Monitoring the Intracellular Fate of Molecular Beacons: The Challenge of False Positive Signals

Molecular beacons (MBs) have been used on surfaces for detecting oligonucleotides. Attempts to use them intracellularly for monitoring mRNA content have been made, however, without any clear conclusion regarding the reliability of the method, mainly due to false positive signals. To reach an understanding of the intracellular fate of MBs, a critical question remains: how long after MB delivery and where in the cell does a false positive signal appear? To answer that question, the MB delivery method should allow for a time-stamped synchronized delivery of MBs to multiple cells, resulting in MBs being distributed in the cytosol immediately after delivery. Herein, nanostraws are used to inject MBs targeting insulin (Ins1) mRNA directly in the cytosol of clonal beta-cells, and the evolution of the MB fluorescence in time and space is monitored. The results show an MB translocation to the nucleus, where MBs are degraded or where they open nonspecifically, before the fluorophore alone is expelled back from the nucleus to the cytosol. The signal translocation to the nucleus and back to the cytosol is faster when scrambled MBs are used. The results shed light on the intracellular fate of MBs and highlight the short time scales before false positive signals become predominant. Nanostraws are used to inject molecular beacons targeting Ins1 mRNA into the cytosol of clonal beta-cells. The results suggest that the beacon signal is mainly false positive, with beacons being transported to the nucleus where they either open nonspecifically, or are degraded before the fluorophores are expelled back to the cytosol.dagger image (c) 2024 WILEY-VCH GmbH