Probing the complete folding trajectory of a DNA hairpin using dual beam fluorescence fluctuation spectroscopy.
The conformational fluctuations of dye-quencher labeled DNA hairpin molecules in aqueous solution were investigated using dual probe beam fluorescence fluctuation spectroscopy. The measurements revealed the flow and diffusion times of the DNA molecules through two spatially offset optical probe regions, the absolute and relative concentrations of each conformational substate of the DNA, and the kinetics of the DNA hairpin folding and unfolding reactions in the 1 micros to 10 ms time range. A DNA hairpin containing a 21-nucleotide polythymine loop and a 4-base pair stem exhibited double exponential relaxation kinetics, with time constants of 84 and 393 micros. This confirms that folding and melting of the DNA hairpin structure is not a two state process but proceeds by way of metastable intermediate states. The fast time constant corresponds to formation and unfolding of an intermediate, and the slow time constant is due to formation and disruption of the fully base-paired stem. This is consistent with a previous study of a similar DNA hairpin with a 5-base pair stem, in which the fast reaction was attributed to the fluctuations of an intermediate DNA conformation [J. Am. Chem. Soc. 2006, 128, 1240-1249]. In that case, reactions involving the native conformation could not be observed directly due to the limited observation time range of the fluorescence correlation spectroscopy experiment. The intermediate states of the DNA hairpins are suggested to be due to a collapsed ensemble of folded hairpins containing various partially folded or misfolded conformations.
Jung
Jaemyeong
J
Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523, USA.
Ihly
Rachelle
R
Scott
Eric
E
Yu
Ming
M
Van Orden
Alan
A
D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D009690:Nucleic Acid Conformation; D013050:Spectrometry, Fluorescence; D044366:Transition Temperature
D004247:DNA