A programmable DNAzyme-activated entropy-driven logic circuit for high-contrast imaging of piRNAs and discrimination of cancer cells.

PIWI-interacting RNAs (piRNAs) are a class of endogenous noncoding RNAs that perform pivotal regulatory functions in numerous physiological activities and pathological progression. Dysregulated piRNA expression is closely associated with the progression and prognosis of cancers. Entropy-driven circuit have been widely designed for the imaging and therapy, but their imaging efficiency is always constrained by the uncontrolled off-site signal leakage and the inefficient circuitry activation that may result in low imaging contrast. Herein, we construct a programmable deoxyribozyme (DNAzyme)-activated entropy-driven logic circuit for the high-contrast imaging of piRNAs and the discrimination of cancer cells. We design a methylation-modified DNAzyme as a fuel strand generator and a hairpin probe that confines the fuel strand in the hairpin structure to suppress the signal leakage from the entropy-driven reaction circuit. The highly expressed fat mass and obesity-associated protein (FTO) in cancer cells can catalyze the demethylation of DNAzyme to activate its cleavage activity, inducing the cleavage of hairpin probes and the release of abundant fuel strands to drive entropy-driven reaction circuit. In the presence of target piRNAs, the fuel strands initiate toehold-assisted branch migration reaction to release signal probes and produce enhanced fluorescence signals. The proposed logic circuit system can profile endogenous piRNAs at the single-cell level, discriminate breast cancer tissues from healthy counterparts, and achieve in situ high-contrast imaging of piRNA in living cells. Importantly, the proposed logic circuit system can distinguish different cell subtypes with high accuracy and good specificity, providing a potential platform for clinical diagnosis.