P1 Stem Research Articles

The Escherichia coli ribB riboswitch senses flavin mononucleotide within a defined transcriptional window.

Riboswitches are metabolite-binding RNA regulators that modulate gene expression at the levels of transcription and translation. One of the hallmarks of riboswitch regulation is that they undergo structural changes upon metabolite binding. While a lot of effort has been put to characterize how the metabolite is recognized by the riboswitch, there is still relatively little information regarding how ligand sensing is performed within a transcriptional context. Here, we study the ligand-dependent cotranscriptional folding of the FMN-sensing ribB riboswitch of Escherichia coli. Using RNase H assays to study nascent ribB riboswitch transcripts, DNA probes targeting the P1 and sequestering stems indicate that FMN binding leads to the protection of these regions from RNase H cleavage, consistent with the riboswitch inhibiting translation initiation when bound to FMN. Our results show that ligand sensing is strongly affected by the position of elongating RNA polymerase, which is defining an FMN binding transcriptional window that is bordered in its 3' extremity by a transcriptional pause site. Also, using successively overlapping DNA probes targeting a subdomain of the riboswitch, our data suggest the presence of a previously unsuspected helical region involving the 3' strand of the P1 stem. Our results show that this helical region is conserved across bacterial species, thus suggesting that this predicted structure, the anti*-P1 stem, is involved in the FMN-free conformation of the ribB riboswitch. Overall, our study further demonstrates that intricate folding strategies may be used by riboswitches to perform metabolite sensing during the transcriptional process.

Bifidobacterium bifidum SAM-VI Riboswitch Conformation Change Requires Peripheral Helix Formation.

The Bifidobacterium bifidum SAM-VI riboswitch undergoes dynamic conformational changes that modulate downstream gene expression. Traditional structural methods such as crystallography capture the bound conformation at high resolution, and additional efforts would reveal details from the dynamic transition. Here, we revealed a transcription-dependent conformation model for Bifidobacterium bifidum SAM-VI riboswitch. In this study, we combine small-angle X-ray scattering, chemical probing, and isothermal titration calorimetry to unveil the ligand-binding properties and conformational changes of the Bifidobacterium bifidum SAM-VI riboswitch and its variants. Our results suggest that the SAM-VI riboswitch contains a pre-organized ligand-binding pocket and stabilizes into the bound conformation upon binding to SAM. Whether the P1 stem formed and variations in length critically influence the conformational dynamics of the SAM-VI riboswitch. Our study provides the basis for artificially engineering the riboswitch by manipulating its peripheral sequences without modifying the SAM-binding core.

A transient intermediate RNA structure underlies the regulatory function of the E. coli thiB TPP translational riboswitch.

Riboswitches are cis-regulatory RNA elements that regulate gene expression in response to ligand binding through the coordinated action of a ligand-binding aptamer domain (AD) and a downstream expression platform (EP). Previous studies of transcriptional riboswitches have uncovered diverse examples that utilize structural intermediates that compete with the AD and EP folds to mediate the switching mechanism on the timescale of transcription. Here we investigate whether similar intermediates are important for riboswitches that control translation by studying the Escherichia coli thiB thiamin pyrophosphate (TPP) riboswitch. Using cellular gene expression assays, we first confirmed that the riboswitch acts at the level of translational regulation. Deletion mutagenesis showed the importance of the AD-EP linker sequence for riboswitch function. Sequence complementarity between the linker region and the AD P1 stem suggested the possibility of an intermediate nascent RNA structure called the antisequestering stem that could mediate the thiB switching mechanism. Experimentally informed secondary structure models of the thiB folding pathway generated from chemical probing of nascent thiB structures in stalled transcription elongation complexes confirmed the presence of the antisequestering stem, and showed it may form cotranscriptionally. Additional mutational analysis showed that mutations to the antisequestering stem break or bias thiB function according to whether the antisequestering stem or P1 is favored. This work provides an important example of intermediate structures that compete with AD and EP folds to implement riboswitch mechanisms.

The function of twister ribozyme variants in non-LTR retrotransposition in Schistosoma mansoni.

