Double-helical RNA Research Articles

Molecular Origin of Distinct Hydration Dynamics in Double Helical DNA and RNA Sequences.

Water molecules are essential to determine the structure of nucleic acids and mediate their interactions with other biomolecules. Here, we characterize the hydration dynamics of analogous DNA and RNA double helices with unprecedented resolution and elucidate the molecular origin of their differences: first, the localization of the slowest hydration water molecules─in the minor groove in DNA, next to phosphates in RNA─and second, the markedly distinct hydration dynamics of the two phosphate oxygen atoms OR and OS in RNA. Using our Extended Jump Model for water reorientation, we assess the relative importance of previously proposed factors, including the local topography, water bridges, and the presence of ions. We show that the slow hydration dynamics at RNA OR sites is not due to bridging water molecules but is caused by both the larger excluded volume and the stronger initial H-bond next to OR, due to the different phosphate orientations in A-form double helical RNA.

Temperature-dependent elasticity of DNA, RNA, and hybrid double helices

Nucleic acid double helices in their DNA, RNA, and DNA-RNA hybrid form play a fundamental role in biology and are main building blocks of artificial nanostructures, but how their properties depend on temperature remains poorly understood. Here, we report thermal dependence of dynamic bending persistence length, twist rigidity, stretch modulus, and twist-stretch coupling for DNA, RNA, and hybrid duplexes between 7°C and 47°C. The results are based on all-atom molecular dynamics simulations using different force field parameterizations. We first demonstrate that unrestrained molecular dynamics can reproduce experimentally known mechanical properties of the duplexes at room temperature. Beyond experimentally known features, we also infer the twist rigidity and twist-stretch coupling of the hybrid duplex. As for the temperature dependence, we found that increasing temperature softens all the duplexes with respect to bending, twisting, and stretching. The relative decrease of the stretch moduli is 0.003–0.004/°C, similar for all the duplex variants despite their very different stretching stiffness, whereas RNA twist stiffness decreases by 0.003/°C, and smaller values are found for the other elastic moduli. The twist-stretch couplings are nearly unaffected by temperature. The stretching, bending, and twisting stiffness all include an important entropic component. Relation of our results to the two-state model of DNA flexibility is discussed. Our work provides temperature-dependent elasticity of nucleic acid duplexes at the microsecond scale relevant for initial stages of protein binding.

Temperature-Dependent Twist of Double-Stranded RNA Probed by Magnetic Tweezer Experiments and Molecular Dynamics Simulations.

RNA plays critical roles in the transmission and regulation of genetic information and is increasingly used in biomedical and biotechnological applications. Functional RNAs contain extended double-stranded regions, and the structure of double-stranded RNA (dsRNA) has been revealed at high resolution. However, the dependence of the properties of the RNA double helix on environmental effects, notably temperature, is still poorly understood. Here, we use single-molecule magnetic tweezer measurements to determine the dependence of the dsRNA twist on temperature. We find that dsRNA unwinds with increasing temperature, even more than DNA, with ΔTwRNA = -14.4 ± 0.7°/(°C·kbp), compared to ΔTwDNA = -11.0 ± 1.2°/(°C·kbp). All-atom molecular dynamics (MD) simulations using a range of nucleic acid force fields, ion parameters, and water models correctly predict that dsRNA unwinds with rising temperature but significantly underestimate the magnitude of the effect. These MD data, together with additional MD simulations involving DNA and DNA-RNA hybrid duplexes, reveal a linear correlation between the twist temperature decrease and the helical rise, in line with DNA but at variance with RNA experimental data. We speculate that this discrepancy might be caused by some unknown bias in the RNA force fields tested or by as yet undiscovered transient alternative structures in the RNA duplex. Our results provide a baseline to model more complex RNA assemblies and to test and develop new parametrizations for RNA simulations. They may also inspire physical models of the temperature-dependent dsRNA structure.

Improved Triplex-Forming Isoorotamide PNA Nucleobases for A-U Recognition of RNA Duplexes.

