Molecular beacon-peptide probe based double recycling amplification for multiplexed detection of serum exosomal microRNAs.

Exosomal microRNAs (exomiRs) have been shown to play crucial roles as biomarkers for early detection and prognosis of cancer. However, simultaneous quantification of multiplex exomiRs is hindered by methods that require additional steps, such as labeling with fluorophores or gel visualization, which are susceptible to various factors. Herein, we developed a mass spectrometry-detectable and target-triggered method for multiplexed exomiR detection using three enzyme-based double recycling amplification in combination with well-designed molecular beacon-peptide (MBP) probes, called molecular beacon-peptide probe-based double recycling amplification (MBPDRA). MBP probes mediated the double recycling amplification reaction and were released as mass-detectable reporter peptides. In particular, the hybridization of the target microRNAs (miRNAs) with the stem-loop of the probe triggers two consecutive processes. The first cycle involved polymerase strand displacement amplification, leading to the production of complementary DNA (cycle I), and the second cycle encompassed the recycling exonuclease cleavage of the MBP probe (cycle II). Subsequently, excess probes were removed by interaction with streptavidin beads biotin-streptavidin binding. The reporter peptides were released using trypsin and subsequently detected by mass spectrometry. Our method enables quantitative detection of multiple exomiRs with a dynamic range from 0.1 fM to 10 pM and a limit of quantification of 0.1 fM. Moreover, the proposed assay was successfully employed for quantification of three exomiRs, exmiR-21, exmiR-191, and exmiR-451a, in the sera of patients with pancreatic cancer. Based on these findings, we believe that the MBPDRA assay holds significant promise as a reliable method for quantifying multiple miRNAs in biomedical research and clinical diagnostics.