Programming Hydrophobic Nanocavity of DNA Frameworks for Ultrastable Fluorescent Imaging

Fluorescent proteins (FPs) are widely used in biological imaging, yet their limited programmability restricts adaptability for advanced nanoscale applications. Synthetic molecular containers offer designable cavities but struggle to simultaneously achieve large dimensions, biocompatible interfaces, and precise microenvironment control. Here we report DNA framework (DNF) dots, highly programmable fluorescent probes constructed by encapsulating Cy3 within three-dimensional DNA-encoded hydrophobic nanocavities. Using amphiphilic tetrahedral DNA frameworks with inward-oriented alkyl chains, we create well-defined nanocavities (4.8-8.9 nm) with tunable size, shape, and internal chemistry. Single-molecule analysis reveals that DNF dots enhance photostability (up to 15-fold) and total photon output (up to 58-fold), as compared to single Cy3 tagged on single-stranded DNA. The exceptional photostability and brightness of DNF dots enable stable binding event frequencies exceeding 7500 frames in DNA-PAINT super-resolution imaging. Furthermore, integration into light-inducible DNA hydrogels yields rewritable information encoding platforms. DNF dots thus establish a versatile platform that combines the environmental shielding of fluorescent proteins with the programmability of synthetic molecular containers, introducing a DNA framework-encoded microenvironment engineering paradigm for ultrastable fluorescent probes.