Four new isoorotamide (Io)-containing PNA nucleobases have been designed for A-U recognition of double helical RNA. New PNA monomers were prepared efficiently and incorporated into PNA nonamers for binding A-U in a PNA:RNA2 triplex. Isothermal titration calorimetry and UV thermal melting experiments revealed slightly improved binding affinity for singly modified PNA compared to known A-binding nucleobases. Molecular dynamics simulations provided further insights into binding of Io bases in the triple helix. Together, the data revealed interesting insights into binding modes including the notion that three Hoogsteen hydrogen bonds are unnecessary for strong selective binding of an extended nucleobase. Cationic monomer Io8 additionally gave the highest affinity observed for an A-binding nucleobase to date. These results will help inform future nucleobase design toward the goal of recognizing any sequence of double helical RNA.

Structure and Dynamics of DNA and RNA Double Helices Formed by d(CTG), d(GTC), r(CUG), and r(GUC) Trinucleotide Repeats and Associated DNA-RNA Hybrids.

Myotonic dystrophy type 1 is the most frequent form of muscular dystrophy in adults caused by an abnormal expansion of the CTG trinucleotide. Both the expanded DNA and the expanded CUG RNA transcript can fold into hairpins. Co-transcriptional formation of stable RNA·DNA hybrids can also enhance the instability of repeat tracts. We performed molecular dynamics simulations of homoduplexes associated with the disease, d(CTG)n and r(CUG)n, and their corresponding r(CAG)n:d(CTG)n and r(CUG)n:d(CAG)n hybrids that can form under bidirectional transcription and of non-pathological d(GTC)n and d(GUC)n homoduplexes. We characterized their conformations, stability, and dynamics and found that the U·U and T·T mismatches are dynamic, favoring anti-anti conformations inside the helical core, followed by anti-syn and syn-syn conformations. For DNA, the secondary minima in the non-expanding d(GTC)n helices are deeper, wider, and longer-lived than those in d(CTG)n, which constitutes another biophysical factor further differentiating the expanding and non-expanding sequences. The hybrid helices are closer to A-RNA, with the A-T and A-U pairs forming two stable Watson-Crick hydrogen bonds. The neutralizing ion distribution around the non-canonical pairs is also described.

Triplex-Forming Peptide Nucleic Acid Controls Dynamic Conformations of RNA Bulges.

RNA folding is driven by the formation of double-helical segments interspaced by loops of unpaired nucleotides. Among the latter, bulges formed by one or several unpaired nucleotides are one of the most common structural motifs that play an important role in stabilizing RNA-RNA, RNA-protein, and RNA-small molecule interactions. Single-nucleotide bulges can fold in alternative structures where the unpaired nucleobase is either looped-out (flexible) in a solvent or stacked-in (intercalated) between the base pairs. In the present study, we discovered that triplex-forming peptide nucleic acids (PNAs) had unusually high affinity for single-purine-nucleotide bulges in double-helical RNA. Depending on the PNA's sequence, the triplex formation shifted the equilibrium between looped-out and stacked-in conformations. The ability to control the dynamic equilibria of RNA's structure will be an important tool for studying structure-function relationships in RNA biology and may have potential in novel therapeutic approaches targeting disease-related RNAs.

Positive Regulation of Splicing of Cellular and Viral mRNA by Intragenic RNA Elements That Activate the Stress Kinase PKR, an Antiviral Mechanism.

The transient activation of the cellular stress kinase, protein kinase RNA-activated (PKR), by double-helical RNA, especially by viral double-stranded RNA generated during replication, results in the inhibition of translation via the phosphorylation of eukaryotic initiation factor 2 α-chain (eIF2α). Exceptionally, short intragenic elements within primary transcripts of the human tumor necrosis factor (TNF-α) and globin genes, genes essential for survival, can form RNA structures that strongly activate PKR and thereby render the splicing of their mRNAs highly efficient. These intragenic RNA activators of PKR promote early spliceosome assembly and splicing by inducing phosphorylation of nuclear eIF2α, without impairing the translation of the mature spliced mRNA. Unexpectedly, excision of the large human immunodeficiency virus (HIV) rev/tat intron was shown to require activation of PKR by the viral RNA and eIF2α phosphorylation. The splicing of rev/tat mRNA is abrogated by viral antagonists of PKR and by trans-dominant negative mutant PKR, yet enhanced by the overexpression of PKR. The TNFα and HIV RNA activators of PKR fold into compact pseudoknots that are highly conserved within the phylogeny, supporting their essential role in the upregulation of splicing. HIV provides the first example of a virus co-opting a major cellular antiviral mechanism, the activation of PKR by its RNA, to promote splicing.