The twister ribozyme is widely distributed over numerous organisms and is especially abundant in Schistosoma mansoni, but has no confirmed biological function. Of the 17 non-LTR retrotransposons known in S. mansoni, none have thus far been associated with ribozymes. Here we report the identification of novel twister variant (T-variant) ribozymes and their function in S. mansoni non-LTR retrotransposition. We show that T-variant ribozymes are located at the 5′ end of Perere-3 non-LTR retrotransposons in the S. mansoni genome. T-variant ribozymes were demonstrated to be catalytically active in vitro. In reporter constructs, T-variants were shown to cleave in vivo, and cleavage of T-variants was sufficient for the translation of downstream reporter genes. Our analysis shows that the T-variants and Perere-3 are transcribed together. Target site duplications (TSDs); markers of target-primed reverse transcription (TPRT) and footmarks of retrotransposition, are located adjacent to the T-variant cleavage site and suggest that T-variant cleavage has taken place inS. mansoni. Sequence heterogeneity in the TSDs indicates that Perere-3 retrotransposition is not site-specific. The TSD sequences contribute to the 5′ end of the terminal ribozyme helix (P1 stem). Based on these results we conclude that T-variants have a functional role in Perere-3 retrotransposition.

Insights into xanthine riboswitch structure and metal ion-mediated ligand recognition.

Riboswitches are conserved functional domains in mRNA that mostly exist in bacteria. They regulate gene expression in response to varying concentrations of metabolites or metal ions. Recently, the NMT1 RNA motif has been identified to selectively bind xanthine and uric acid, respectively, both are involved in the metabolic pathway of purine degradation. Here, we report a crystal structure of this RNA bound to xanthine. Overall, the riboswitch exhibits a rod-like, continuously stacked fold composed of three stems and two internal junctions. The binding-pocket is determined by the highly conserved junctional sequence J1 between stem P1 and P2a, and engages a long-distance Watson–Crick base pair to junction J2. Xanthine inserts between a G–U pair from the major groove side and is sandwiched between base triples. Strikingly, a Mg2+ ion is inner-sphere coordinated to O6 of xanthine and a non-bridging oxygen of a backbone phosphate. Two further hydrated Mg2+ ions participate in extensive interactions between xanthine and the pocket. Our structure model is verified by ligand binding analysis to selected riboswitch mutants using isothermal titration calorimetry, and by fluorescence spectroscopic analysis of RNA folding using 2-aminopurine-modified variants. Together, our study highlights the principles of metal ion-mediated ligand recognition by the xanthine riboswitch.

The conserved structure of plant telomerase RNA provides the missing link for an evolutionary pathway from ciliates to humans

Telomerase is essential for maintaining telomere integrity. Although telomerase function is widely conserved, the integral telomerase RNA (TR) that provides a template for telomeric DNA synthesis has diverged dramatically. Nevertheless, TR molecules retain 2 highly conserved structural domains critical for catalysis: a template-proximal pseudoknot (PK) structure and a downstream stem-loop structure. Here we introduce the authentic TR from the plant Arabidopsis thaliana, called AtTR, identified through next-generation sequencing of RNAs copurifying with Arabidopsis TERT. This RNA is distinct from the RNA previously described as the templating telomerase RNA, AtTER1. AtTR is a 268-nt Pol III transcript necessary for telomere maintenance in vivo and sufficient with TERT to reconstitute telomerase activity in vitro. Bioinformatics analysis identified 85 AtTR orthologs from 3 major clades of plants: angiosperms, gymnosperms, and lycophytes. Through phylogenetic comparisons, a secondary structure model conserved among plant TRs was inferred and verified using in vitro and in vivo chemical probing. The conserved plant TR structure contains a template-PK core domain enclosed by a P1 stem and a 3' long-stem P4/5/6, both of which resemble a corresponding structural element in ciliate and vertebrate TRs. However, the plant TR contains additional stems and linkers within the template-PK core, allowing for expansion of PK structure from the simple PK in the smaller ciliate TR during evolution. Thus, the plant TR provides an evolutionary bridge that unites the disparate structures of previously characterized TRs from ciliates and vertebrates.

Molecular dynamics simulation of the binding process of ligands to the add adenine riboswitch aptamer.

