Structure±function relationships of shared-stem and conventional molecular beacons

Molecular beacons are oligonucleotide probes cap-able of forming a stem±loop hairpin structure with a reporter dye at one end and a quencher at the other end. Conventional molecular beacons are designed with a target-binding domain ¯anked by two com-plementary short arm sequences that are indepen-dent of the target sequence. Here we report the design of shared-stem molecular beacons with one arm participating in both stem formation when the beacon is closed and target hybridization when it is open. We performed a systematic study to compare the behavior of conventional and shared-stem molecular beacons by conducting thermodynamic and kinetic analyses. Shared-stem molecular beacons form more stable duplexes with target molecules than conventional molecular beacons; however, conventional molecular beacons may dis-criminate between targets with a higher speci®city. For both conventional and shared-stem molecular beacons, increasing stem length enhanced the ability to differentiate between wild-type and mutant targets over a wider range of temperatures. Interestingly, probe±target hybridization kinetics were similar for both classes of molecular beacons and were in¯uenced primarily by the length and sequence of the stem. These ®ndings should enable better design of molecular beacons for various applications