Linoleic acid binds to SARS-CoV-2 RdRp and represses replication of seasonal human coronavirus OC43

Fatty acids belong to a group of compounds already acknowledged for their broad antiviral efficacy. However, little is yet known about their effect on replication of human coronaviruses. To shed light on this subject, we first screened 15 fatty acids, three lipid-soluble vitamins, and cholesterol, on SARS-CoV-2 RdRp, and identified the four fatty acids with the highest RdRp inhibitory potential. Among them, linoleic acid was found to have the greatest interaction with SARS-CoV-2 RdRp, with its direct binding to the cavity formed by the RNA double helix and protein. Linoleic acid forms hydrophobic interactions with multiple residues, and at the same time forms electrostatic interactions including the hydrogen bond with Lys593 and Asp865. In line with these results, a dose-dependent inhibition of HCoV-OC43 replication in vitro was observed, additionally strengthened by data from in vivo study, which also confirmed anti-inflammatory potential of linoleic acid. Based on these results, we concluded that our study provides a new understanding of the antiviral properties of fatty acids against human coronaviruses including the SARS-CoV-2 strain. Particularly, they lays down a new prospect for linoleic acid’s RdRp-inhibitory activity, as a candidate for further studies, which are warranted to corroborate the results presented here.

Structure of angiogenin dimer bound to double-stranded RNA.

Angiogenin is an unusual member of the RNase A family and is of great interest in multiple pathological contexts. Although it has been assigned various regulatory roles, its core catalytic function is that of an RNA endonuclease. However, its catalytic efficiency is comparatively low and this has been linked to a unique C-terminal helix which partially blocks its RNA-binding site. Assuming that binding to its RNA substrate could trigger a conformational rearrangement, much speculation has arisen on the topic of the interaction of angiogenin with RNA. To date, no structural data on angiogenin-RNA interactions have been available. Here, the structure of angiogenin bound to a double-stranded RNA duplex is reported. The RNA does not reach the active site of angiogenin and nostructural arrangement of the C-terminal domain is observed. However, angiogenin forms a previously unobserved crystallographic dimer that makes several backbone interactions with the major and minor grooves of the RNA double helix.

Mechanical properties of nucleic acids and the non-local twistable wormlike chain model.

Mechanical properties of nucleic acids play an important role in many biological processes that often involve physical deformations of these molecules. At sufficiently long length scales (say, above ∼20-30 base pairs), the mechanics of DNA and RNA double helices is described by a homogeneous Twistable Wormlike Chain (TWLC), a semiflexible polymer model characterized by twist and bending stiffnesses. At shorter scales, this model breaks down for two reasons: the elastic properties become sequence-dependent and the mechanical deformations at distal sites get coupled. We discuss in this paper the origin of the latter effect using the framework of a non-local Twistable Wormlike Chain (nlTWLC). We show, by comparing all-atom simulations data for DNA and RNA double helices, that the non-local couplings are of very similar nature in these two molecules: couplings between distal sites are strong for tilt and twist degrees of freedom and weak for roll. We introduce and analyze a simple double-stranded polymer model that clarifies the origin of this universal distal couplings behavior. In this model, referred to as the ladder model, a nlTWLC description emerges from the coarsening of local (atomic) degrees of freedom into angular variables that describe the twist and bending of the molecule. Different from its local counterpart, the nlTWLC is characterized by a length-scale-dependent elasticity. Our analysis predicts that nucleic acids are mechanically softer at the scale of a few base pairs and are asymptotically stiffer at longer length scales, a behavior that matches experimental data.

RNABPDB: Molecular Modeling of RNA Structure-From Base Pair Analysis in Crystals to Structure Prediction.

