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

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

Systematic screens of proteins binding to synthetic microRNA precursors

We describe a new, broadly applicable methodology for screening in parallel interactions of RNA-binding proteins (RBPs) with large numbers of microRNA (miRNA) precursors and for determining their affinities in native form in the presence of cellular factors. The assays aim at identifying pre-miRNAs that are potentially affected by the selected RBP during their biogenesis. The assays are carried out in microtiter plates and use chemiluminescent readouts. Detection of bound RBPs is achieved by protein or tag-specific antibodies allowing crude cell lysates to be used as a source of RBP. We selected 70 pre-miRNAs with phylogenetically conserved loop regions and 25 precursors of other well-characterized miRNAs for chemical synthesis in 3′-biotinylated form. An equivalent set in unmodified form served as inhibitors in affinity determinations. By testing three RBPs known to regulate miRNA biogenesis on this set of pre-miRNAs, we demonstrate that Lin28 and hnRNP A1 from cell lysates or as recombinant protein domains recognize preferentially precursors of the let-7 family, and that KSRP binds strongly to pre-miR-1-

New methods for the investigation of RNA refolding by NMR spectroscopy

A detailed characterization of the time scales and mechanisms by which RNA molecules change their folding structure upon binding of metabolites or during catalysis is important for the understanding of the different biological functions of RNA. The folding from an unfolded, denaturated state has been extensively studied, establishing hierarchical pathways, in which fast formation of secondary structural elements precedes the folding of tertiary elements. In contrast, only a few examples of time-resolved RNA refolding in the native state have been reported so far. Specifically, NMR spectroscopy has not yet been employed for the investigation of RNA refolding despite its intrinsic sensibility to dynamic structural changes. In the project presented here, new tools for the identification of RNA secondary structures and the quantification of RNA refolding processes by NMR spectroscopy have been developed and employed. Preparation of 15N-labeled phosphoramidites and their sequence-selective introduction into RNA sequences allowed a straightforward identification of base pairs and imino protons by HNN-COSY and 15N HSQC experiments, respectively. Synthesis and introduction of NPE-protected uridines and guanosines into bistable RNA sequences provided a powerful method for the photoinduced preparation of selected RNA folds far from equilibrium. When introduced into bistable RNA sequences they allow to disrupt selected base pairs, thereby destabilizing the associated secondary structures. As a consequence, one out of usually two coexisting fold could be prepared selectively in its caged form. Upon photolysis, the native sequence was released under physiological conditions with completely retained, preselected secondary structure arrangement. Refolding into the thermodynamic equilibrium was subsequently followed by real-time imino proton NMR spectroscopy, providing quantitative, time-resolved and structural information for the refolding processes of three bistable RNA sequences and a Mg2+-induced secondary and tertiary structure refolding. Depending on their topology, these RNA sequences refold either via a dissociative mechanism in which the disruption of existing base pairs precedes the formation of new ones or via an associative mechanism in which new base pairs are formed from initially unpaired sequence regions simultaneously to the detachement of existing base pairs. In the transition state approximately half of the base pairs are disrupted. The investigation of a bistable RNA sequence designed to undergo a topologically favored refolding processes was carried out by exchange sensitive NMR experiments. The introduction of sequence specific 15N-labeles into RNA sequences allowed the implementation of exchange sensitive 1D and 2D 1H/15N-heteronuclear NMR methods which were designed to map very slow exchange. We found that a 34mer RNA sequence exhibits two folds which exchange on the observable time scale (τobs = 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, however, are high and largely independent on the topology of the system. Interestingly, the activation energies determined for the secondary structure rearrangement of these quite small RNA sequences are similar to the values reported for secondary or tertiary structure rearrangements of much larger and more complex systems. The refolding rate constants are in the range of k = 0.002 - 90 min-1 at 25°C and therefore at least four orders of magnitude smaller than the rates observed for hairpin formation of similar systems from an unfolded state, which occur with k = 105-107 s-1. Thus, uncatalyzed secondary structure rearrangements of already folded RNAs happen at rates that are in the range of rate-limiting steps for biological reactions

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

Kinetics of photoinduced RNA refolding by real-time NMR spectroscopy

By introducing a photolabile group in the Watson-Crick base-pairing site of a guanosine residue, a bistable 20-base RNA sequence was forced into a less stable conformation. A single laser pulse released the native sequence, and the subsequent refolding equilibration was monitored with time-resolved NMR spectroscopy. This permitted a quant. description of the refolding process. [on SciFinder (R)

Conformational dynamics of bistable RNAs studied by time-resolved NMR spectroscopy

The structural transition between two alternate conformations of bistable RNAs has been characterized by time-resolved NMR spectroscopy. The mechanism, kinetics, and thermodynamics underlying the global structural transition of bistable RNAs were delineated. Both bistable RNA conformations and a partial unstructured RNA of identical sequence could be trapped using photolabile protecting groups. This trapping allowed for an investigation of the initial folding from an unfolded RNA to one of the preferred conformations of the bistable RNA and of the structural transitions involved. Folding of the secondary structure elements occurs rapidly, while the global structural transition of the bistable RNA occurs on a time scale of minutes and shows marked temperature dependence. Comparison of these results with bistable systems previously investigated leads to the prediction of activation enthalpies (Delta H-double dagger) associated with global structural transitions in RNA