Molecular Beacons

Oliver Seitz

A growing number of fluorescence-based assays makes use of distance-dependent interactions between a fluorophore and a chromophore [1]. For example, once excited by absorption of light a fluorescent group can pass its energy by radiationless fluorescence resonance energy-transfer (FRET) to an acceptor group, assuming donor emission overlaps (at least partially) with acceptor absorption. The acceptor group dissipates the energy as heat, or emits light if the acceptor is fluorescent itself. As a result donor emission becomes quenched. At very short distances a second quenching mechanism can operate - energy is transferred by contact. This so-called contact-mediated quenching can occur even with spectrally non-matched fluo-rophores. Irrespective of the quenching mechanism, donor fluorescence increases with the spatial separation of the two interacting fluorophores.

In molecular beacons fluorescence dequenching serves for detection of specific DNA segments in homogeneous solution, for example in real-time n—-n—:-rn- rr

O - fluoresce rice donor 0 = ilouiesuence quer-chei

Fig. B.18.1. Molecular beacons are virtually non-fluorescent in the unhybridized form 1. Target binding opens the molecular beacon to form 2 and fluorescence occurs.

PCR analysis and RNA detection in living cells [2]. Molecular beacons 1 are oligonucleotides designed to form hairpin structures with a target-unrelated double-stranded stem sequence that holds two reporter groups in close proximity (Figure B.18.1). In the closed form, 1, the fluorescence is quenched because of FRET and contact-mediated quenching. The loop portion is a probe sequence that is complementary to a segment of the nucleic acid target. When the target-probe duplex is longer and more stable than the double-stranded stem, structural reorganization occurs. Target annealing rigidifies the loop segment, and, because stem duplex and target-probe duplex cannot co-exist, the hairpin is opened to form hybrid 2. The disruption of the double-stranded stem separates the fluorescence donor from the acceptor, quenching becomes less likely, and fluorescence can occur. Hybridization of the target sequence to the probe is, therefore, accompanied by strong fluorescence enhancement (up to fiftyfold). A host of reporter groups can be used. Commonly, a fluorophore is combined with a non-fluorescent acceptor. It seems that contact-mediated quenching is the predominant quenching mechanism in the closed form 1 which facilitates the selection of fluorophores, particularly in multiplex formats [3].

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