Riboswitches are RNA-structured elements that modulate gene expression through changing their conformation in response to specific metabolite binding. However, the regulation mechanisms of riboswitches by ligand binding are still not well understood. At present two possible ligand-binding mechanisms have been proposed: conformational selection and induced fit. Based on explicit-solvent molecular dynamics (MD) simulations, we have studied the process of the binding of ligands (adenines) to add adenine riboswitch aptamer (AARA) in detail. Our results show that the relative high flexibility of the junction J23 of AARA allows the ligands to be captured by the binding pocket of AARA in a near-native state, which may be driven by hydrophobic and base-stacking interactions. In addition, the binding of a ligand makes the stem P1 and the junction J23 of AARA more stable than in the absence of the ligand. Generally, our analyses show that the ligand-binding process of the add adenine riboswitch may be partially embodied by a conformational selection mechanism.

The NTP binding site of the polymerase ribozyme.

A previously developed RNA polymerase ribozyme uses nucleoside triphosphates (NTPs) to extend a primer 3′-terminus, templated by an RNA template with good fidelity, forming 3′-5′-phosphordiester bonds. Indirect evidence has suggested that the ribozyme's accessory domain binds the NTP with a highly conserved purine-rich loop. To determine the NTP binding site more precisely we evolved the ribozyme for efficient use of 6-thio guanosine triphosphate (6sGTP). 6sGTP never appeared in the evolutionary history of the ribozyme, therefore it was expected that mutations would appear at the NTP binding site, adapting to more efficient binding of 6sGTP. Indeed, the evolution identified three mutations that mediate 200-fold improved incorporation kinetics for 6sGTP. A >50-fold effect resulted from mutation A156U in the purine-rich loop, identifying the NTP binding site. This mutation acted weakly cooperative with two other beneficial mutations, C113U in the P2 stem near the catalytic site, and C79U on the surface of the catalytic domain. The preference pattern of the ribozyme for different NTPs changed when position 156 was mutated, confirming a direct contact between position 156 and the NTP. The results suggest that A156 stabilizes the NTP in the active site by a hydrogen bond to the Hoogsteen face of the NTP.

Sensitive and specific detection of ligands using engineered riboswitches

Riboswitches are RNA elements found in non-coding regions of messenger RNAs that regulate gene expression through a ligand-triggered conformational change. Riboswitches typically bind tightly and specifically to their ligands, so they have the potential to serve as highly effective sensors in vitro. In B. subtilis and other gram-positive bacteria, purine nucleotide synthesis is regulated by riboswitches that bind to guanine. We modified the xpt-pbuX guanine riboswitch for use in a fluorescence quenching assay that allowed us to specifically detect and quantify guanine in vitro. Using this assay, we reproducibly detected as little as 5 nM guanine. We then produced sensors for 2′-deoxyguanosine and cyclic diguanylate (c-diGMP) by appending the P1 stem of the guanine riboswitch to the ligand-binding domains of a 2′-deoxyguanosine riboswitch and a c-diGMP riboswitch. These hybrid sensors could detect 15 nM 2′-deoxyguanosine and 3 nM c-diGMP, respectively. Each sensor retained the ligand specificity of its corresponding natural riboswitch. In order to extend the utility of our approach, we developed a strategy for the in vitro selection of sensors with novel ligand specificity. Here we report a proof-of-principle experiment that demonstrated the feasibility of our selection strategy.

Pseudoknot Formation Seeds the Twister Ribozyme Cleavage Reaction Coordinate.

The twister RNA is a recently discovered nucleolytic ribozyme that is present in both bacteria and eukarya. While its biological role remains unclear, crystal structure analyses and biochemical approaches have revealed critical features of its catalytic mechanism. Here, we set out to explore dynamic aspects of twister RNA folding along the cleavage reaction coordinate. To do so, we have employed both bulk and single-molecule fluorescence resonance energy transfer (FRET) methods to investigate a set of twister RNAs with labels strategically positioned at communicating segments. The data reveal that folding of the central pseudoknot (T1), the most crucial structural determinant to promote cleavage, exhibits reversible opening and closing dynamics at physiological Mg2+ concentration. Uncoupled folding, in which T1 formation precedes structuring for closing of stem P1, was confirmed using pre-steady-state three-color smFRET experiments initiated by Mg2+ injection. This finding suggests that the folding path of twister RNA requires proper orientation of the substrate prior to T1 closure such that the U5-A6 cleavage site becomes embraced to achieve its cleavage competent conformation. We also find that the cleaved 3'-fragment retains its compacted pseudoknot fold, despite the absence of the phylogenetically conserved stem P1, rationalizing the poor turnover efficiency of the twister ribozyme.

