Additional Base Pairs Research Articles

Role of RNA structural plasticity in modulating HIV-1 genome packaging and translation

HIV-1 transcript function is controlled in part by twinned transcriptional start site usage, where 5' capped RNAs beginning with a single guanosine (1G) are preferentially packaged into progeny virions as genomic RNA (gRNA) whereas those beginning with three sequential guanosines (3G) are retained in cells as mRNAs. In 3G transcripts, one of the additional guanosines base pairs with a cytosine located within a conserved 5' polyA element, resulting in formation of an extended 5' polyA structure as opposed to the hairpin structure formed in 1G RNAs. To understand how this remodeling influences overall transcript function, we applied in vitro biophysical studies with in-cell genome packaging and competitive translation assays to native and 5' polyA mutant transcripts generated with promoters that differentially produce 1G or 3G RNAs. We identified mutations that stabilize the 5' polyA hairpin structure in 3G RNAs, which promote RNA dimerization and Gag binding without sequestering the 5' cap. None of these 3G transcripts were competitively packaged, confirming that cap exposure is a dominant negative determinant of viral genome packaging. For all RNAs examined, conformations that favored 5' cap exposure were both poorly packaged and more efficiently translated than those that favored 5' cap sequestration. We propose that structural plasticity of 5' polyA and other conserved RNA elements place the 5' leader on a thermodynamic tipping point for low-energetic (~3 kcal/mol) control of global transcript structure and function.

An Effective Model for Capturing the Role of Excitonic Interactions in the Wave-Packet Dynamics of DNA Nucleobases

Investigating exciton dynamics within DNA nucleobases is essential for comprehensively understanding how inherent photostability mechanisms function at the molecular level, particularly in the context of life’s resilience to solar radiation. In this paper, we introduce a mathematical model that effectively simulates the photoexcitation and deactivation dynamics of nucleobases within an ultrafast timeframe, particularly focusing on wave-packet dynamics under conditions of strong nonadiabatic coupling. Employing the hierarchy equation of motion, we simulate two-dimensional electronic spectra (2DES) and calibrate our model by comparing it with experimentally obtained spectra. This study also explores the effects of base stacking on the photo-deactivation dynamics in DNA. Our results demonstrate that, while strong excitonic interactions between nucleobases are present, they have a minimal impact on the deactivation dynamics of the wave packet in the electronic excited states. We further observe that the longevity of electronic excited states increases with additional base stacking and pairing, a phenomenon accurately depicted by our excitonic model. This model enables a detailed examination of the wave packet’s motion on electronic excited states and its rapid transition to the ground state. Additionally, using this model, we studied base stacks in DNA hairpins to effectively capture the primary exciton dynamics at a reasonable computational scale. Overall, this work provides a valuable framework for studying exciton dynamics from single nucleobases to complex structures such as DNA hairpins.

Base pair dynamics, electrostatics, and thermodynamics at the LTR-III quadruplex:duplex junction

G-quadruplexes (GQs) play key regulatory roles within the human genome and have also been identified to play similar roles in other eukaryotes, bacteria, archaea, and viruses. Human immunodeficiency virus 1, the etiological agent of acquired immunodeficiency syndrome, can form two GQs in its long terminal repeat (LTR) promoter region, each of which act to regulate viral gene expression in opposing manners. The major LTR GQ, called LTR-III, is a distinct hybrid GQ containing a 12-nucleotide duplex loop attached to the quadruplex motif. The resulting quadruplex:duplex junction (QDJ) has been hypothesized to serve as a selective drug targeting site. To better understand the dynamics of this QDJ, we performed conventional and enhanced-sampling molecular dynamics simulations using the Drude-2017 force field. We observed unbiased and reversible formation of additional base pairs in the QDJ, between Ade4:Thy14 and Gua3:Thy14. Both base pairs were electrostatically favored, but geometric constraints within the junction may drive the formation of, and preference for, the Ade4:Thy14 base pair. Finally, we demonstrated that the base pairs are separated only by small energy barriers that may enable transitions between both base-paired states. Together, these simulations provide new insights into the dynamics, electrostatics, and thermodynamics of the LTR-III QDJ.

Intramolecular aptamer switches.

