Kinetics of RNA refolding in dynamic equilibrium by 1H-detected 15N exchange NMR spectroscopy.
By implementing new NMR methods that were designed to map very slow exchange processes we have investigated and characterized the refolding kinetics of a thermodynamically stable 34mer RNA sequence in dynamic equilibrium. The RNA sequence was designed to undergo a topologically favored conformational exchange between different hairpin folds, serving as a model to estimate the minimal time required for more complex RNA folding processes. Chemically prepared RNA sequences with sequence-selective (15)N labels provided the required signal separation and allowed a straightforward signal assignment of the imino protons by HNN correlation experiments. The 2D version of the new (1)H-detected (15)N exchange spectroscopy (EXSY) pulse sequence provided cross-peaks for resonances belonging to different folds that interchange on the time scale of longitudinal relaxation of (15)N nuclei bound to imino protons. The 34mer RNA sequence exhibits two folds which exchange on the observable time scale (tau(obs) approximately T(1){(15)Nu} < 5 s) and a third fold which is static on this time scale. A 1D version of the (15)N exchange experiment allowed the measurement of the exchange rates between the two exchanging folds as a function of temperature and the determination of the corresponding activation energies E(a) and frequency factors A. We found that the refolding rates are strongly affected by an entropically favorable preorientation of the replacing strand. The activation energies are comparable to values obtained for the slow refolding of RNA sequences of similar thermodynamic stability but less favorable topology.
Wenter
Philipp
P
Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, EPFL, Batochime, 1015 Lausanne, Switzerland.
Bodenhausen
Geoffrey
G
Dittmer
Jens
J
Pitsch
Stefan
S
D001483:Base Sequence; D003903:Deuterium / Q000737:chemistry*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry*; D013816:Thermodynamics
D009587:Nitrogen Isotopes; D012313:RNA; D003903:Deuterium