Structural basis for ligand binding to the guanidine-II riboswitch.

The guanidine-II riboswitch, also known as mini-ykkC, is a conserved mRNA element with more than 800 examples in bacteria. It consists of two stem–loops capped by identical, conserved tetraloops that are separated by a linker region of variable length and sequence. Like the guanidine-I riboswitch, it controls the expression of guanidine carboxylases and SugE-like genes. The guanidine-II riboswitch specifically binds free guanidinium cations and functions as a translationally controlled on-switch. Here we report the structure of a P2 stem–loop from the Pseudomonas aeruginosa guanidine-II riboswitch aptamer bound to guanidine at 1.57 Å resolution. The hairpins dimerize via the conserved tetraloop, which also contains the binding pocket. Two guanidinium molecules bind near the dimerization interface, one in each tetraloop. The guanidinium cation is engaged in extensive hydrogen bonding to the RNA. Contacts include the Hoogsteen face of a guanine base and three nonbridging phosphate oxygens. Cation–π interactions and ionic interactions also stabilize ligand binding. The guanidine-II riboswitch utilizes the same recognition strategies as the guanidine-I riboswitch while adopting an entirely different and much smaller RNA fold.

Unwinding the twister ribozyme: from structure to mechanism.

The twister ribozyme motif has been identified by bioinformatic means very recently. Currently, four crystal structures with ordered active sites together with a series of chemical and biochemical data provide insights into how this RNA accomplishes its efficient self‐cleavage. Of particular interest for a mechanistic proposal are structural distinctions observed in the active sites that concern the conformation of the U‐A cleavage site dinucleotide (in‐line alignment of the attacking 2′‐O nucleophile to the to‐be‐cleaved P—O5′ bond versus suboptimal alignments) as well as the presence/absence of Mg2+ ions at the scissile phosphate. All structures support the notion that an active site guanine and the conserved adenine at the cleavage site are important contributors to cleavage chemistry, likely being involved in general acid base catalysis. Evidence for innersphere coordination of a Mg2+ ion to the pro‐S nonbridging oxygen of the scissile phosphate stems from two of the four crystal structures. Together with the finding of thio/rescue effects for phosphorothioate substrates, this suggests the participation of divalent ions in the overall catalytic strategy employed by twister ribozymes. In this context, it is notable that twister retains wild‐type activity when the phylogenetically conserved stem P1 is deleted, able to cleave a single nucleotide only. WIREs RNA 2017, 8:e1402. doi: 10.1002/wrna.1402For further resources related to this article, please visit the WIREs website.

Biophysical properties, thermal stability and functional impact of 8-oxo-7,8-dihydroguanine on oligonucleotides of RNA-a study of duplex, hairpins and the aptamer for preQ1 as models.

A better understanding of the effects that oxidative lesions have on RNA is of importance to understand their role in the development/progression of disease. 8-oxo-7,8-dihydroguanine was incorporated into RNA to understand its structural and functional impact on RNA:RNA and RNA:DNA duplexes, hairpins and pseudoknots. One to three modifications were incorporated into dodecamers of RNA [AAGAGGGAUGAC] resulting in thermal destabilization (ΔTm – 10°C per lesion). Hairpins with tetraloops c-UUCG*-g* (8-10), a-ACCG-g* (11-12), c-UUG*G*-g* (13-16) and c-ACG*G*-g* (17-20) were modified and used to determine thermal stabilities, concluding that: (i) modifying the stem leads to destabilization unless adenosine is the opposing basepair of 8-oxoGua; (ii) modification at the loop is position- and sequence-dependent and varies from slight stabilization to large destabilization, in some cases leading to formation of other secondary structures (hairpin→duplex). Functional effects were established using the aptamer for preQ1 as model. Modification at G5 disrupted the stem P1 and inhibited recognition of the target molecule 7-methylamino-7-deazaguanine (preQ1). Modifying G11 results in increased thermal stability, albeit with a Kd 4-fold larger than its canonical analog. These studies show the capability of 8-oxoG to affect structure and function of RNA, resulting in distinct outcomes as a function of number and position of the lesion.

Molecular Dynamics Study of Twister Ribozyme: Role of Mg(2+) Ions and the Hydrogen-Bonding Network in the Active Site.

