RNA folding thermodynamics are crucial for structure prediction, which requires characterization of both enthalpic and entropic contributions of tertiary motifs to conformational stability. We explore the temperature dependence of RNA folding due to the ubiquitous GAAA tetraloop-receptor docking interaction, exploiting immobilized and freely diffusing single-molecule fluorescence resonance energy transfer (smFRET) methods. The equilibrium constant for intramolecular docking is obtained as a function of temperature (T = 21-47 °C), from which a van't Hoff analysis yields the enthalpy (ΔH°) and entropy (ΔS°) of docking. Tetraloop-receptor docking is significantly exothermic and entropically unfavorable in 1 mM MgCl 2 and 100 mM NaCl, with excellent agreement between immobilized (ΔH° = −17.4 ± 1.6 kcal/mol, and ΔS° = −56.2 ± 5.4 cal mol −1 K −1 ) and freely diffusing (ΔH° = −17.2 ± 1.6 kcal/mol, and ΔS° = −55.9 ± 5.2 cal mol −1 K −1 ) species. Kinetic heterogeneity in the tetraloop-receptor construct is unaffected over the temperature range investigated, indicating a large energy barrier for interconversion between the actively docking and nondocking subpopulations. Formation of the tetraloop-receptor interaction can account for ~60% of the ΔH°a nd ΔS° of P4-P6 domain folding in the Tetrahymena ribozyme, suggesting that it may act as a thermodynamic clamp for the domain. Comparison of the isolated tetraloop-receptor and other tertiary folding thermodynamics supports a theme that enthalpy-versus entropy-driven folding is determined by the number of hydrogen bonding and base stacking interactions.RNA folding is generally hierarchical, with tertiary structure occurring through interactions of preformed secondary elements (1-3). As a result, the kinetics and thermodynamics of tertiary interactions are crucial to understanding RNA folding and functionality as well for accurate structural predictions (1,4). Toward this end, individual folding motifs must be characterized both in isolation and in combination for a unifying thermodynamic description of RNA folding † This work was supported in part by the NSF, NIST, the W. M. Keck Foundation initiative in RNA sciences at the University of Colorado, Boulder, and PicoQuant, GmbH. J.L.F. was supported in part by Optical Science and Engineering Program NSF-IGERT and CU Biophysics Training (T32 GM-065103) grants.© 2009 American Chemical Society * To whom correspondence should be addressed: JILA, University of Colorado, 440 UCB, Boulder, CO 80309-0440. djn@jila.colorado.edu. Phone: (303) . SUPPORTING INFORMATION AVAILABLEPrediction of the effect of donor quantum yield (Q D ) on the observed FRET efficiency (E FRET ) as a function of Cy3-Cy5 distance (R) ( Figure S1) and mean cross correlations of donor and acceptor channels for the same sample containing tetraloop-receptor constructs under freely diffusing single-molecule conditions at 21 and 45 °C ( Figure S2). This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ...

Stimulated Emission Depletion (STED) Microscopy has evolved into a well established method offering optical superresolution below 50 nm. Running both excitation and depletion lasers in picosecond pulsed modes allows for highest optical resolution as well as fully exploiting the photon arrival time information using time-resolved single photon counting (TCSPC). Non-superresolved contributions can be easily dismissed through time-gated detection (gated STED) or a more detailed fluorescence decay analysis (FLIM-STED), both leading to an even further improved imaging resolution. Furthermore, these methods allow for accurate separation of different fluorescent species, especially if subtle differences in the excitation and emission spectra as well as the fluorescence decay are taken into account in parallel. STED can also be used to shrink the observation volume while studying the dynamics of diffusing species in Fluorescence Correlation Spectroscopy (FCS) to overcome averaging issues along long transit paths. A further unique advantage of STED-FCS is that the observation spot diameter can be tuned in a gradual manner enabling, for example, determining the type of hindered diffusion in lipid membrane studies. Our completely pulsed illumination scheme allows realizing an improved STED-FCS data acquisition using pulsed interleaved excitation (PIE). PIE-STED-FCS allows for a straightforward online check whether the STED laser has an influence on the investigated diffusion dynamics.

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