Folding and unfolding kinetics of DNA hairpins in flowing solution by multiparameter fluorescence correlation spectroscopy.
Dynamic equilibrium between the folded and unfolded conformations of single stranded DNA hairpin molecules containing polythymine hairpin loops was investigated using simultaneous two-beam fluorescence cross-correlation spectroscopy and single beam autocorrelation spectroscopy. The hairpins were end-labeled with a fluorescent dye and a quencher, such that folding and unfolding of the DNA hairpin primary structure caused the dye fluorescence to fluctuate on the same characteristic time scale as the folding and unfolding reaction. These fluctuations were observed as the molecules flowed sequentially between two spatially offset, microscopic detection volumes. Cross-correlation analysis of fluorescence from the two detection volumes revealed the translational diffusion and flow properties of the hairpins, as well as the average molecular occupancy of the two volumes. Autocorrelation analysis of the fluorescence from the individual detection volumes revealed the kinetics of hairpin folding and unfolding, with the parameters relating to diffusion, flow, and molecular occupancy constrained to the values determined from the cross-correlation analysis. This allowed unambiguous characterization of the folding and unfolding kinetics, without the need to determine the hydrodynamic properties by analyzing a separate control sample. The analysis revealed nonexponential relaxation kinetics and DNA size-dependent folding times characteristic of dynamic heterogeneity in the DNA hairpin-forming mechanism.
Jung
Jaemyeong
J
Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523, USA.
Van Orden
Alan
A
D002627:Chemistry, Physical / Q000379:methods*; D004247:DNA / Q000737:chemistry*; D004277:DNA, Single-Stranded / Q000737:chemistry; D005456:Fluorescent Dyes / Q000494:pharmacology; D007700:Kinetics; D015233:Models, Statistical; D008968:Molecular Conformation; D016011:Normal Distribution; D009690:Nucleic Acid Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013696:Temperature; D013997:Time Factors
D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D004247:DNA