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    Higher-order fluorescence correlation spectroscopy (FCS) uniquely identified DNA hairpin conformational states. This advanced technique revealed distinct brightness levels and populations, clarifying folding dynamics beyond conventional FCS capabilities.

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    Area of Science:

    • Biophysics
    • Molecular Biology
    • Spectroscopy

    Background:

    • Previous fluorescence correlation spectroscopy (FCS) studies indicated at least three DNA hairpin conformational states.
    • These states were distinguished by fluorescent dye-quencher label brightness, with rapid fluctuations and a static state observed.
    • Conventional FCS limitations in uniquely distinguishing these states and their roles in folding dynamics were noted.

    Purpose of the Study:

    • To investigate DNA hairpin conformational states using novel higher-order FCS techniques.
    • To overcome the limitations of conventional FCS in uniquely identifying and characterizing these states.
    • To directly measure the brightnesses and populations of all observed conformational states.

    Main Methods:

    • Application of newly developed higher-order fluorescence correlation spectroscopy (FCS) techniques.
    • Analysis of rapid fluctuations and static states observed in DNA hairpin folding dynamics.
    • Quantification of brightness and population distributions for distinct conformational states.

    Main Results:

    • Higher-order FCS uniquely identified static and rapidly fluctuating DNA hairpin conformational states.
    • Direct measurement of brightnesses and populations for all three observed states was achieved.
    • Rapid fluctuations were attributed to a reversible reaction between folded and random-coil conformations.
    • A third, brightest state, potentially representing extended unfolded conformations, was identified.

    Conclusions:

    • Higher-order FCS provides crucial information unattainable by conventional FCS for DNA hairpin dynamics.
    • The study elucidates the mechanism of rapid fluctuations and identifies distinct conformational states.
    • Extended unfolded conformations represent a significant, albeit minor, population under physiological conditions.