Researchers often use fluorescence correlation spectroscopy to study conformational changes such as DNA hairpin folding -- labeling the DNA with a dye-quencher pair, for example, so that folding results in the quenching of the dye, and unfolding in the recovery of the fluorescence. The technique may produce only limited findings, however, if the reaction under study takes place on a timescale similar to the transit time of molecules as they pass through the optical probe region.For this reason, a team at Colorado State University in Fort Collins developed a dual-beam fluorescence fluctuation spectroscopy method with which to monitor the reaction kinetics. “We had reason to believe the reaction mechanism for DNA hairpin folding was more complicated than previously thought,” explained Alan Van Orden, principal investigator of the study. Researchers have developed a dual-beam fluorescence fluctuation spectroscopy method with which to monitor the kinetics of DNA hairpin folding. The technique measures the cross-correlation function of the fluorescence fluctuations while molecules flow between two spatially offset probe regions. By adjusting the flow velocity of the solution in the capillary, the researchers monitored the changes in the cross-correlation vs. flow velocity, which provided the relaxation properties of the reaction in the time range of about 100 μs to 10 ms -- thus shedding light on the folding and unfolding kinetics of a DNA hairpin molecule. D = detector. Folding occurs when all of the complementary base pairs in the stem region of the DNA hairpin form a stable double helix. Researchers had long believed this to be a straightforward two-step process in which only the unfolded DNA and the fully folded hairpin structures are present. “However, recent results from our lab and others suggested the reaction occurred in a multistep process involving fast and slow reactions,” Van Orden said. “Previous experiments had observed only the fast, intermediate steps.”In the Jan. 10 issue of the Journal of Physical Chemistry B, the investigators reported the use of the new method to observe the complete multistep reaction in a single experiment. The method combines single-beam fluorescence autocorrelation spectroscopy, dual-beam fluorescence cross-correlation spectroscopy and photon-counting histogram analysis to probe the chemical relaxation properties of molecules in solution.In the study, the researchers measured the cross-correlation function of the fluorescence fluctuations while the molecules flowed between two spatially offset probe regions, created by focusing two spatially offset beams from an air-cooled 514.5-nm Ar+ laser made by Melles Griot (now CVI Melles Griot) of Carlsbad, Calif., inside a flow chamber using a 100×, 1.25-NA oil-immersion objective made by Edmund Optics of Barrington, N.J. The fluorescence was collected by the objective and monitored using two single-photon-counting avalanche photodiode detectors made by PerkinElmer Optoelectronics of Wellesley, Mass.The scientists adjusted the flow velocity of the solution and monitored the changes in the cross-correlation function vs. flow velocity. “These changes revealed the time-dependent properties of the reaction in the time range of about 100 μs to 10 ms,” Van Orden said. “Simultaneous single-beam measurements probed the reactions occurring between 1 and 200 μs. This made it possible to observe the complete folding of a DNA hairpin containing four base pairs in the stem region.”Challenges arose along the way. The researchers had to resolve the forward and reverse rate constants of the fast and slow reactions -- that is, the rates at which the forward chemical processes and their reverse reactions occur. They derived these from the reaction times and the equilibrium distributions. They obtained the former from the auto- and cross-correlation measurements and developed a means to determine the equilibrium distributions using photon-counting histogram analysis, a method that measures the number of molecules with different fluorescence intensities. Each intensity represents a different conformational state of the hairpin. By measuring the number of molecules in each state, they determined the equilibrium constants for the reactions.Using the technique, the researchers observed the complete folding trajectory of a DNA hairpin molecule containing a 21-nucleotide polythymine loop and a four-base-pair stem. These experiments confirmed a three-state reaction with extended, intermediate and native hairpin conformations. The researchers observed both fast and slow fluctuations, attributing the former to the folding and unfolding of an intermediate and the latter to the formation and disruption of the native stem structure. Thus the study provided insight into the mechanisms of intramolecular DNA duplex formation by highlighting the role of the intermediate conformations.Other methods -- single-molecule fluorescence, laser-induced temperature jump spectroscopy and single-molecule optical trapping -- are available for the study of base-pairing, although they each have disadvantages, according to Van Orden. The time resolution of single-molecule fluorescence is 1 ms, whereas that of fluorescence correlation spectroscopy can be as low as 10 ns. Laser-induced temperature jump spectroscopy has a time resolution of less than 1 ns but is not as sensitive to a broad range of times as fluorescence correlation spectroscopy, and the hairpin is generally unstable under the conditions during which temperature jumps are performed. Finally, optical trapping produces millisecond time resolution and, therefore, is not sensitive to the fast reactions; because the technique destabilizes the intermediate states of the reaction, the intermediate reactions are not present.As the researchers continue to work with the technique, they would like to investigate how the interaction of DNA hairpins with proteins affects the dynamics of the DNA. “For example, DNA helicase is an enzyme that unwinds base-paired regions of DNA. Our experiments could provide novel insight into how this enzyme performs its function,” Van Orden said.