Aptamer switches as effective biosensing tools have become a focal point of research in engineered aptasensors. Intramolecular aptamer switches are more versatile, affordable, and simpler than classical "open-close" and strand displacement-based aptamer switches. Recently, many new aptamers with an overall hairpin structure have been reported. In this study, intramolecular aptamer switches were developed by adding new base pairs to the end of aptamers. The additional nucleotides can pair with the internal domains of the aptamer, causing a change in its conformation from the original secondary structure without a target. When a target binds to an aptamer, a marked change in the structure of the aptamer is expected. As models for testing this intramolecular aptamer switch idea, aptamers of oxytetracycline (OTC), 17β-estradiol (E2), and adenosine were employed. When the additional base pairs are too long, binding the target to the aptamer becomes more challenging. This research offers valuable insights into the development of intramolecular aptamer switches and their potential applications in biosensor design.

Structure of a DNA G-quadruplex that Modulates SP1 Binding Sites Architecture in HIV-1 Promoter

Nucleic acid sequences containing guanine tracts are able to form non-canonical DNA or RNA structures known as G-quadruplexes (or G4s). These structures, based on the stacking of G-tetrads, are involved in various biological processes such as gene expression regulation. Here, we investigated a G4 forming sequence, HIVpro2, derived from the HIV-1 promoter. This motif is located 60 nucleotides upstream of the proviral Transcription Starting Site (TSS) and overlaps with two SP1 transcription factor binding sites. Using NMR spectroscopy, we determined that HIVpro2 forms a hybrid type G4 structure with a core that is interrupted by a single nucleotide bulge. An additional reverse-Hoogsteen AT base pair is stacked on top of the tetrad. SP1 transcription factor is known to regulate transcription activity of many genes through the recognition of Guanine-rich duplex motifs. Here, the formation of HIVpro2 G4 may modulate SP1 binding sites architecture by competing with the formation of the canonical duplex structure. Such DNA structural switch potentially participates to the regulation of viral transcription and may also interfere with HIV-1 reactivation or viral latency.

A draft human pangenome reference

Here the Human Pangenome Reference Consortium presents a first draft of the human pangenome reference. The pangenome contains 47 phased, diploid assemblies from a cohort of genetically diverse individuals1. These assemblies cover more than 99% of the expected sequence in each genome and are more than 99% accurate at the structural and base pair levels. Based on alignments of the assemblies, we generate a draft pangenome that captures known variants and haplotypes and reveals new alleles at structurally complex loci. We also add 119 million base pairs of euchromatic polymorphic sequences and 1,115 gene duplications relative to the existing reference GRCh38. Roughly 90 million of the additional base pairs are derived from structural variation. Using our draft pangenome to analyse short-read data reduced small variant discovery errors by 34% and increased the number of structural variants detected per haplotype by 104% compared with GRCh38-based workflows, which enabled the typing of the vast majority of structural variant alleles per sample.

Determining the confromational ensemble of the HIV-1 LTR G-quadruplexes through gaussian-accelerated molecular dynamics using the drude polarizable force field.

Human immunodeficiency virus (HIV) impacts nearly 37.6 million people globally, but there is no effective cure for the infections it causes. HIV-1 is a retrovirus containing a single-stranded RNA genome. After reverse transcription, the 5’ long terminal repeat (LTR) acts as a promoter for viral replication. Two DNA G-quadruplexes (GQs), known as LTR-III and LTR-IV, form within this promoter and inversely regulate viral gene expression. The LTR-III GQ downregulates viral expression and is a quadruplex: duplex hybrid whereas LTR-IV upregulates viral expression and is a parallel GQ containing a single-nucleotide bulge. Their distinct topologies and functional roles suggest their potential to act as selective drug targets for HIV-1.To gain a greater understanding of structure and dynamics of the LTR-III and LTR-IV GQs, we performed conventional molecular dynamics simulations using the Drude polarizable force field. The loop nucleotides in both structures were highly flexible, sampling a variety of conformational states. The LTR-IV loop also formed base pair interactions that limited ion access to its loop cavity and further highlighted two distinct ion binding sites near the backbone tetrad guanines. Within the LTR-III duplex loop, an additional Watson-Crick base pair formed between Ade4:Thy14 and persisted for 40% of the snapshots collected in the total 4-μs simulation time. Analysis of the electric field exerted on Thy14 indicates a preference for base pairing with Ade4 compared to being unpaired as in the NMR ensemble. Gaussian-accelerated molecular dynamics simulations were also performed to identify high and low energy conformational states for subsequent drug targeting. Our results emphasize the importance of electronic polarization in GQ dynamics and can guide future development of antiviral therapeutics.