The stable three-dimensional structure of RNA is known to play several important biochemical roles, from post-transcriptional gene regulation to enzymatic action. These structures contain double-helical regions, which often have different types of non-canonical base pairs in addition to Watson-Crick base pairs. Hence, it is important to study their structures from experimentally obtained or even predicted ones, to understand their role, or to develop a drug against the potential targets. Molecular Modeling of RNA double helices containing non-canonical base pairs is a difficult process, particularly due to the unavailability of structural features of non-Watson-Crick base pairs. Here we show a composite web-server with an associated database that allows one to generate the structure of RNA double helix containing non-canonical base pairs using consensus parameters obtained from the database. The database classification is followed by an evaluation of the central tendency of the structural parameters as well as a quantitative estimation of interaction strengths. These parameters are used to construct three-dimensional structures of double helices composed of Watson-Crick and/or non-canonical base pairs. Our benchmark study to regenerate double-helical fragments of many experimentally derived RNA structures indicate very high accuracy. This composite server is expected to be highly useful in understanding functions of various pre-miRNA by modeling structures of the molecules and estimating binding efficiency. The database can be accessed from http://hdrnas.saha.ac.in/rnabpdb .

Tuning Exciton Coupling of Merocyanine Nucleoside Dimers by RNA, DNA and GNA Double Helix Conformations.

Exciton coupling between two or more chromophores in a specific environment is a key mechanism associated with color tuning and modulation of absorption energies. This concept is well exemplified by natural photosynthetic proteins, and can also be achieved in synthetic nucleic acid nanostructures. Here we report the coupling of barbituric acid merocyanine (BAM) nucleoside analogues and show that exciton coupling can be tuned by the double helix conformation. BAM is a nucleobase mimic that was incorporated in the phosphodiester backbone of RNA, DNA and GNA oligonucleotides. Duplexes with different backbone constitutions and geometries afforded different mutual dye arrangements, leading to distinct optical signatures due to competing modes of chromophore organization via electrostatic, dipolar, π–π‐stacking and hydrogen‐bonding interactions. The realized supramolecular motifs include hydrogen‐bonded BAM–adenine base pairs and antiparallel as well as rotationally stacked BAM dimer aggregates with distinct absorption, CD and fluorescence properties.

Tuning Exciton Coupling of Merocyanine Nucleoside Dimers by RNA, DNA and GNA Double Helix Conformations

AbstractExciton coupling between two or more chromophores in a specific environment is a key mechanism associated with color tuning and modulation of absorption energies. This concept is well exemplified by natural photosynthetic proteins, and can also be achieved in synthetic nucleic acid nanostructures. Here we report the coupling of barbituric acid merocyanine (BAM) nucleoside analogues and show that exciton coupling can be tuned by the double helix conformation. BAM is a nucleobase mimic that was incorporated in the phosphodiester backbone of RNA, DNA and GNA oligonucleotides. Duplexes with different backbone constitutions and geometries afforded different mutual dye arrangements, leading to distinct optical signatures due to competing modes of chromophore organization via electrostatic, dipolar, π–π‐stacking and hydrogen‐bonding interactions. The realized supramolecular motifs include hydrogen‐bonded BAM–adenine base pairs and antiparallel as well as rotationally stacked BAM dimer aggregates with distinct absorption, CD and fluorescence properties.

Fluorobenzene Nucleobase Analogues for Triplex-Forming Peptide Nucleic Acids.

2,4-Difluorotoluene is a nonpolar isostere of thymidine that has been used as a powerful mechanistic probe to study the role of hydrogen bonding in nucleic acid recognition and interactions with polymerases. In the present study, we evaluated five fluorinated benzenes as nucleobase analogues in peptide nucleic acids designed for triple helical recognition of double helical RNA. We found that analogues having para and ortho fluorine substitution patterns (as in 2,4-difluorotoluene) selectively stabilized Hoogsteen triplets with U-A base pairs. The results were consistent with attractive electrostatic interactions akin to non-canonical F to H-N and C-H to N hydrogen bonding. The fluorinated nucleobases were not able to stabilize Hoogsteen-like triplets with pyrimidines in either G-C or A-U base pairs. Our results illustrate the ability of fluorine to engage in non-canonical base pairing and provide insights into triple helical recognition of RNA.

