A Caged Uridine for the Selective Preparation of an RNA Fold and Determination of its Refolding Kinetics by Real‐Time NMR

The biological function of many RNAs is linked to a reversible conformational switching between active and inactive folds that differ in their secondary and/or tertiary structures. Regulatory RNAs, such as riboswitches, 3] and catalytic RNAs, such as ribozymes, undergo this conformational switching upon binding of metabolites or during catalysis. In general, the energetic differences between the alternative conformations are only small, and therefore the equilibrium distribution is strongly affected by ligand or substrate binding, or by small structural modifications, such as methylation of the nucleobases. Recently, we have introduced a method for studying the kinetics of RNA-conformational switching by time-resolved NMR spectroscopy. The method is based on RNA sequences modified with 1-(2-nitrophenyl)ethyl-substituted (NPE-substituted) nucleobases, which are designed to impair the formation of selected base pairs. When introduced at proper sequence position within the oligonucleotide fold, these photocleavable modifications lead to a specific destabilization of preselected folds. Rapid and traceless removal of the photolabile groups, carried out by coupling laser optics into the NMR tube, then allows NMR detection of the refolding process. The induced structural transition can be monitored from the time-dependent increase and decrease in intensity of imino proton resonances, which belong to specific base pairs present in specific folds. For medium-sized oligonucleotides, these resonances often show sufficient resolution in chemical shifts to be resolved in 1D experiments. In this context, we have already reported the refolding kinetics and a plausible refolding mechanism of the bistable 20-mer RNA sequence 5’-r[GACCGGAAGGUCCGCCUUCC]-3’. For this study, an (S)-NPE-substituted guanosine was introduced at position 6. The activation energy was Ea=26 kcalmol 1 and the frequency factor was A=10 s 1 for the forward process, or 31 kcalmol 1 and 10 s 1 for the backward process, respectively. These refolding rates are surprisingly low compared to the folding of similar systems from an unfolded state and are in agreement with a transient disruption of half of the base pairs. Together with an additional dynamical characterization by comparative water-exchange experiments, an associative mechanism was deduced, in which refolding is initiated by base-pair contacts between previously unpaired nucleotides. In this communication, we extend our real-time-NMR investigation of RNA secondary structural transitions to another bistable, 20-mer RNA sequence 5’-r(GAAGGGCAACCUUCGGGUUG)-3’ that was designed by Hçbartner et al. This RNA sequence adopts two almost equally stable hairpin folds A and B, each consisting of a five-base-pair stem, a stable tetraloop and six unpaired nucleotides (Figure 1C, D). In order to selectively destabilize fold B, we introduced an (R)-NPE-protected uridine at position U18 (Figure 1B). Structural and kinetic characterization were carried out by analyzing the imino proton NMR signals, each representing a specific base pair. The unmodified sequence shows 12 signals in the iminoproton region (Figure 1D), which were assigned to the base pairs of the two coexisting folds A and B by H,H-NOESY experiments. The signals assigned to fold A agreed well with the signals of the corresponding truncated 15-mer RNA hairpin (Figure 1A). After introduction of the (R)-NPE-protected U18 into the 20-mer RNA sequence, only the set of six imino proton signals characteristic of fold A were detected (Figure 1B). Importantly, this single NPE-modification completely suppressed the formation of fold B by preventing the formation of the associated U18–A9 base pair. Removal of the NPE group by photolysis and equilibration resulted in a NMR spectrum (Figure 1C) that was practically identical to the spectrum of the parent sequence (Figure 1D). To determine the kinetic parameters for refolding, the NPE group was released by a short continuous-wave laser pulse (duration 1.5 s) guided by an optical fibre into the NMR tube. Relaxation of the system towards equilibrium was monitored by the subsequent recording of 1D H spectra. 11] The refolding rate constants were obtained by fitting the time-dependent intensities of two selected pairs of imino proton signals according to the equation for a reversible first-order process (Figure 2A–D). The independently determined equilibrium constant KA! B was employed as an additional fit parameter. The thermodynamic parameters DHA! B=1.6 0.3 kcalmol 1 and DSA! B=6.0 1.0 calmol K 1 were determined from the temperature dependence of these K values (Figure S1 in the Supporting Information). The directly determined rate constants kA! B ranged between 0.005 0.001 s 1 at 283 K and 0.091 0.020 s 1 at 298 K; the rate constants kB! A were calculated from the kA! B and K values, and ranged between 0.005 0.002 s 1 at 283 K and 0.076 0.020 s 1 at 298 K. In each experiment, rates that were identical within the error range were determined for the two independently analyzed pairs of imino proton signals (all rates are given in Figure S2 in the Supporting Information). Arrhenius analysis revealed an activation energy of Ea,A! B=32.7 3.0 kcalmol 1 (Ea,B! A=31.0 [a] P. Wenter, A. Hainard, Prof. Dr. S. Pitsch Laboratory of Nucleic Acid Chemistry Ecole Polyt! chnique F! d! rale de Lausanne (EPFL) 1015 Lausanne (Switzerland) Fax: (+41)21-693-9355 E-mail : stefan.pitsch@epfl.ch [b] B. F6rtig, Prof. Dr. H. Schwalbe Johann Wolfgang Goethe University Institute for Organic Chemistry and Chemical Biology Center for Biomolecular Magnetic Resonance Marie-Curie Strasse 11, Frankfurt, 60439 Frankfurt/Main (Germany) Fax: (+49)69-7982-9515 E-mail : schwalbe@nmr.uni-frankfurt.de Supporting information for this article is available on the WWW under http://www.chembiochem. org or from the author.