| Summary: | Abstract Stimuli‐responsive designs with exogenous stimuli enable remote and reversible control of DNA nanostructures, which break many limitations of static nanostructures and inspired development of dynamic DNA nanotechnology. Moreover, the introduction of various types of organic molecules, polymers, chemical bonds, and chemical reactions with stimuli‐responsive properties development has greatly expand the application scope of dynamic DNA nanotechnology. Here, DNA assembly‐based stimuli‐responsive systems are reviewed, with the focus on response units and mechanisms that depend on different exogenous stimuli (DNA strand, pH, light, temperature, electricity, metal ions, etc.), and their applications in fields of nanofabrication (DNA architectures, hybrid architectures, nanomachines, and constitutional dynamic networks) and biomedical research (biosensing, bioimaging, therapeutics, and theranostics) are discussed. Finally, the opportunities and challenges for DNA assembly‐based stimuli‐responsive systems are overviewed and discussed.
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