The recently discovered twister ribozyme is thought to utilize general acid–base catalysis in its self-cleavage mechanism, but the roles of nucleobases and metal ions in the mechanism are unclear. Herein, molecular dynamics simulations of the env22 twister ribozyme are performed to elucidate the structural and equilibrium dynamical properties, as well as to examine the role of Mg2+ ions and possible candidates for the general base and acid in the self-cleavage mechanism. The active site region and the ends of the pseudoknots were found to be less mobile than other regions of the ribozyme, most likely providing structural stability and possibly facilitating catalysis. A purported catalytic Mg2+ ion and the closest neighboring Mg2+ ion remained chelated and relatively immobile throughout the microsecond trajectories, although removal of these Mg2+ ions did not lead to any significant changes in the structure or equilibrium motions of the ribozyme on the microsecond time scale. In addition, a third metal ion, a Na+ ion remained close to A1(O5′), the leaving group atom, during the majority of the microsecond trajectories, suggesting that it might stabilize the negative charge on A1(O5′) during self-cleavage. The locations of these cations and their interactions with key nucleotides in the active site suggest that they may be catalytically relevant. The P1 stem is partially melted at its top and bottom in the crystal structure and further unwinds in the trajectories. The simulations also revealed an interconnected network comprised of hydrogen-bonding and π-stacking interactions that create a relatively rigid network around the self-cleavage site. The nucleotides involved in this network are among the highly conserved nucleotides in twister ribozymes, suggesting that this interaction network may be important to structure and function.

Structural and Dynamic Basis for Low-Affinity, High-Selectivity Binding of L-Glutamine by the Glutamine Riboswitch.

Naturally occurring L-glutamine riboswitches occur in cyanobacteria and marine metagenomes, where they reside upstream of genes involved in nitrogen metabolism. By combining X-ray, NMR, and MD, we characterized an L-glutamine-dependent conformational transition in the Synechococcus elongatus glutamine riboswitch from tuning fork to L-shaped alignment of stem segments. This transition generates an open ligand-binding pocket with L-glutamine selectivity enforced by Mg(2+)-mediated intermolecular interactions. The transition also stabilizes the P1 helix through a long-range "linchpin" Watson-Crick G-C pair-capping interaction, while melting a short helix below P1 potentially capable of modulating downstream readout. NMR data establish that the ligand-free glutamine riboswitch in Mg(2+) solution exists in a slow equilibrium between flexible tuning fork and a minor conformation, similar, but not identical, to the L-shaped bound conformation. We propose that an open ligand-binding pocket combined with a high conformational penalty for forming the ligand-bound state provide mechanisms for reducing binding affinity while retaining high selectivity.

A Mini-Twister Variant and Impact of Residues/Cations on the Phosphodiester Cleavage of this Ribozyme Class.

Nucleolytic ribozymes catalyze site-specific cleavage of their phosphodiester backbones. A minimal version of the twister ribozyme is reported that lacks the phylogenetically conserved stem P1 while retaining wild-type activity. Atomic mutagenesis revealed that nitrogen atoms N1 and N3 of the adenine-6 at the cleavage site are indispensable for cleavage. By NMR spectroscopy, a pKa value of 5.1 was determined for a (13) C2-labeled adenine at this position in the twister ribozyme, which is significantly shifted compared to the pKa of the same adenine in the substrate alone. This finding pinpoints at a potential role for adenine-6 in the catalytic mechanism besides the previously identified invariant guanine-48 and a Mg(2+) ion, both of which are directly coordinated to the non-bridging oxygen atoms of the scissile phosphate; for the latter, additional evidence stems from the observation that Mn(2+) or Cd(2+) accelerated cleavage of phosphorothioate substrates. The relevance of this metal ion binding site is further emphasized by a new 2.6 Å X-ray structure of a 2'-OCH3 -U5 modified twister ribozyme.

High-affinity RNA Aptamers Against the HIV-1 Protease Inhibit Both In Vitro Protease Activity and Late Events of Viral Replication.

