Stem-loop formation drives RNA folding in mechanical unzipping experiments.

Accurate knowledge of RNA hybridization is essential for understanding RNA structure and function. Here we mechanically unzip and rezip a 2-kbp RNA hairpin and derive the 10 nearest-neighbor base pair (NNBP) RNA free energies in sodium and magnesium with 0.1 kcal/mol precision using optical tweezers. Notably, force-distance curves (FDCs) exhibit strong irreversible effects with hysteresis and several intermediates, precluding the extraction of the NNBP energies with currently available methods. The combination of a suitable RNA synthesis with a tailored pulling protocol allowed us to obtain the fully reversible FDCs necessary to derive the NNBP energies. We demonstrate the equivalence of sodium and magnesium free-energy salt corrections at the level of individual NNBP. To characterize the irreversibility of the unzipping-rezipping process, we introduce a barrier energy landscape of the stem-loop structures forming along the complementary strands, which compete against the formation of the native hairpin. This landscape correlates with the hysteresis observed along the FDCs. RNA sequence analysis shows that base stacking and base pairing stabilize the stem-loops that kinetically trap the long-lived intermediates observed in the FDC. Stem-loops formation appears as a general mechanism to explain a wide range of behaviors observed in RNA folding.

Tertiary RNA Folding-Targeted Drug Screening Strategy Using a Protein Nanopore.

Bacterial riboswitch RNAs are attractive targets for novel antibiotics against antibiotic-resistant superbacteria. Their binding to cognate metabolites is essential for the regulation of bacterial gene expression. Despite the importance of RNAs as therapeutic targets, the development of RNA-targeted, small-molecule drugs is limited by current biophysical methods. Here, we monitored the specific interaction between the adenine-sensing riboswitch aptamer domain (ARS) and adenine at the single-molecule level using α-hemolysin (αHL) nanopores. During adenine-induced tertiary folding, adenine-bound ARS intermediates exhibited characteristic nanopore events, including a two-level ionic current blockade and a ∼ 5.6-fold longer dwell time than that of free RNA. In a proof-of-concept experiment, tertiary RNA folding-targeted drug screening was performed using a protein nanopore, which resulted in the discovery of three new ARS-targeting hit compounds from a natural compound library. Taken together, these results reveal that αHL nanopores are a valuable platform for ultrasensitive, label-free, and single-molecule-based drug screening against therapeutic RNA targets.

Design of a small molecule that stimulates vascular endothelial growth factor A enabled by screening RNA fold-small molecule interactions.

Vascular endothelial growth factor A (VEGFA) stimulates angiogenesis in human endothelial cells, and increasing its expression is a potential treatment for heart failure. Here, we report the design of a small molecule (TGP-377) that specifically and potently enhances VEGFA expression by the targeting of a non-coding microRNA that regulates its expression. A selection-based screen, named two-dimensional combinatorial screening, revealed preferences in small-molecule chemotypes that bind RNA and preferences in the RNA motifs that bind small molecules. The screening program increased the dataset of known RNA motif-small molecule binding partners by 20-fold. Analysis of this dataset against the RNA-mediated pathways that regulate VEGFA defined that the microRNA-377 precursor, which represses Vegfa messenger RNA translation, is druggable in a selective manner. We designed TGP-377 to potently and specifically upregulate VEGFA in human umbilical vein endothelial cells. These studies illustrate the power of two-dimensional combinatorial screening to define molecular recognition events between 'undruggable' biomolecules and small molecules, and the ability of sequence-based design to deliver efficacious structure-specific compounds.

Characterization of a transcriptional TPP riboswitch in the human pathogen Neisseria meningitidis.

Increasing evidence has demonstrated that regulatory RNA elements such as riboswitches (RS) play a pivotal role in the fine-tuning of bacterial gene expression. In this study, we investigated and characterized a novel transcriptional thiamine pyrophosphate (TPP) RS in the obligate human pathogen N. meningitidis MC58 (serogroup B). This RS is located in the 5´ untranslated region upstream of thiC gene, encoding a protein involved in TPP biosynthesis, an essential cofactor for all living beings. Primer extension revealed the transcriptional start site of thiC. Northern blot analysis of thiC mRNA and reporter gene studies confirmed the presence of an active TPP-sensing RS. Expression patterns of the wild-type RS and site-specific mutants showed that it is an OFF switch that controls transcription elongation of thiC mRNA. Interestingly, the regulatory mechanism of the meningococcal thiC RS resembles the Gram-positive Bacillus subtilis thiC RS rather than the Gram-negative Escherichia coli thiC RS. Therefore, the meningococcal thiC RS represents a rare example of transcriptional RS in a Gram-negative bacterium. We further observed that the RS is actively involved in modulating gene expression in response to different growth media and to supplemented bacterial and eukaryotic cell lysates as possible sources of nutrients in the nasopharynx. Our results suggest that RS-mediated gene regulation could influence meningococcal fitness, through the fine-tuning of biosynthesis and scavenging of nutrients and cofactors, such as thiamine.

