Enzymatic cascade amplification-modulated Thermus thermophilus Argonaute biosensor for simultaneous monitoring of multiple Piwi-interacting RNAs

PIWI-interacting RNAs (piRNAs) play critical regulatory roles in a variety of physiological and pathological process, and their aberrant expression levels are implicated in the progression and prognosis of cancers. Herein, we construct an enzymatic cascade amplification-modulated Thermus thermophilus Argonaute (TtAgo) biosensor for simultaneous monitoring of multiple piRNAs (i.e., piR-36026 and piR-36743) in breast tissues. Targets piR-36026 and piR-36743 can initiate enzymatic cascade amplification events to produce two corresponding amplicons with 5′-phosphate termini (i.e., gDNAs 1 and 2), respectively. The gDNAs 1 and 2 can serve as the DNA guides to activate TtAgo-dependent cyclic cleavage of reporters 1 and 2, respectively, liberating numerous Cy3 and Cy5 fluorophores. Taking advantage of the high efficiency of enzymatic cascade amplification, and the precise recognition and multi-turnover cleavage activity of TtAgo, this TtAgo biosensor achieves high sensitivity, good selectivity, and multiplex analysis capability. Moreover, it can be employed for simultaneous quantification of endogenous piR-36026 and piR-36743 with single-cell sensitivity, and differentiation of piRNA levels in the tissues of breast cancer patients and healthy individuals, offering a promising platform for bioanalytical and biomedical researches.