A comprehensive update to the Mycobacterium tuberculosis H37Rv reference genome

H37Rv is the most widely used Mycobacterium tuberculosis strain, and its genome is globally used as the M. tuberculosis reference sequence. Here, we present Bact-Builder, a pipeline that uses consensus building to generate complete and accurate bacterial genome sequences and apply it to three independently cultured and sequenced H37Rv aliquots of a single laboratory stock. Two of the 4,417,942 base-pair long H37Rv assemblies are 100% identical, with the third differing by a single nucleotide. Compared to the existing H37Rv reference, the new sequence contains ~6.4 kb additional base pairs, encoding ten new regions that include insertions in PE/PPE genes and new paralogs of esxN and esxJ, which are differentially expressed compared to the reference genes. New sequencing and de novo assemblies with Bact-Builder confirm that all 10 regions, plus small additional polymorphisms, are also present in the commonly used H37Rv strains NR123, TMC102, and H37Rv1998. Thus, Bact-Builder shows promise as an improved method to perform accurate and reproducible de novo assemblies of bacterial genomes, and our work provides important updates to the primary M. tuberculosis reference genome.

Ecology, genetics and distribution of Punctoribates zachvatkini, an oribatid mite so far overlooked in Germany

Punctoribates is one of few genera in Poronota (Acari: Oribatida) containing species with porose areas and species with saccules, the two types of the octotaxic system. These porose organs are the main difference between two morphologically similar species, P. punctum with porose areas and P. zachvatkini with saccules. As the octotaxic system can vary within species, species separation solely based on this trait might be insufficient. To assess the species status of P. zachvatkini, we investigated additional differences from P. punctum by comparing habitat preferences of the two species regarding nature reserves and agricultural landscapes during a field study in the German Eifel region, and by examining Punctoribates material from four large German natural history museums. We also performed scanning electron microscopy (SEM) and a genetic analysis using the D3 marker of the nuclear 28S rDNA gene. In the field study, P. zachvatkini had higher densities in the nature reserves and P. punctum in the agricultural landscapes. Evaluation of the museum material revealed P. punctum occurred more regularly in disturbed sites such as urban, agricultural and post-mining areas compared to P. zachvatkini. Pairwise distances of the 28S D3 genetic marker as well as an additional base pair in P. zachvatkini further support the separation of the two species, and SEM investigations revealed new details regarding the punctulation of P. zachvatkini. The review of the museum material showed that P. zachvatkini already occurred in Germany in 1967 and has a wider distribution than previously known.

Thermodynamic analysis of SL1 1x2 internal loop in SARS‐CoV‐2

SARS‐CoV‐2 utilizes a positive‐sense single stranded RNA as its genomic material. This single stranded mRNA hijacks the host cell’s ribosome to translate its own proteins. Upon infection of a host cell, SARS‐CoV‐2 produces polyprotein 1ab which is then cleaved into 16 non‐structural proteins. Non‐structural protein 1 (Nsp1) inhibits host cell translation by binding to the 40S ribosomal subunit. Stem loop 1(SL1) in the 5’‐UTR of the mRNA mediates the binding of Nsp1 to the ribosome. SARS‐CoV‐2 specifically contains a 1x2 internal loop within the SL1 that is not seen in other coronaviruses. In this study, we are examining the thermodynamic properties of the 1x2 internal loop of SL1. UV‐visible thermal melts and isothermal titration calorimetry (ITC) were performed on various DNA and RNA constructs containing the 1x2 internal loop in SL1 in the presence of 1 M KCl or 10 mM magnesium chloride at two different pHs. Initial results show additional base pairing at lower pH in 1 M KCl buffer, indicating the formation of an A+•C base pair.

