Bidirectional\nPolymerization-Transcription Amplification-Encoded\nDual-Color Fluorescent Biosensor for Label-Free and Primer-Free Detection\nof Multiple piRNAs

PIWI-interacting RNAs (piRNAs) are a type of endogenous\nnoncoding\nRNAs with a length of 24–31 nucleotides, and they can specifically\nbind with PIWI proteins to form the piRNA/PIWI complexes for regulating\nmultiple physiological and pathological processes. Herein, we develop\na bidirectional polymerization-transcription amplification-encoded\ndual-color fluorescent biosensor for label-free and primer-free measurements\nof multiple piRNAs. The designed hairpin probe contains a palindromic\ntail, and it can serve as the target recognition unit, polymerization\nprimer, and transcription template. In the presence of target piRNAs,\nthe hairpin probes are opened to expose a palindromic sequence that\ncan trigger bidirectional polymerization and transcription reaction\nwith the assistance of KF polymerase and T7 RNA polymerase for the\nproduction of numerous RNA aptamers. The aptamers subsequently bind\nwith the corresponding fluorophores (DFHBI-1T/MG) to form the RNA\naptamer-fluorophore complexes for the generation of enhanced fluorescence\nsignals. This biosensor can sensitively detect piR-36026 with a limit\nof detection (LOD) of 82.08 aM and piR-36743 with a LOD of 44.44 aM.\nMoreover, it can quantify cellular piRNAs with single-cell sensitivity\nand distinguish cancer cells from normal cells. Furthermore, it has\nthe capability of distinguishing the expression of piRNAs in the tissues\nof breast cancer patients and healthy individuals. By simply altering\nthe target recognition site of the hairpin probe, this biosensor can\nbe extended to detect various piRNAs, offering a powerful platform\nfor piRNA-related clinical diagnostics and therapeutics.