Visualizing single-base RNA mutations in living cells through DNA nanostructure-mediated amplification

Abstract Capturing RNA dynamics in living cells would provide critical insights into transcriptional control and cellular adaptation, but remains technically formidable — particularly at single-base precision. Here, we introduce a DNA tetrahedron based three-dimensional catalytic hairpin assembly (3D@CHA) nanoplatform that couples target recognition with catalytic activation in a spatially organized framework. Three cascaded hairpins (H-AN, H1, and H2) then enable localized and efficient signal amplification. Without external carriers or transfection, the platform exhibits robust biocompatibility, distinguishing highly homologous insulin I (Ins1) and insulin II (Ins2) mRNAs in living cells and tracking their redistribution and intercellular transfer during metabolic changes. Introducing a single-base mismatch site into H1 and coupling it with a Förster resonance energy transfer (FRET) readout yielded a KRAS-3D@CHA probe capable of detecting KRASG12D mutations at the RNA level with single-base resolution. This platform establishes a programmable framework for precise RNA imaging and mutation discrimination, opening new avenues for RNA-level diagnostics and precision oncology. Competing Interest Statement The authors have declared no competing interest.