Reversible ratiometric probe for quantitative DNA measurements

We have designed a reversible fluorescent DNA probe that can be used to determine the concentration of single-stranded DNA in solution by a ratiometric fluorescence measurement. The probe consists of a single-stranded dual fluorescently labeled DNA molecule that adopts a stem-loop conformation in its nonhybridized state. The stem length and the length of the loop region complementary to the target were chosen to allow for reversible binding. The excitation and emission wavelengths of the two labels Cy3 and Cy5 allow for fluorescence resonance energy transfer in the closed state. Upon hybridization, the probe opens up resulting in a fluorescence intensity increase of die donor and a fluorescence intensity decrease of the acceptor. The ratio of the acceptor-to-donor fluorescence intensities is independent of the amount of probe and provides a quantitative measure of the free target concentration.