A thymidine-terminated molecular beacon for selective Hg2+ or sequence-specific DNA assay

A new molecular beacon (MB) in which fluorescein (FAM) attached to its 3' end acts as a fluorophore and a bridged thymidine-Hg-thymidine (T-Hg-T) complex acts as a quencher is designed. The fluorescence resonance energy transfer (FRET) between the fluorophore and the quencher results in annihilation of the FAM fluorescence. Experimental conditions that govern the fluorescence quenching, such as number of thymidine bases, pH value, and salt concentration, have been optimized. The MB was found to be highly selective for Hg(2+) among a number of metal ions investigated. In the presence of single-stranded (ss-) target oligonucleotides (ODNs) with bases complementary to those in the loop of MB, the FAM fluorescence can be largely restored due to DNA duplex formation. The present method for DNA assay is also sequence-specific and can determine target ODN concentration at a nanomolar level. The substitution of the quencher group in a conventional MB molecule with simple thymidine bases affords an inexpensive ODN that retains the unique property of the MB molecule.