CONGENITAL MYOPATHIES 1 – NEMALINE: P.26 A new intronic mutation in nebulin myopathy

The aim of this study was to demonstrate functional and clinical presentation of a new homozygous intronic mutation in nebulin. Nebulin encodes for a giant protein component of the cytoskeletal matrix that coexists with the thick and thin filaments within the sarcomeres of skeletal muscle. Of the 183 exons in the nebulin gene, at least 43 are alternatively spliced, although exons 143 and 144 are not found in the same transcript. We report the case of a 6 yo boy, born from consanguineous parents with typical phenotypic presentation for nemaline myopathy, proven on muscle biopsy. Muscle biopsy showed a significant amount of nemaline bodies. Muscle MRI was strongly suggestive of nebulin related myopathy but gene panel for congenital myopathies was negative. Exome sequencing was performed and did not reveal specific findings. Approach with SNP to show loss of heterozygosity demonstrates that nebulin gene was of interest. Deep sequencing of nebulin revealed an intronic homozygous splicing mutation between exon 144 and exon 145. Cloning of the biopsy PCR fragment revealed that a large proportion of the isoform with exon 144 contained an 117 additional base pairs probably due to the creation of a new splicing site as predicted by in silico analysis. Exon 143 and 144 cannot coexist in the same isoform but in our patient functional studies demonstrated an altered exon 144/143 isoform ratio. Nebulin protein sequence encoded by exon 143 is highly conserved across species, suggesting that it may have a special function, such as species interaction with another protein. For example, the binding site for KLHL40 is within S21, but its precise location is not known. Therefore this intronic mutation alters nebulin protein by direct functional alteration of the transcript and by destabilizing other transcripts, particularly exon 143, whose interaction in KLHL40 binding site could explain the high amount of nemaline inclusion bodies in the muscle biopsy as seen in KLHL40 related nemaline myopathy. Some unresolved cases of nemaline myopathy could be explained by intronic mutations in nebulin. New diagnostic approaches could help to explain these cases.

Expanding the Genetic Code: Unnatural Base Pairs in Biological Systems

The genetic code is considered to use five nucleic bases (adenine, guanine, cytosine, thymine and uracil), which form two pairs for encoding information in DNA and two pairs for encoding information in RNA. Nevertheless, in recent years several artificial base pairs have been developed in attempts to expand the genetic code. Employment of these additional base pairs increases the information capacity and variety of DNA sequences, and provides a platform for the site-specific, enzymatic incorporation of extra functional components into DNA and RNA. As a result, of the development of such expanded systems, many artificial base pairs have been synthesized and tested under various conditions. Following many stages of enhancement, unnatural base pairs have been modified to eliminate their weak points, qualifying them for specific research needs. Moreover, the first attempts to create a semi-synthetic organism containing DNA with unnatural base pairs seem to have been successful. This further extends the possible applications of these kinds of pairs. Herein, we describe the most significant qualities of unnatural base pairs and their actual applications.

Mechanisms of improved specificity of engineered Cas9s revealed by single-molecule FRET analysis.

Cas9 (from Streptococcus pyogenes) in complex with a guide RNA targets complementary DNA for cleavage. Here, we developed a single-molecule FRET analysis to study the mechanisms of specificity enhancement of two engineered Cas9s (eCas9 and Cas9-HF1). A DNA-unwinding assay showed that mismatches affect cleavage reactions through rebalancing the unwinding-rewinding equilibrium. Increasing PAM-distal mismatches facilitates rewinding, and the associated cleavage impairment shows that cleavage proceeds from the unwound state. Engineered Cas9s depopulate the unwound state more readily with mismatches. The intrinsic cleavage rate is much lower for engineered Cas9s, preventing cleavage from transiently unwound off-targets. Engineered Cas9s require approximately one additional base pair match for stable binding, freeing them from sites that would otherwise sequester them. Therefore, engineered Cas9s achieve their improved specificity by inhibiting stable DNA binding to partially matching sequences, making DNA unwinding more sensitive to mismatches and slowing down the intrinsic cleavage reaction.

Backbone conformation affects duplex initiation and duplex propagation in hybridisation of synthetic H-bonding oligomers.

