Short, synthetic and selectively 13C-labeled RNA sequences for the NMR structure determination of protein-RNA complexes

We report an optimized synthesis of all canonical 2′-O-TOM protected ribonucleoside phosphoramidites and solid supports containing [13C5]-labeled ribose moieties, their sequence-specific introduction into very short RNA sequences and their use for the structure determination of two protein-RNA complexes. These specifically labeled sequences facilitate RNA resonance assignments and are essential to assign a high number of sugar-sugar and intermolecular NOEs, which ultimately improve the precision and accuracy of the resulting structures. This labeling strategy is particularly useful for the study of protein-RNA complexes with single-stranded RNA in solution, which is rapidly an increasingly relevant research area in biolog

Short, synthetic and selectively 13C-labeled RNA sequences for the NMR structure determination of protein–RNA complexes

We report an optimized synthesis of all canonical 2′-O-TOM protected ribonucleoside phosphoramidites and solid supports containing [13C5]-labeled ribose moieties, their sequence-specific introduction into very short RNA sequences and their use for the structure determination of two protein–RNA complexes. These specifically labeled sequences facilitate RNA resonance assignments and are essential to assign a high number of sugar–sugar and intermolecular NOEs, which ultimately improve the precision and accuracy of the resulting structures. This labeling strategy is particularly useful for the study of protein–RNA complexes with single-stranded RNA in solution, which is rapidly an increasingly relevant research area in biology

Synthesis of selectively 15N-labeled 2'-O-{[(triisopropylsilyl)oxy]methyl}(=tom)-protected ribonucleoside phosphoramidites and their incorporation into a bistable 32mer RNA sequence

We present optimized reaction conditions for the conversion of 2'-O-{[(triisopropylsilyl)oxy]methyl}(tom) protected uridine and adenosine nucleosides into the corresponding protected (3-15N)-labeled uridine and cytidine and (1-15N)-labeled adenosine and guanosine nucleosides. On a DNA synthesizer, the resulting 15N-labeled 2'-O-tom-protected phosphoramidite building blocks were efficiently incorporated into five selected positions of a hairpin bi-stable 32mer RNA sequence. By 2D-HSQC and HNN-COSY expts. in H2O/D2O 9:1, the 15N-signals of all base-paired 15N-labeled nucleotides could be identified and attributed to one of the two coexisting structures of 32mer RNA sequence. [on SciFinder (R)

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 15N 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 1H-detected 15N exchange spectroscopy (EXSY) pulse sequence provided cross-peaks for resonances belonging to different folds that interchange on the time scale of longitudinal relaxation of 15N nuclei bound to imino protons. The 34mer RNA sequence exhibits two folds which exchange on the observable time scale (tau_obs T1{15N} < 5 s) and a third fold which is static on this time scale. A 1D version of the 15N 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 Ea 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

Probing Mechanism and Transition State of RNA Refolding

Kinetics and the atomic detail of RNA refolding are only poorly understood. It has been proposed that conformations with transient base pairing interaction are populated during RNA refolding, but a detailed description of those states is lacking. By NMR and CD spectroscopy, we examined the refolding of a bistable RNA and the influence of urea, Mg2+, and spermidine on its refolding kinetics. The bistable RNA serves as a model system and exhibits two almost equally stable ground-state conformations. We designed a photolabile caged RNA to selectively stabilize one of the two ground-state conformations and trigger RNA refolding by in situ light irradiation in the NMR spectrometer. We can show that the refolding kinetics of the bistable RNA is modulated by urea, Mg2+, and spermidine by different mechanisms. From a statistical analysis based on elementary rate constants, we deduce the required number of base pairs that need to be destabilized during the refolding transition and propose a model for the transition state of the folding reaction