A smart multi-activatable DNAzyme nanoamplifier for spatiotemporal imaging of multiple piRNAs and on-demand gene...

PIWI-interacting RNAs (piRNAs) are pivotal regulators of physiological and pathological processes, and their aberrant expression is tightly linked to cancer development and prognosis. Nevertheless, their ubiquitous presence in both tumor and normal cells poses a major challenge to achieving cancer-specific detection and regulation of cancer-associated piRNAs. Herein, we design a smart multi-activatable DNAzyme nanoamplifier for spatiotemporal imaging of multiple piRNAs and on-demand gene therapy. This nanoamplifier integrates three functional modules: an APE1-gated recognition module, a target-activated orthogonally controlled catalytic DNA (CCD) circuitry module, and a DNAzyme-mediated module with the integration of signal output and gene therapy. Upon cellular internalization of the nanoamplifier, APE1 within the tumor microenvironment triggers the release of intelligent-responsive elements. These elements can specifically bind to piRNAs, initiating the CCD circuitry to generate two active double-ended DNAzyme assemblies. Leveraging their high-turnover efficiency to cleave molecular beacons (MBs) and specifically knock down VEGF mRNA, these double-ended DNAzyme assemblies can simultaneously restore Cy5/Cy3 fluorescence signals in MB1/MB2 and achieve target-stimulated in situ on-demand gene therapy. Notably, the liberated quencher-labeled fragments can serve as piRNAs analogues to re-activate the CCD circuitry, significantly improving detection sensitivity. This nanoamplifier can overcome the kinetic bottleneck of traditional CHA systems, facilitating sensitive detection and in situ visualization of attomolar-level multiple piRNAs without pre-amplification. Moreover, this nanoamplifier can not only quantify piRNAs levels across various human cells and precisely discriminate expression differences between breast cancer and healthy tissues, but also induce cancer cell apoptosis through synergistic gene therapy, providing a robust integrated theranostic platform.