Synthetic oligomers equipped with complementary H-bond donor and acceptor side chains form multiply H-bonded duplexes in organic solvents. Comparison of the duplex forming properties of four families of oligomers with different backbones shows that formation of an extended duplex with three or four inter-strand H-bonds is more challenging than formation of complexes that make only two H-bonds. The stabilities of 1 : 1 complexes formed between length complementary homo-oligomers equipped with either phosphine oxide or phenol recognition modules were measured in toluene. When the backbone is very flexible (pentane-1,5-diyl thioether), the stability increases uniformly by an order of magnitude for each additional base-pair added to the duplex: the effective molarities for formation of the first intramolecular H-bond (duplex initiation) and subsequent intramolecular H-bonds (duplex propagation) are similar. This flexible system is compared with three more rigid backbones that are isomeric combinations of an aromatic ring and methylene groups. One of the rigid systems behaves in exactly the same way as the flexible backbone, but the other two do not. For these systems, the effective molarity for formation of the first intramolecular H-bond is the same as that found for the other two backbones, but additional H-bonds are not formed between the longer oligomers. The effective molarities are too low for duplex propagation in these systems, because the oligomer backbones cannot adopt conformations compatible with formation of an extended duplex.

Structural Insight into the Unbound State of the DNA Analogue of the PreQ1 Riboswitch: A Thermodynamic Approach.

The preQ1 riboswitch aptamer domain is very dynamic in its unbound state with the ability to form multiple structures: a hairpin, kissing hairpins, and pseudoknot-like structure. The aim of this study is to determine whether the DNA analogue (PreQ1) is able to form structures similar to that of the reported RNA aptamer. Using a thermodynamic approach, we report on structural determination using differential scanning calorimetry under different salt conditions. Further analysis of the primary sequence allowed us to design modified molecules to determine what potential structures are forming in this single-stranded DNA analogue. We found, in a 16 mM Na+ solution, PreQ1 has three transitions with TM values of 14.8, 19.4, and 26.2 °C and a total ΔH of -44.7 kcal/mol. With the increase in salt concentration to 116 mM, there are TM values of 22.3, 28.7, and 38.9 °C and a ΔH of -69.1 kcal/mol, while at 216 mM, the three transitions have TM values of 24.4, 31.6, and 42.9 °C with a total ΔH of -71.5 kcal/mol. Therefore, the increase in enthalpy is due to the formation of additional base-pair stacks. The modified molecules, which would inhibit pseudoknot formation, kissing hairpins, and internal loop interactions, were fully characterized and compared to the native DNA analogue. The analysis of the enthalpy and differential binding of counterions allows us to conclude this single-stranded DNA analogue under physiological conditions is not forming a pseudoknot-like structure. Instead, two potential structures, Compact-Hairpin and Kissing-Complex, are more likely and could be in equilibrium.

Mesoscopic modelling of Cy3 and Cy5 dyes attached to DNA duplexes

Cy3 and Cy5 dyes linked to the 5′ end of a double stranded DNA molecule are known to attach to both strands in a way that is very similar to an additional base pair and has a stabilizing effect on the oligonucleotide. Here we adapt the Peyrard-Bishop mesoscopic model to incorporate cyanine dyes and use the technique of thermal equivalence to obtain the appropriate parameters from existing melting temperatures. We have found that the stacking parameters are in the same range of ordinary AT and CG base pairs, in particular Cy3-A was found to be most rigidly stacked. While the cyanines stabilize the AT hydrogen bonds quite strongly the CG bonds are mostly unaffected.

Lattice model of oligonucleotide hybridization in solution. II. Specificity and cooperativity.

Because oligonucleotides are short sequences of nucleic acid bases, their association in solution with complementary strands (hybridization) is often seen to conform to a simple two-state model. However, experimental evidence suggests that, despite their short length, oligonucleotides may hybridize through multiple states involving intermediates. We investigate whether these apparently contradictory scenarios are possible by imposing different levels of sequence specificity on a lattice model of oligonucleotides in solution, which we introduced in Part I [J. C. Araque et al., J. Chem. Phys. 134, 165103 (2011)]. We find that both multiple-intermediate (weakly cooperative) and two-state (strongly cooperative) transitions are possible and that these are directly linked to the level of sequence specificity. Sequences with low specificity hybridize (base-by-base) by way of multiple stable intermediates with increasing number of paired bases. Such intermediate states are weakly cooperative because the energetic gain from adding an additional base pair is outweighed by the conformational entropy loss. Instead, sequences with high specificity hybridize through multiple metastable intermediates which easily bridge the configurational and energetic gaps between single- and double-stranded states. These metastable intermediates interconvert with minimal loss of conformational entropy leading to a strongly cooperative hybridization. The possibility of both scenarios, multiple- and two-states, is therefore encoded in the specificity of the sequence which in turn defines the level of cooperativity.

Force-Induced Rupture of a DNA Duplex: From Fundamentals to Force Sensors.