Maturation of siRNA by strand separation: Steered molecular dynamics study

RNA interference, particularly siRNA induced gene silencing is becoming an important avenue of modern therapeutics. The siRNA is delivered to the cells as short double helical RNA which becomes single stranded for forming the RISC complex. Significant experimental evidence is available for most of the steps except the process of the separation of the two strands. We have attempted to understand the pathway for double stranded siRNA (dsRNA) to single stranded (ssRNA) molecules using steered molecular dynamics simulations. As the process is completely unexplored we have applied force from all possible directions restraining all possible residues to convert dsRNA to ssRNA. We found pulling one strand along the helical axis direction restraining the far end of the other strand demands excessive force for ssRNA formation. Pulling a central residue of one strand, in a direction perpendicular to the helix axis, while keeping the base paired residue fixed requires intermediate force for strand separation. Moreover, we found that in this process the force requirement is quite high for the first bubble formation (nucleation energy) and the bubble propagation energies are quite small. We believe the success rate of the design of siRNA sequences for gene silencing may increase if this mechanistic knowledge is utilized for such a design process. Communicated by Ramaswamy H. Sarma

Comparative studies on the binding interaction of two chiral Ru(II) polypyridyl complexes with triple- and double-helical forms of RNA

Two chiral Ru(II) polypyridyl complexes, Δ-[Ru(bpy)2(6-F-dppz)]2+ (Δ-1; bpy = 2,2′-bipyridine, 6-F-dppz = 6-fluorodipyrido[3,2-a:2′,3′-c]phenazine) and Λ-[Ru(bpy)2(6-F-dppz)]2+ (Λ-1), have been synthesized and characterized as binders for the RNA poly(U)•poly(A)*poly(U) triplex and poly(A)•poly(U) duplex in this work. Analysis of the UV–Vis absorption spectra and fluorescence emission spectra indicates that the binding of intercalating Δ-1 with the triplex and duplex RNA is greater than that of Λ-1, while the binding affinities of the two enantiomers to triplex structure is stronger than that of duplex structure. Fluorescence titrations show that the two enantiomers can act as molecular “light switches” for triple- and double-helical RNA. Thermal denaturation studies revealed that that the two enantiomers are more stable to Watson-Crick base-paired double strand of the triplex than the Hoogsteen base-paired third strand, but their stability and selectivity are different. For Δ-enantiomer, the increase of the thermal stability of the Watson-Crick base-paired duplex (13 °C) is slightly stronger than of the Hoogsteen base-paired strand (10 °C), displaying no obvious selectivity. However, compared to the Hoogsteen base-paired strand (5 °C), the stability of the Λ-enantiomer to the Watson-Crick base-paired duplex (13 °C) is more significant, which has obvious selectivity. The overall increase in viscosity of the RNA-(Λ-1) system and its curve shape are similar to that of the RNA-(Δ-1) system, suggesting that the binding modes of two enantiomers with RNA are intercalation. The obtained results in this work may be useful for understanding the binding differences in chiral Ru(II) polypyridyl complexes toward RNA triplex and duplex.

Aspects of Nucleic Acid Structure and Function

Despite decades of intense study, certain physico-chemical aspects of the nucleic acids–the genetic repository of life–remain enigmatic. Thus, solutions of DNA are apparently constituted of varying amounts of double and single-stranded forms, with melting studies being inconclusive about the effect of temperature on the composition. Consequently, this casts doubt on current estimates of the thermodynamic stability of the base pairs. However, the overwhelming stability of the Watson-Crick model is adumbrated by a kinetic analysis of the action of DNA polymerase, microscopic reversibility indicating that polymerase action is kinetically controlled, whereas proof reading-excision is thermodynamically controlled.
 The structural basis for the differing roles played by DNA and RNA in the sustenance and propagation of life also remains to be clarified. It appears that formation of the RNA double helix is sterically inhibited by the 2’ ribose hydroxyl group, which is also relatively inaccessible to external base, thus enhancing hydrolytic stability. The in vivo conformation of RNA is likely determined by the requirements of its appointed biological role, the large size of tRNAs being particularly significant in possibly leading to enhanced specificity in its interaction with the corresponding tRNA synthetase. The proclivity of RNA generally to remain single stranded may indeed be the reason for the existence of viruses as stable RNA-protein complexes.
 The preeminent role of nucleic acids as “genetic guardians” is, however, blurred by the fact that the translation of the genetic code is contingent on the action of the various tRNA synthetases. This implies that the genetic code is manifested via codon-anticodon specificity only by the involvement of protein molecules that are unique to each codon-anticodon pair. This has intriguing implications for the origin and evolution of the genetic code, possibly indicating that the tRNA synthetases are a relic of prebiotic protein-nucleic acid hybrids (thus also raising doubts about the RNA world hypothesis).

