Development of a Single-Molecule Biosensor Based on Polymerization-Transcription-Mediated Target Regeneration for Simultaneously One-Pot Detection of Multiple piRNAs.

PIWI-interacting RNAs (piRNAs) are a class of small noncoding RNAs associated with PIWI proteins within the male germline, and they play significant roles in maintaining genome stability via the modulation of gene expression. The piRNAs are implicated in the progression of various cancers, but the simultaneous monitoring of multiple piRNAs remains a challenge. Herein, we construct a single-molecule biosensor based on polymerization-transcription-mediated target regeneration for the simultaneous one-pot detection of multiple piRNAs. This assay involves two cycles. In step 1, target piRNAs hybridize with the template probe complexes to yield three-way junction (3WJ) structures. Then, KF DNA polymerase initiates the extension to generate a complete T7 promoter, and the extension product can act as an invading strand to displace signal probes, resulting in the release of fluorophores. Then, in step 2, the T7 promoter can be recognized by T7 RNA polymerase to initiate transcription, producing abundant transcripts with 3'-OH that are identical to piRNAs. The resultant transcripts can hybridize with free template probe complexes to obtain new 3WJ structures that can be elongated by KF polymerase for the recovery of fluorescence signals. This assay can be performed homogeneously in a one-pot format within 30 min, and it exhibits high sensitivity, with a limit of detection (LOD) of 19.26 aM for piRNA-36026 and 41.88 aM for piRNA-36743. Moreover, it can simultaneously detect endogenous piRNAs at the single-cell level and differentiate piRNA expression in the tissues of healthy individuals and breast cancer patients, offering a prospective platform for clinic diagnosis.