloides

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Mushroom foraging is very popular in some regions of the world. Sometimes toxic and edible mushrooms are mistaken by mushroom collectors, leading to serious human poisoning. The group of mushrooms highly dangerous for human health includes Amanita phalloides. This mushroom produces a toxic octapeptide called α-amanitin which is an inhibitor of nuclear RNA polymerase II. The inhibition of this polymerase results in the abortion of mRNA synthesis. The ingestion of A. phalloides causes liver failure due to the fact that most of the toxin is uptaken by hepatocytes. The hospitalization of poisoned patients involves the removal of the toxin from the digestive tract, its dilution in the circulatory system and the administration of therapeutic adjuvants. Since there is no effective antidote against amanitin poisoning, in this study we developed a DNA aptamer exhibiting specific binding to α-amanitin. This aptamer was selected using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method. Next, its ability of toxin removal from aqueous solution was confirmed by pull-down assay. The aptamer region sufficient for α-amanitin binding was determined. Finally, the dissociation constant of the α-amanitin/DNA aptamer complex was calculated.
Physical description
  • Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland
  • Department of Comparative Biochemistry and Bioanalytics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland
  • Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland
  • Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland
  • Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland
  • Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland
  • Bioorganic Research Group, Max Planck Institute for Medical Research, Heidelberg, Germany
  • Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland
  • Avci-Adali M, Paul A, Wilhelm N, Ziemer G, Wendel HP (2010) Upgrading SELEX technology by using lambda exonuclease digestion for single-stranded DNA generation. Molecules 15: 1-11. doi: 10.3390/molecules15010001.
  • Bartnicki F, Bonarek P2, Kowalska E1, Strzalka W (2017) The Argi system: one-step purification of proteins tagged with arginine-rich cell-penetrating peptides. Sci Rep 7: 2619. doi: 10.1038/s41598-017-02432-6.
  • Bartnicki F, Kowalska E, Pels K, Strzalka W (2015) Imidazole-free purification of His3-tagged recombinant proteins using ssDNA aptamer-based affinity chromatography. J Chromatogr A 1418: 130-139. doi: 10.1016/j.chroma.2015.09.055.
  • Bergis D, Friedrich-Rust M, Zeuzem S, Betz C, Sarrazin C, Bojunga J (2012) Treatment of Amanita phalloides intoxication by fractionated plasma separation and adsorption (Prometheus®). J Gastrointest Liver 21: 171-176. doi: 10.1016/S0168-8278(11)60913-6
  • Bonnet MS, Basson PW (2002) The toxicology of Amanita phalloides. Homeopathy 91: 249-254. doi: doi: 10.1054/homp.2002.0056.
  • Bushnell DA, Cramer P, Kornberg RD (2002) Structural basis of transcription: α-amanitin-RNA polymerase II cocrystal at 2.8 Å resolution. Proc Natl Acad Sci USA 99: 1218–1222. doi: 10.1073/pnas.251664698.
  • Dwivedi HP, Smiley RD, Jaykus LA (2013) Selection of DNA aptamers for capture and detection of Salmonella Typhimurium using a whole-cell SELEX approach in conjunction with cell sorting. Appl Microbiol Biotechnol 97: 3677-3686. doi: 10.1007/s00253-013-4766-4.
  • Ellington A, Szostak J (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346: 818-22. doi: 10.1038/346818a0.
  • Enjalbert F, Rapior S, Nouguier-Soulé J, Guillon S, Amouroux N, Cabot C (2002) Treatment of amatoxin poisoning: 20-year retrospective analysis. J Toxicol Clin Toxicol 40: 715-757. doi: 10.1081/CLT-120014646.
  • Ferenc T, Lukasiewicz B, Ciecwierz J (2009) Poisonings with Amanita phalloides. Medycyna pracy 60: 415-426 (in Polish).
  • Garcia J, Costa VM, Carvalho A, Baptista P, de Pinho PG, de Lourdes Bastos M, Carvalho F (2015) Amanita phalloides poisoning: Mechanisms of toxicity and treatment. Food Chem Toxicol 86: 41-55. doi: 10.1016/j.fct.2015.09.008.
  • Hermann T, Patel DJ (2000) Adaptive recognition by nucleic acid aptamers. Science 287: 820-825. doi: 10.1126/science.287.5454.820.
  • Hong KL, Sooter LJ (2015) Single-stranded DNA aptamers against pathogens and toxins: identification and biosensing applications. Biomed Res Int 2015: 419318. doi: 10.1155/2015/419318.
  • Huizenga DE, Szostak JW (1995) A DNA aptamer that binds adenosine and ATP. Biochemistry 34: 656-665. doi: 10.1021/bi00002a033.
  • James W (2000) Aptamers. In Encyclopedia of Analytical Chemistry. Meyers RA ed, pp 4848-4871. John Wiley & Sons Ltd, Chichester
  • Kaplan CD, Larsson KM, Kornberg RD (2008) The RNA polymerase II trigger loop functions in substrate selection and is directly targeted by α-amanitin. Mol Cell 30: 547-556. doi: 10.1016/j.molcel.2008.04.023.
