This study introduces a screening method to design the new aptamers (mutants) with an improved target specificity by using a joint of theoretical and experimental study. The proposed method consists of some steps: 1) selecting the most specific aptamer toward the target among the SELEX-introduced aptamers through the screening by using the molecular dynamics (MD) simulation, 2) designing the mutants by changing the nucleotides of the selected aptamer, 3) selecting the mutants with the improved specificity toward the target via the screening by using the MD simulation, 4) examining the specificity of the designed mutants toward the target by using an experimental technique. The approach has been used to introduce some mutants toward Pb2+ as a case study. The T30695 aptamer has been chosen theoretically as the most specific aptamer to the ion among the previously obtained aptamers through the SELEX process. All possible mutants have been designed through the mutation of the T30695 aptamer. The MD results prove that the M4, M16, and M4-16 possess the improved specificity toward Pb2+ among the designed mutants. The specificity of the mutants has been examined by using a simple colorimetric assay based on the gold nanoparticles (AuNPs). According to the experimental results, the detection limits of 192, 177, and 65 pM have been obtained for the T30695, M4, and M4-16 aptamers, respectively. The colorimetric results also prove the selectivity of the mutants to Pb2+ in comparison with the other metal ions. The study clarifies the excellent ability of the approach to introduce new aptamers for detection of various targets.

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