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Abstract Controlling the activity of DNAzymes by external triggers is an important task. Here a temporal control over DNAzyme activity through a mechanochemical pathway with the help of ultrasound (US) is demonstrated. The deactivation of the DNAzyme is achieved by hybridization to a complementary strand generated through rolling circle amplification (RCA), an enzymatic polymerization process. Due to the high molar mass of the resulting polynucleic acids, shear force can be applied on the RCA strand through inertial cavitation induced by US. This exerts mechanical force and leads to the cleavage of the base pairing between RCA strand and DNAzyme, resulting in the recovery of DNAzyme activity. This is the first time that this release mechanism is applied for the activation of catalytic nucleic acids, and it has multiple advantages over other stimuli. US has higher penetration depth into tissues compared to light, and it offers a more specific stimulus than heat, which has also limited use in biological systems due to cell damage caused by hyperthermia. This approach is envisioned to improve the control over DNAzyme activity for the development of reliable and specific sensing applications.