  • Kaymaz MB, Kandemir FM, Pamukҫu E, Eröksüz Y, Özdemir N (2016) Alfa-Amanitin İle Oluşturulmuş Karaciğer Hasari Üzerine Enginar (Cynara scolymus) Sulu Yaprak Ekstresinin Etkileri. Kafkas Univ Vet Fak 23: 155-160 (In Turkisz). doi: 10.9775/kvfd.2016.16094
  • Keefe AD, Pai S, Ellington A (2010) Aptamers as therapeutics. Nat Rev Drug Discov 9: 537-550. doi: 10.1038/nrd3141.
  • Kostek H, Szponar J, Tchórz M, Majewska M, Lewandowska-Stanek H (2012) Silibinin and its hepatoprotective action from the perspective of a toxicologist. Przegl Lek 69: 541-543 (In Polish).
  • Kowalska E, Bartnicki F, Pels K, Strzalka W (2014) The impact of immobilized metal affinity chromatography (IMAC) resins on DNA aptamer selection. Anal Bioanal Chem 406: 5495-5499. doi: 10.1007/s00216-014-7937-y.
  • Lee SC, Gedi V, Ha NR, Cho JH, Park HC, Yoon MY (2015) Development of receptor-based inhibitory RNA aptamers for anthrax toxin neutralization. Int J Biol Macromol 77: 293-302. doi: 10.1016/j.ijbiomac.2015.03.043.
  • Ng EW, Shima DT, Calias P, Cunningham ET Jr, Guyer DR, Adamis AP (2006) Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. Nat Rev Drug Discov 5: 123-132. doi: 10.1038/nrd1955.
  • Pei X, Zhang J, Liu J (2014) Clinical applications of nucleic acid aptamers in cancer. Mol Clin Oncol 2: 341-348. doi: 10.3892/mco.2014.255.
  • Poucheret P, Fons F, Doré JC, Michelot D, Rapior S (2010) Amatoxin poisoning treatment decision-making: Pharmaco-therapeutic clinical strategy assessment using multidimensional multivariate statistic analysis. Toxicon 55: 1338-1345. doi: 10.1016/j.toxicon.2010.02.005.
  • Santi L, Maggioli C, Mastroroberto M, Tufoni M, Napoli L, Caraceni P (2012) Acute liver failure caused by Amanita phalloides poisoning. Int J Hepatol 2012: 487480. doi: 10.1155/2012/487480.
  • Smith MR, Davis RL (2016) Mycetismus: a review. Gastroenterology Report 4: 107-112. doi: 10.1093/gastro/gov062.
  • Tan L, He R, Li Y, Liang Y, Li H, Tang Y (2016) Fabrication of a biomimetic adsorbent imprinted with a common specificity determinant for the removal of α- and β-amanitin from plasma. J Chromatogr A 1459: 1-8. doi: 10.1016/j.chroma.2016.06.072.
  • Tian RY, Lin C, Yu SY, Gong S, Hu P, Li YS, Wu ZC, Gao Y, Zhou Y, Liu ZS, Ren HL, Lu SY (2016) Preparation of a specific ssDNA aptamer for Brevetoxin-2 using SELEX. J Anal Methods Chem 2016: 9241860. doi: 10.1155/2016/9241860.
  • Tuerk C, Gold L (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249: 505-510.
  • Vavvas D, D'Amico DJ (2006) Pegaptanib (Macugen): Treating neovascular age-related macular degeneration and current role in clinical practice. Ophthalmology Clinics of North America 19: 353-360. doi: 10.1016/j.ohc.2006.05.008.
  • Vivekananda J, Salgado C, Millenbaugh NJ (2014) DNA aptamers as a novel approach to neutralize Staphylococcus aureus α-toxin. Biochem Biophys Res Commun 444: 433-438. doi: 10.1016/j.bbrc.2014.01.076.
  • Wang C, Zhang J, Zhang Y, Peng Z, Xueqiang X, Bian P, Ma W, Qin C (2014) Experience of treatments of Amanita phalloides-induced fulminant liver failure with molecular adsorbent recirculating system and therapeutic plasma exchange. ASAIO J 60: 407-412. doi: 10.1097/MAT.0000000000000083.
  • Wang P, Yang Y, Hong H, Zhang Y, Cai W, Fang D (2011) Aptamers as therapeutics in cardiovascular diseases. Curr Med Chem 18: 4169-4174. doi: BSP/CMC/E-Pub/2011/ 314a [pii].
  • Ward J, Kapadia K, Brush E, Salhanick SD (2013) Amatoxin poisoning: case reports and review of current therapies. J Emerg Med 44: 116-21. doi: 10.1016/j.jemermed.2012.02.020.
  • Zhu Q, Liu G, Kai M (2015) DNA aptamers in the diagnosis and treatment of human diseases. Molecules 20: 20979-20997. doi: 10.3390/molecules201219739.
  • Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31: 3406-3415.
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