Alexidine, identified as a Z-DNA inducer by the NanoZ screening platform, acts as a transcriptional regulator.

Z-DNA is a left-handed DNA helix implicated in gene regulation, genome stability, and immune responses, yet effective small-molecule modulators in cells remain scarce. To address this gap, we developed NanoZ, a two-step screening platform designed to identify Z-DNA-inducing molecules. The first step, a DNA condensation assay, detects ligand-induced DNA condensation using gold nanoparticles functionalized with Z-DNA-forming sequences, providing a rapid optical readout through localized surface plasmon resonance. The second step, a biophysical validation pipeline, employs circular dichroism, 2-aminopurine fluorescence, and NMR spectroscopy to confirm Z-DNA formation. Using this workflow, we screened 2000 compounds from the Natural Product and Prestwick Chemical Libraries, identifying 18 positive hits in the condensation assay and selecting Alexidine dihydrochloride as a Z-DNA inducer. Alexidine efficiently promoted the B-to-Z transition and DNA condensation in vitro, and markedly increased Z-DNA formation in cells, as confirmed by immunofluorescence and ChIP-seq. Genome-wide analysis revealed that Alexidine-induced Z-DNA localized to transcriptionally active, purine-pyrimidine repeat-rich loci, leading to gene repression. Molecular dynamics simulations showed that Alexidine bridges adjacent Z-DNA helices, facilitating condensation. Collectively, our findings highlight Alexidine as the first small-molecule Z-DNA inducer that modulates transcription in cells and establish NanoZ as a versatile platform for discovering Z-DNA modulators.