The rupture of double-stranded DNA under stress is a key process in biophysics and nanotechnology. In this article, we consider the shear-induced rupture of short DNA duplexes, a system that has been given new importance by recently designed force sensors and nanotechnological devices. We argue that rupture must be understood as an activated process, where the duplex state is metastable and the strands will separate in a finite time that depends on the duplex length and the force applied. Thus, the critical shearing force required to rupture a duplex depends strongly on the time scale of observation. We use simple models of DNA to show that this approach naturally captures the observed dependence of the force required to rupture a duplex within a given time on duplex length. In particular, this critical force is zero for the shortest duplexes, before rising sharply and then plateauing in the long length limit. The prevailing approach, based on identifying when the presence of each additional base pair within the duplex is thermodynamically unfavorable rather than allowing for metastability, does not predict a time-scale-dependent critical force and does not naturally incorporate a critical force of zero for the shortest duplexes. We demonstrate that our findings have important consequences for the behavior of a new force-sensing nanodevice, which operates in a mixed mode that interpolates between shearing and unzipping. At a fixed time scale and duplex length, the critical force exhibits a sigmoidal dependence on the fraction of the duplex that is subject to shearing.

Sampled ensemble neutrality as a feature to classify potential structured RNAs.

BackgroundStructured RNAs have many biological functions ranging from catalysis of chemical reactions to gene regulation. Yet, many homologous structured RNAs display most of their conservation at the secondary or tertiary structure level. As a result, strategies for structured RNA discovery rely heavily on identification of sequences sharing a common stable secondary structure. However, correctly distinguishing structured RNAs from surrounding genomic sequence remains challenging, especially during de novo discovery. RNA also has a long history as a computational model for evolution due to the direct link between genotype (sequence) and phenotype (structure). From these studies it is clear that evolved RNA structures, like protein structures, can be considered robust to point mutations. In this context, an RNA sequence is considered robust if its neutrality (extent to which single mutant neighbors maintain the same secondary structure) is greater than that expected for an artificial sequence with the same minimum free energy structure.ResultsIn this work, we bring concepts from evolutionary biology to bear on the structured RNA de novo discovery process. We hypothesize that alignments corresponding to structured RNAs should consist of neutral sequences. We evaluate several measures of neutrality for their ability to distinguish between alignments of structured RNA sequences drawn from Rfam and various decoy alignments. We also introduce a new measure of RNA structural neutrality, the structure ensemble neutrality (SEN). SEN seeks to increase the biological relevance of existing neutrality measures in two ways. First, it uses information from an alignment of homologous sequences to identify a conserved biologically relevant structure for comparison. Second, it only counts base-pairs of the original structure that are absent in the comparison structure and does not penalize the formation of additional base-pairs.ConclusionWe find that several measures of neutrality are effective at separating structured RNAs from decoy sequences, including both shuffled alignments and flanking genomic sequence. Furthermore, as an independent feature classifier to identify structured RNAs, SEN yields comparable performance to current approaches that consider a variety of features including stability and sequence identity. Finally, SEN outperforms other measures of neutrality at detecting mutational robustness in bacterial regulatory RNA structures.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-014-1203-8) contains supplementary material, which is available to authorized users.

Computational methods toward accurate RNA structure prediction using coarse-grained and all-atom models.

Computational methods can provide significant insights into RNA structure and dynamics, bridging the gap in our understanding of the relationship between structure and biological function. Simulations enrich and enhance our understanding of data derived on the bench, as well as provide feasible alternatives to costly or technically challenging experiments. Coarse-grained computational models of RNA are especially important in this regard, as they allow analysis of events occurring in timescales relevant to RNA biological function, which are inaccessible through experimental methods alone. We have developed a three-bead coarse-grained model of RNA for discrete molecular dynamics simulations. This model is efficient in de novo prediction of short RNA tertiary structure, starting from RNA primary sequences of less than 50 nucleotides. To complement this model, we have incorporated additional base-pairing constraints and have developed a bias potential reliant on data obtained from hydroxyl probing experiments that guide RNA folding to its correct state. By introducing experimentally derived constraints to our computer simulations, we are able to make reliable predictions of RNA tertiary structures up to a few hundred nucleotides. Our refined model exemplifies a valuable benefit achieved through integration of computation and experimental methods.