Dynamic nuclear polarization on a hybridized hammerhead ribozyme: An explorative study of RNA folding and direct DNP with a paramagnetic metal ion cofactor.

While uniform isotope labeling of ribonucleic acids (RNA) can simply and efficiently be achieved by in-vitro transcription, the specific introduction of nucleotides in larger constructs is non-trivial and often ineffective. Here, we demonstrate how a medium-sized (67-mer), biocatalytically relevant RNA (hammerhead ribozyme, HHRz) can be formed by spontaneous hybridization of two differently isotope-labeled strands, each individually synthesized by in-vitro transcription. This allows on the one hand for a significant reduction in the number of isotope-labeled nucleotides and thus spectral overlap particularly under magic-angle spinning (MAS) dynamic nuclear polarization (DNP) NMR conditions, on the other hand for orthogonal 13C/15N-labeling of complementary strands and thus for specific investigation of structurally or functionally relevant inter-strand and/or inter-stem contacts. By this method, we are able to confirm a non-canonical interaction due to single-site resolution and unique spectral assignments by two-dimensional 13C-13C (PDSD) as well as 15N-13C (TEDOR) correlation spectroscopy under "conventional" DNP enhancement. This contact is indicative of the ribozyme's functional conformation, and is present in frozen solution irrespective of the presence or absence of a Mg2+ co-factor. Finally, we use different isotope-labeling schemes in order to investigate the distance dependence of paramagnetic interactions and direct metal-ion DNP if the diamagnetic Mg2+ is substituted by paramagnetic Mn2+.

Distributed biotin-streptavidin transcription roadblocks for mapping cotranscriptional RNA folding.

RNA folding during transcription directs an order of folding that can determine RNA structure and function. However, the experimental study of cotranscriptional RNA folding has been limited by the lack of easily approachable methods that can interrogate nascent RNA structure at nucleotide resolution. To address this, we previously developed cotranscriptional selective 2΄-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) to simultaneously probe all intermediate RNA transcripts during transcription by stalling elongation complexes at catalytically dead EcoRIE111Q roadblocks. While effective, the distribution of elongation complexes using EcoRIE111Q requires laborious PCR using many different oligonucleotides for each sequence analyzed. Here, we improve the broad applicability of cotranscriptional SHAPE-Seq by developing a sequence-independent biotin-streptavidin (SAv) roadblocking strategy that simplifies the preparation of roadblocking DNA templates. We first determine the properties of biotin-SAv roadblocks. We then show that randomly distributed biotin-SAv roadblocks can be used in cotranscriptional SHAPE-Seq experiments to identify the same RNA structural transitions related to a riboswitch decision-making process that we previously identified using EcoRIE111Q. Lastly, we find that EcoRIE111Q maps nascent RNA structure to specific transcript lengths more precisely than biotin-SAv and propose guidelines to leverage the complementary strengths of each transcription roadblock in cotranscriptional SHAPE-Seq.

Ensemble simulations: folding, unfolding and misfolding of a high-efficiency frameshifting RNA pseudoknot.

Massive all-atom molecular dynamics simulations were conducted across a distributed computing network to study the folding, unfolding, misfolding and conformational plasticity of the high-efficiency frameshifting double mutant of the 26 nt potato leaf roll virus RNA pseudoknot. Our robust sampling, which included over 40 starting structures spanning the spectrum from the extended unfolded state to the native fold, yielded nearly 120 µs of cumulative sampling time. Conformational microstate transitions on the 1.0 ns to 10.0 µs timescales were observed, with post-equilibration sampling providing detailed representations of the conformational free energy landscape and the complex folding mechanism inherent to the pseudoknot motif. Herein, we identify and characterize two alternative native structures, three intermediate states, and numerous misfolded states, the latter of which have not previously been characterized via atomistic simulation techniques. While in line with previous thermodynamics-based models of a general RNA folding mechanism, our observations indicate that stem-strand-sequence-separation may serve as an alternative predictor of the order of stem formation during pseudoknot folding. Our results contradict a model of frameshifting based on structural rigidity and resistance to mechanical unfolding, and instead strongly support more recent studies in which conformational plasticity is identified as a determining factor in frameshifting efficiency.

