Grégoire Altan‐Bonnet

Fluorescence correlation spectroscopy (FCS) is an experimental technique using statistical analysis of the fluctuations of fluorescence in a system in order to decipher dynamic molecular events, such as diffusion or conformational fluctuations of biomolecules. First introduced by Magde et al to measure the diffusion and binding of ethidium bromide onto double-stranded DNA, the technique has been undergoing a renaissance since 1993 with the implementation of confocal microscopy FCS. Since then, a flurry of experiments has implemented FCS to characterize the photochemistry of dyes, the translational and rotational mobilities of fluorescent molecules, as well as to monitor conformational fluctuations of green fluorescent proteins and DNA molecules.

Molecular beacons are DNA probes that form a stem-and-loop structure and possess an internally quenched fluorophore. When they bind to complementary nucleic acids, they undergo a conformational transition that switches on their fluorescence. These probes recognize their targets with higher specificity than probes that cannot form a hairpin stem, and they easily discriminate targets that differ from one another by only a single nucleotide. Our results show that molecular beacons can exist in three different states: bound to a target, free in the form of a hairpin structure, and free in the form of a random coil. Thermodynamic analysis of the transitions between these states reveals that enhanced specificity is a general feature of conformationally constrained probes.

The kinetics of DNA hairpin-loop fluctuations has been investigated by using a combination of fluorescence energy transfer and fluorescence correlation spectroscopy. We measure the chemical rates and the activation energies associated with the opening and the closing of the hairpin for different sizes and sequences of the loop and for various salt concentrations. The rate of unzipping of the hairpin stem is essentially independent of the characteristics of the loop, whereas the rate of closing varies greatly with the loop length and sequence. The closing rate scales with the loop length, with an exponent 2.6 +/- 0.3. The closing rate is increased at higher salt concentrations. For hairpin closing, a loop of adenosine repeats leads to smaller rates and higher activation energies than a loop with thymine repeats.