Bidirectional Polymerization-Transcription Amplification-Encoded Dual-Color Fluorescent Biosensor for Label-Free and Primer-Free Detection of Multiple piRNAs

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