In silico maturation of affinity and selectivity of DNA aptamers against aflatoxin B₁ for biosensor development

A high affinity and selectivity DNA aptamer for aflatoxin B₁ (AFB₁) was designed through Genetic Algorithm (GA) based in silico maturation (ISM) strategy. The sequence of a known AFB₁ aptamer (Patent: PCT/CA2010/001292, Apt1) applied as a probe in many aptasensors was modified using seven GA rounds to generate an initial library and three different generations of ss DNA oligonucleotides as new candidate aptamers. Molecular docking methodology was used to screen and analyze the best aptamer-AFB₁ complexes. Also, a new pipeline was proposed to faithfully predict the tertiary structure of all single stranded DNA sequences. By the second generation, aptamer Apt1 sequence was optimized in the local search space and five aptamers including F20, g12, C52, C32 and H1 were identified as the best aptamers for AFB₁. The selected aptamers were applied as probes in an unmodified gold nanoparticles-based aptasensor to evaluate their binding affinity to AFB₁ and their selectivity against other mycotoxins (aflatoxins B₂, G₁, G₂, M₁, ochratoxin A and zearalenone). In addition, a novel direct fluorescent anisotropy aptamer assay was developed to confirm the binding interaction of the selected aptamers over AFB_1. The ISM allowed the identification of an aptamer, F20, with up to 9.4 and 2 fold improvement in affinity and selectivity compared to the parent aptamer, respectively.