A m6 A Sensing Method by Its Impact on the Stability of RNA Double Helix.

N6 -Methyladenosine (m6 A) is one of the most important RNA modifications in epigenetics. The development of detection method for m6 A is limited by its abundance and structure. Although it has been previously reported that its presence has an impact on the complementary pairing of RNA, few assays have been developed using this finding. We used this discovery and designed a detection method based on Cas13a system, which has different fluorescence signals for target RNAs containing m6 A modification and target RNAs without m6 A modification. We verified the fact that the presence of m6 A could cause the instability of dsRNA using the Cas13a system and provided a new direction and strategy for the development of m6 A detection methods in the future.

Unraveling the structure and biological functions of RNA triple helices.

It has been nearly 63 years since the first characterization of an RNA triple helix in vitro by Gary Felsenfeld, David Davies, and Alexander Rich. An RNA triple helix consists of three strands: A Watson–Crick RNA double helix whose major‐groove establishes hydrogen bonds with the so‐called “third strand”. In the past 15 years, it has been recognized that these major‐groove RNA triple helices, like single‐stranded and double‐stranded RNA, also mediate prominent biological roles inside cells. Thus far, these triple helices are known to mediate catalysis during telomere synthesis and RNA splicing, bind to ligands and ions so that metabolite‐sensing riboswitches can regulate gene expression, and provide a clever strategy to protect the 3′ end of RNA from degradation. Because RNA triple helices play important roles in biology, there is a renewed interest in better understanding the fundamental properties of RNA triple helices and developing methods for their high‐throughput discovery. This review provides an overview of the fundamental biochemical and structural properties of major‐groove RNA triple helices, summarizes the structure and function of naturally occurring RNA triple helices, and describes prospective strategies to isolate RNA triple helices as a means to establish the “triplexome”.This article is categorized under:RNA Structure and Dynamics > RNA Structure and DynamicsRNA Structure and Dynamics > RNA Structure, Dynamics and ChemistryRNA Structure and Dynamics > Influence of RNA Structure in Biological Systems

Symmetry in Nucleic-Acid Double Helices

In nature and in the test tube, nucleic acids occur in many different forms. Apart from single-stranded, coiled molecules, DNA and RNA prefer to form helical arrangements, in which the bases are stacked to shield their hydrophobic surfaces and expose their polar edges. Focusing on double helices, we describe the crucial role played by symmetry in shaping DNA and RNA structure. The base pairs in nucleic-acid double helices display rotational pseudo-symmetry. In the Watson–Crick base pairs found in naturally occurring DNA and RNA duplexes, the symmetry axis lies in the base-pair plane, giving rise to two different helical grooves. In contrast, anti-Watson–Crick base pairs have a dyad axis perpendicular to the base-pair plane and identical grooves. In combination with the base-pair symmetry, the syn/anti conformation of paired nucleotides determines the parallel or antiparallel strand orientation of double helices. DNA and RNA duplexes in nature are exclusively antiparallel. Watson–Crick base-paired DNA or RNA helices display either right-handed or left-handed helical (pseudo-) symmetry. Genomic DNA is usually in the right-handed B-form, and RNA double helices adopt the right-handed A-conformation. Finally, there is a higher level of helical symmetry in superhelical DNA in which B-form double strands are intertwined in a right- or left-handed sense.