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Oriented tetrahedron-mediated protection of catalytic DNA mo-lecular-scale detector against in vivo degradation for intracellular miRNA detection.

We report a tetrahedron-based DNAzyme probe (Tetra-ES) for intracellular miRNA detection. Two DNA tetrahedrons (Tetra) were arranged at the different positions of the enzyme (E)/substrate (S) complex in a unique direction. A Na+-dependent DNAzme was designed to be initially locked to inhibit the activity of the DNAzyme. Fluorescence imaging and gel electrophoresis analyses demonstrated that the silenced DNAzyme could be specifically initiated by intracellular tar-get miRNA. The activated DNAzyme repeatedly cleaved the substrates, allowing the controllable signal transduction and amplification effect. The combination of spatially controlled arrangement of DNA tetrahedrons with the stimuli-responsive behavior of the locked DNAzyme improved cell permeability and desirable nuclease resistance. Tetra-ES detec-tor exhibited at least 10 times higher detection sensitivity (LOD of 16 pM) than that of the non-amplification molecular beacon counterpart and was capable of discriminating miRNA target from the corresponding family members. The ex-pression level of target miRNA inside the cells of interest as well as different miRNAs inside the same type of cell lines were reliably screened utilizing the Tetra-ES detector. As an intracellular probe, Tetra-ES may provide valuable insight into developing a homogeneous DNA nanostructure-based controllable signal transduction strategy suitable for detection of miRNA and potential application to cancer diagnosis, prognosis and therapeutics.