Molecular beacons have become a very useful tool for many homogeneous single-stranded nucleic acid detection assays due to their ability to differentiate between bound and unbound states and their improved specificity over linear probes. However, to optimize the performance of molecular beacons for different applications, it is necessary to understand their structure–function relationships. This may become critical in certain assays since the addition (or deletion) of just a single nucleotide to the stem can dramatically change the behavior of molecular beacons. Here we describe a new design of molecular beacons, i.e. the shared-stem molecular beacons, of which the stem-arm nearest the reporter dye participates in both hairpin formation and target hybridization. In contrast to conventional molecular beacons whose stems are independent of the target sequence and thus can freely rotate around the probe–target duplex, this new design helps immobilize the fluorophores of molecular beacons when they hybridize to the target, which is desirable when two molecular beacons are used in a FRET assay (19
). Specifically, with shared-stem molecular beacons, there is a better control of the distance between the donor dye on one beacon and the acceptor dye on the other beacon, since the rotational motion of the fluorophore-attached stem-arm is constrained, as illustrated in Figure B. To facilitate the design and application of, and to reveal the differences between, shared-stem and conventional molecular beacons, we performed a systematic study of the thermodynamic and kinetic parameters that control the hybridization of these molecular beacons with complementary and mismatched targets.
In general, it was found that compared with shared-stem molecular beacons, conventional molecular beacons form less stable duplexes with single-stranded nucleic acid targets but have a slightly improved ability to discriminate between wild-type and mutant targets. The difference in the duplex stability may be understood by considering the thermal-driven interactions between the two stem-forming arms after the molecular beacon hybridized to a target molecule. Unlike linear oligonucleotide probes, a molecular beacon can have two stable conformations: bound to target, and as a stem–loop hairpin. These two stable states compete with each other, giving rise to an improved specificity. The additional freedom inherent in both arms of conventional molecular beacon increases the likelihood that, when a beacon–target complex is partially denatured due to thermal fluctuations, these arms will have a higher probability of encountering each other, allowing the molecular beacon to dissociate from the target. There might exist other explanations for the difference in the stability of probe–target duplexes of conventional and shared-stem molecular beacons and this possibility needs to be further explored.
The reduced stability for conventional beacon hybrids also corresponds to a smaller value in the free energy difference between bound and unbound states of the probe–target duplex. The change in free energy due to any mismatch between the probe and target, therefore, will have a more profound effect on the preference of the stem–loop hairpin conformation of the conventional molecular beacons, leading to an improved ability to differentiate between wild-type and mutated targets. However, this improvement was found to be marginal.
With any given probe length and sequence, the hybridization kinetics of molecular beacons appears to be primarily dependent on the length and sequence of the stem, regardless of whether they are designed in the conventional or shared-stem configuration. Both types of molecular beacon exhibited comparable hybridization rates when the dissociation constants describing the thermal fluctuation induced opening of the stem–loop structure, K23, were similar. When the difference in K23 for the shared-stem and conventional molecular beacons was increased, so was the difference in the hybridization on-rate.
In addition to the above-mentioned differences in the behavior of shared-stem and conventional molecular beacons, it is worth mentioning that, with the same probe length (i.e. bases that hybridize to the target) of a conventional beacon, a shared-stem molecular beacon has a shorter loop length, thus reducing the potential for secondary structure within the loop portion of the beacon. Furthermore, the choice of the stem length is independent of the probe length for conventional molecular beacons, whereas there are certain constraints on the stem-length and probe-length combinations in designing the share-stem molecular beacons. This, together with the dependence of the thermodynamic and kinetic properties on the probe and stem lengths demonstrated in this study, should be considered in the design of molecular beacons for specific applications.