Molecular crowders and cosolutes promote folding cooperativity of RNA under physiological ionic conditions.

Folding mechanisms of functional RNAs under idealized in vitro conditions of dilute solution and high ionic strength have been well studied. Comparatively little is known, however, about mechanisms for folding of RNA in vivo where Mg(2+) ion concentrations are low, K(+) concentrations are modest, and concentrations of macromolecular crowders and low-molecular-weight cosolutes are high. Herein, we apply a combination of biophysical and structure mapping techniques to tRNA to elucidate thermodynamic and functional principles that govern RNA folding under in vivo-like conditions. We show by thermal denaturation and SHAPE studies that tRNA folding cooperativity increases in physiologically low concentrations of Mg(2+) (0.5-2 mM) and K(+) (140 mM) if the solution is supplemented with physiological amounts (∼ 20%) of a water-soluble neutral macromolecular crowding agent such as PEG or dextran. Low-molecular-weight cosolutes show varying effects on tRNA folding cooperativity, increasing or decreasing it based on the identity of the cosolute. For those additives that increase folding cooperativity, the gain is manifested in sharpened two-state-like folding transitions for full-length tRNA over its secondary structural elements. Temperature-dependent SHAPE experiments in the absence and presence of crowders and cosolutes reveal extent of cooperative folding of tRNA on a nucleotide basis and are consistent with the melting studies. Mechanistically, crowding agents appear to promote cooperativity by stabilizing tertiary structure, while those low molecular cosolutes that promote cooperativity stabilize tertiary structure and/or destabilize secondary structure. Cooperative folding of functional RNA under physiological-like conditions parallels the behavior of many proteins and has implications for cellular RNA folding kinetics and evolution.

Conserved arginines on the rim of Hfq catalyze base pair formation and exchange.

The Sm-like protein Hfq is required for gene regulation by small RNAs (sRNAs) in bacteria and facilitates base pairing between sRNAs and their mRNA targets. The proximal and distal faces of the Hfq hexamer specifically bind sRNA and mRNA targets, but they do not explain how Hfq accelerates the formation and exchange of RNA base pairs. Here, we show that conserved arginines on the outer rim of the hexamer that are known to interact with sRNA bodies are required for Hfq's chaperone activity. Mutations in the arginine patch lower the ability of Hfq to act in sRNA regulation of rpoS translation and eliminate annealing of natural sRNAs or unstructured oligonucleotides, without preventing binding to either the proximal or distal face. Stopped-flow FRET and fluorescence anisotropy show that complementary RNAs transiently form a ternary complex with Hfq, but the RNAs are not released as a double helix in the absence of rim arginines. RNAs bound to either face of Hfq quench the fluorescence of a tryptophan adjacent to the arginine patch, demonstrating that the rim can simultaneously engage two RNA strands. We propose that the arginine patch overcomes entropic and electrostatic barriers to helix nucleation and constitutes the active site for Hfq's chaperone function.

The HIV-2 Rev-response element: determining secondary structure and defining folding intermediates.

Interaction between the viral protein Rev and the RNA motifs known as Rev response elements (RREs) is required for transport of unspliced and partially spliced human immunodeficiency virus (HIV)-1 and HIV-2 RNAs from the nucleus to the cytoplasm during the later stages of virus replication. A more detailed understanding of these nucleoprotein complexes and the host factors with which they interact should accelerate the development of new antiviral drugs targeting cis-acting RNA regulatory signals. In this communication, the secondary structures of the HIV-2 RRE and two RNA folding precursors have been identified using the SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemical probing methodology together with a novel mathematical approach for determining the secondary structures of RNA conformers present in a mixture. A complementary chemical probing technique was also used to support these secondary structure models, to confirm that the RRE2 RNA undergoes a folding transition and to obtain information about the relative positioning of RRE2 substructures in three dimensions. Our analysis collectively suggests that the HIV-2 RRE undergoes two conformational transitions before assuming the energetically most favorable conformer. The 3D models for the HIV-2 RRE and folding intermediates are also presented, wherein the Rev-binding stem-loops (IIB and I) are located coaxially in the former, which is in agreement with previous models for HIV-1 Rev-RRE binding.