Spatial Confinement of a Dual Activatable DNAzyme Sensor in the Cavity of a DNA Nanocage for Logic‐Gated Molecular Imaging

Abstract Despite intense interest in design of DNAzyme sensors for molecular detection and imaging in living cells, their intracellular applications are still hampered by limited spatial control and poor bio‐stability. Here we present controlled spatial confinement of a rationally designed, microRNA (miRNA)‐activatable DNAzyme sensor probe (mDz) within the cavity of DNA nanocage, enabling efficient intracellular delivery with improved bio‐stability for AND‐gate molecular imaging. The mDz that possesses inactive DNAzyme activity is designed by the introduction of a blocking DNA strand, while miR‐21 mediated strand displacement reaction allows for the formation of an intact DNAzyme structure for metal‐ion‐mediated catalytic reaction. Furthermore, the DNA nanocage serves as a nanocarrier for intracellular delivery of mDz, in which the cavity is accessible to the dual targets for logic‐gated molecular imaging, while the confinement effect can provide steric protection of mDz by obstructing nuclease from entering the cavity of the DNA nanocage, resulting in enhanced bio‐stability and improved molecular imaging precision. This strategy paves a way for the engineering of activatable DNA nanosensors with both self‐delivery and self‐protection capabilities to detect diverse intracellular targets.