Rational design of Cr(VI) aptamer guided by revealed cation-mediated binding mechanism and construction of a...

Aptasensors are widely recognized as practical and rapid detection tools for heavy metal pollution due to their high specificity and sensitivity. However, screening aptamers for small molecules (including ions) is significantly challenging. To date, only two Cr(VI) aptamers have been reported, which cannot meet the demand of aptasensor development for Cr(VI) detection. In this work, aiming at rational design of Cr(VI) aptamers, a new pattern of cation-mediated binding mechanism of aptamer Cr-62mer with target CrO₄^2- was revealed for the first time by combined molecular dynamics (MD) simulation and experimental methods at the atomic level. It was found that CrO₄^2- binds indirectly at A15 and C42 base sites, which was mediated by two Ca²⁺ cations with coordination interaction. Accordingly, based on the original Cr-62mer aptamer, a series of sequences were designed to form more binding sites by reasonable nucleotide fragment cutting and insertion strategy. The binding affinity of obtained aptamers was significantly increased by up to 76%. Furthermore, a label-free fluorescent aptasensor detection method was built, which can realize a sensitive detection of Cr(VI) level with limit of detection of 2.924 nM and detection range of 10-160 nm in real samples. This work not only revealed a new pattern of DNA aptamers binding to Cr(VI), but also offers a generalizable strategy for aptamer development.