Electrochemical DNA hairpin sensors for differentiating small molecule intercalation from minor groove binding

Small molecule double-stranded DNA intercalators have significant potential for therapeutic applications. However, screening for and confirming a drug candidate's intercalative behavior remains labor-intensive and costly. To address this, we investigated the sequence and biophysical parameters that affect the performance of electrochemical DNA hairpin sensors for streamlined identification of structural intercalators. These sensors utilize oligonucleotide (oligo) sequences that form hairpins upon intercalator binding. The 3' end of the oligo is modified with alkylthiol linkers for gold electrode surface monolayer self-assembly, while the 5' end carries a methylene blue redox reporter. Hairpin formation enhances electron transfer between methylene blue and the gold electrode, which can be detected via voltammetry. We tested seven hairpin structures varying in stem length and sequence. Our optimal oligo, HP4, features a four-base-pair stem and responds to five DNA intercalators over a broad detection range, with EC₅₀ in close agreement with published affinity (K _D) values for these interactions. We further demonstrate HP4's ability to discriminate intercalator binding from a series of minor groove binders through significant differences in signal gain upon incubation. Altogether, our strategy establishes a platform for identifying intercalative compounds that should support the development of DNA-targeting therapeutics.