Pyrene Excimer Signaling Molecular Beacons for Probing Nucleic Acids

TLDR

Multiple‑pyrene labeled molecular beacons adopt a stem‑closed conformation that quenches pyrene excimer fluorescence in the absence of target nucleic acids. The engineered beacons, bearing 1–4 pyrene monomers at the 5′ end and a DABCYL quencher at the 3′ end, switch to a stem‑open state upon target binding, separating the pyrene from the quencher and producing an excimer emission proportional to target concentration. Steady‑state assays achieved subnanomolar detection limits in buffer and low‑nanomolar limits in cell‑growth media, with excimer intensity, quantum yield, and lifetime increasing with each added pyrene, yielding a ~40‑ns lifetime, 130‑nm Stokes shift, and tunable signal that offers a superior alternative to conventional fluorophores.

Abstract

Molecular beacon DNA probes, containing 1-4 pyrene monomers on the 5' end and the quencher DABCYL on the 3' end, were engineered and employed for real-time probing of DNA sequences. In the absence of a target sequence, the multiple-pyrene labeled molecular beacons (MBs) assumed a stem-closed conformation resulting in quenching of the pyrene excimer fluorescence. In the presence of target, the beacons switched to a stem-open conformation, which separated the pyrene label from the quencher molecule and generated an excimer emission signal proportional to the target concentration. Steady-state fluorescence assays resulted in a subnanomolar limit of detection in buffer, whereas time-resolved signaling enabled low-nanomolar target detection in cell-growth media. It was found that the excimer emission intensity could be scaled by increasing the number of pyrene monomers conjugated to the 5' terminal. Each additional pyrene monomer resulted in substantial increases in the excimer emission intensities, quantum yields, and excited-state lifetimes of the hybridized MBs. The long fluorescence lifetime ( approximately 40 ns), large Stokes shift (130 nm), and tunable intensity of the excimer make this multiple-pyrene moiety a useful alternative to traditional fluorophore labeling in nucleic acid probes.