معرفی DNAآپتامر کوتاه شده به عنوان پروب مولکولی جدید برای ردیابی آفلاتوکسین B1 به کمک تکنیک های شبیه سازی محاسباتی

نوع مقاله : مقاله کامل پژوهشی

نویسندگان

1 گروه گیاهپزشکی. پردیس کشاورزی و منابع طبیعی-دانشگاه تهران- بخش بیوتکنولوژی میکروبی- پژوهشگاه بیوتکنولوژِی کشاورزی ایران

2 عضو هیئت علمی دانشگاه تهران

3 مرکز تحقیقات چشم. بیمارستان رسول اکرم. دانشگاه علوم پزشکی ایران. تهران. ایران

4 دانشکده شیمی، دانشگاه تورین ایتالیا

5 گروه گیاهپزشکی، پردیس کشاورزی و منابع طبیعی، دانشگاه تهران

چکیده

توالی های تک رشته DNA یا RNA که تحت عنوان آپتامر شناخته می شوند قادرند مولکول هدف را با اختصاصیتی در حد آنتی بادی های مونوکلونال ردیابی نمایند. با این وجود قرار گیری مایکوتوکسین ها در گروه مولکول های کوچک و تفاوت بالای وزن مولکولی آنها با توالی های آپتامری تبدیل به چالشی جدی در معرفی پروب های آپتامری برای آنها شده است. در بررسی حاضر با هدف دستیابی به توالی آپتامری جدید و کوتاه تر برای آفلاتوکسین B1 (AFB1)، ابتدا به کمک الگوریتم ژنتیک یک کتابخانه الیگونوکلئوتیدی اولیه(Lib1) براساس توالی آپتامری اختصاصی آفلاتوکسین B1 (bp 50 Apt1,) طراحی گردید. قدرت اتصال توالی های آپتامری کتابخانه Lib1 به مولکول AFB1 با روش داکینگ مولکولی ارزیابی و بهترین توالی آپتامری با هدف ایجاد کتابخانه جدید(Lib2) و با استراتژی کوتاه کردن تغییر داده شد. غربال مجازی توالی های آپتامری کتابخانه Lib2 از نظرقدرت اتصال به مولکول AFB1 منجر به معرفی توالی آپتامری کوتاه شده C52-T با طول bp 19 گردید. پایداری و نوع تعاملات درکمپلکس آپتامری C52-T و مولکول AFB1 با روش های شبیه سازی دینامیک مولکولی و MM-PBSA مورد بررسی قرار گرفت. نتایج حاصل از تخمین ثابت اتصال برای توالی های آپتامری C52، C52-T و Apt1 با روش رنگ سنجی مبتنی بر نانوذرات تغییر نیافته طلا در مطابقت کامل با نتایج مطالعات درون رایانه ای بود. به نظر می رسد که تکنیک های محاسباتی از پتانسیل بسیار خوبی درمعرفی پروب های مولکولی و حساس جهت طراحی آپتاسنسورهای اختصاصی مایکوتوکسین ها برخوردار باشند.

کلیدواژه‌ها

عنوان مقاله [English]

Introducing truncated DNA aptamer as a new molecular probe for aflatoxin B1 detection using computational simulation techniques

نویسندگان [English]

  • Maryam Mousivand 1
  • Mohammad Javan-Nikkhah 2
  • Kowsar Bagherzadeh 3
  • Laura Anfossi 4
  • Amir Mirzadi Gohari 5
1 Department of Plant Protection, College of Agricultural Sciences & Engineering, University of Tehran, Karaj 31587-77871, Iran. Microbial Biotechnology Department, Agricultural Biotechnology Research Institute of Iran,
2 Department of Plant Protection, College of Agricultural Sciences & Engineering, University of Tehran, Karaj
3 Eye Research Center, the Five Senses Institute, Rassoul Akram Hospital, Iran University of Medical Sciences, Tehran, Iran
4 Department of Chemistry, University of Turin, Via Giuria, 5, I-10125 Turin, Italy.
5 Department of Plant Protection, College of Agricultural Sciences & Engineering, University of Tehran, Karaj 31587-77871, Iran.
چکیده [English]

Single strand DNA or RNA known as aptamers, are able to detect the target molecule with specificity at the monoclonal antibody level. However, the placement of mycotoxins in small molecule groups and their high molecular weight differences with aptamers have become a serious challenge in introducing aptameric probes for them. In the present study, in order to achieve a new and shorter aptamers sequence for aflatoxin B1 (AFB1), an initial oligonucleotide library (Lib1) was designed based on the sequence of a known AFB1 aptamer (named Apt1, 50 bp) using the genetic algorithm. The best aptamer from the Lib1 library was selected based on the molecular docking results and has been modified to create a new library (Lib2) using the truncating strategy. Virtual screening of the Lib2 library in terms of their binding affinity over AFB1 molecule led to obtain the truncated aptamer, C52-T, with 19 bp in length. Type of interaction and stability of C52-T-AFB1 complex were investigated using molecular dynamics simulations (MD) and MM-PBSA method. The affinity constant of C52, C52-T and Apt1 aptamers over AFB1 were estimated through unmodified gold nanoparticle-based colorimetric assay. The experimentally founding and in silico results were completely consistent. It seems that the computational techniques have the great potential to introduce sensitive molecular probes to design specific mycotoxins aptasensors.

کلیدواژه‌ها [English]

  • mycotoxin
  • genetic algorithm
  • molecular docking
  • molecular dynamics
  • gold nanoparticles

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بیماریهای گیاهی
دوره 56، شماره 1
خرداد 1399
صفحه 99-117

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