HIV-1 aspartyl protease (PR) plays a key role in virion morphogenesis, underscoring the effectiveness of protease inhibitors (PI). Despite their utility, side effects and drug-resistance remains a problem. We report the development of RNA aptamers as inhibitors of HIV-1 PR for potential use in anti-HIV gene therapy. Employing Systematic Evolution of Ligands by Exponential Enrichment (SELEX), we isolated four unique families of anti-HIV-1 PR RNA aptamers displaying moderate binding affinities (Kd = 92–140 nmol/l) and anti-PR inhibitory activity (Kis = 138–647 nmol/l). Second-generation RNA aptamers selected from partially randomized pools based on two of the aptamer sequences displayed striking enhancements in binding (Kds = 2–22 nmol/l) and inhibition (Kis = 31–49 nmol/l). The aptamers were specific in that they did not bind either the related HIV-2 protease, or the cellular aspartyl protease, Cathepsin D. Site-directed mutagenesis of a second-generation aptamer to probe the predicted secondary structure indicated that the stem-loops SL2 and SL3 and the stem P1 were essential for binding and that only the 3'-most 17 nucleotides were dispensable. Anti-PR aptamers inhibited HIV replication in vitro and the degree of inhibition was higher for second-generation aptamers with greater affinity and the inhibition was abrogated for a nonbinding aptamer variant.

Ligand binding by the tandem glycine riboswitch depends on aptamer dimerization but not double ligand occupancy.

The glycine riboswitch predominantly exists as a tandem structure, with two adjacent, homologous ligand-binding domains (aptamers), followed by a single expression platform. The recent identification of a leader helix, the inclusion of which eliminates cooperativity between the aptamers, has reopened the debate over the purpose of the tandem structure of the glycine riboswitch. An equilibrium dialysis-based assay was combined with binding-site mutations to monitor glycine binding in each ligand-binding site independently to understand the role of each aptamer in glycine binding and riboswitch tertiary interactions. A series of mutations disrupting the dimer interface was used to probe how dimerization impacts ligand binding by the tandem glycine riboswitch. While the wild-type tandem riboswitch binds two glycine equivalents, one for each aptamer, both individual aptamers are capable of binding glycine when the other aptamer is unoccupied. Intriguingly, glycine binding by aptamer-1 is more sensitive to dimerization than glycine binding by aptamer-2 in the context of the tandem riboswitch. However, monomeric aptamer-2 shows dramatically weakened glycine-binding affinity. In addition, dimerization of the two aptamers in trans is dependent on glycine binding in at least one aptamer. We propose a revised model for tandem riboswitch function that is consistent with these results, wherein ligand binding in aptamer-1 is linked to aptamer dimerization and stabilizes the P1 stem of aptamer-2, which controls the expression platform.

Role of Magnesium Ions and Ligand Stacking in the Adenine Riboswitch Folding

Riboswitches are RNA structured elements that modulate fundamental pathways in bacteria and plants. Depending on the availability of their sensed metabolite they undergo a conformational change that turns on or off gene expression. In the add adenine riboswitch from Vibrio Vulnificus there are three crucial spots for the switch between the two conformations: the P1 stem, the kissing-loops and the binding site for adenine. Our work is based on advanced in silico techniques, including atomistic molecular simulations, steered molecular dynamics, umbrella sampling, metadynamics and Hamiltonian replica exchange. First we investigated the P1-ligand dependent stabilization quantifying this effect in terms of free energy (Di Palma et al doi:10.1261/rna.040493.113). Then we evaluated the different contributions that Mg2+ and adenine give to the formation of the tertiary interactions between the two loops and we analysed the influence of these interactions on the binding site. The obtained data are in quantitative agreement with experiments. The atomistic description of metabolite binding, RNA-Mg2+ interaction, and tertiary-contact formation clarifies the details of the ligand-aptamer interactions and of the role of divalent cations in the global aptamer folding.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Ligand-induced stabilization of the aptamer terminal helix in the add adenine riboswitch

Riboswitches are structured mRNA elements that modulate gene expression. They undergo conformational changes triggered by highly specific interactions with sensed metabolites. Among the structural rearrangements engaged by riboswitches, the forming and melting of the aptamer terminal helix, the so-called P1 stem, is essential for genetic control. The structural mechanisms by which this conformational change is modulated upon ligand binding mostly remain to be elucidated. Here, we used pulling molecular dynamics simulations to study the thermodynamics of the P1 stem in the add adenine riboswitch. The P1 ligand-dependent stabilization was quantified in terms of free energy and compared with thermodynamic data. This comparison suggests a model for the aptamer folding in which direct P1-ligand interactions play a minor role on the conformational switch when compared with those related to the ligand-induced aptamer preorganization.