Structure of an internal loop motif with three consecutive U•U mismatches from stem-loop 1 in the 3'-UTR of the SARS-CoV-2 genomic RNA.

The single-stranded RNA genome of SARS-CoV-2 is highly structured. Numerous helical stem-loop structures interrupted by mismatch motifs are present in the functionally important 5'- and 3'-UTRs. These mismatches modulate local helical geometries and feature unusual arrays of hydrogen bonding donor and acceptor groups. However, their conformational and dynamical properties cannot be directly inferred from chemical probing and are difficult to predict theoretically. A mismatch motif (SL1-motif) consisting of three consecutive U•U base pairs is located in stem-loop 1 of the 3'-UTR. We combined NMR-spectroscopy and MD-simulations to investigate its structure and dynamics. All three U•U base pairs feature two direct hydrogen bonds and are as stable as Watson-Crick A:U base pairs. Plasmodium falciparum 25S rRNA contains a triple U•U mismatch motif (Pf-motif) differing from SL1-motif only with respect to the orientation of the two closing base pairs. Interestingly, while the geometry of the outer two U•U mismatches was identical in both motifs the preferred orientation of the central U•U mismatch was different. MD simulations and potassium ion titrations revealed that the potassium ion-binding mode to the major groove is connected to the different preferred geometries of the central base pair in the two motifs.

Structural and dynamic effects of pseudouridine modifications on noncanonical interactions in RNA.

Pseudouridine is the most frequently naturally occurring RNA modification, found in all classes of biologically functional RNAs. Compared to uridine, pseudouridine contains an additional hydrogen bond donor group and is therefore widely regarded as a structure stabilizing modification. However, the effects of pseudouridine modifications on the structure and dynamics of RNAs have so far only been investigated in a limited number of different structural contexts. Here, we introduced pseudouridine modifications into the U-turn motif and the adjacent U:U closing base pair of the neomycin-sensing riboswitch (NSR)-an extensively characterized model system for RNA structure, ligand binding, and dynamics. We show that the effects of replacing specific uridines with pseudouridines on RNA dynamics crucially depend on the exact location of the replacement site and can range from destabilizing to locally or even globally stabilizing. By using a combination of NMR spectroscopy, MD simulations and QM calculations, we rationalize the observed effects on a structural and dynamical level. Our results will help to better understand and predict the consequences of pseudouridine modifications on the structure and function of biologically important RNAs.

Mechanical Stability and Unfolding Pathways of Parallel Tetrameric G-Quadruplexes Probed by Pulling Simulations.

Guanine quadruplex (GQ) is a noncanonical nucleic acid structure formed by guanine-rich DNA and RNA sequences. Folding of GQs is a complex process, where several aspects remain elusive, despite being important for understanding structure formation and biological functions of GQs. Pulling experiments are a common tool for acquiring insights into the folding landscape of GQs. Herein, we applied a computational pulling strategy─steered molecular dynamics (SMD) simulations─in combination with standard molecular dynamics (MD) simulations to explore the unfolding landscapes of tetrameric parallel GQs. We identified anisotropic properties of elastic conformational changes, unfolding transitions, and GQ mechanical stabilities. Using a special set of structural parameters, we found that the vertical component of pulling force (perpendicular to the average G-quartet plane) plays a significant role in disrupting GQ structures and weakening their mechanical stabilities. We demonstrated that the magnitude of the vertical force component depends on the pulling anchor positions and the number of G-quartets. Typical unfolding transitions for tetrameric parallel GQs involve base unzipping, opening of the G-stem, strand slippage, and rotation to cross-like structures. The unzipping was detected as the first and dominant unfolding event, and it usually started at the 3'-end. Furthermore, results from both SMD and standard MD simulations indicate that partial spiral conformations serve as a transient ensemble during the (un)folding of GQs.

Spontaneous binding of single-stranded RNAs to RRM proteins visualized by unbiased atomistic simulations with a rescaled RNA force field.

Recognition of single-stranded RNA (ssRNA) by RNA recognition motif (RRM) domains is an important class of protein-RNA interactions. Many such complexes were characterized using nuclear magnetic resonance (NMR) and/or X-ray crystallography techniques, revealing ensemble-averaged pictures of the bound states. However, it is becoming widely accepted that better understanding of protein-RNA interactions would be obtained from ensemble descriptions. Indeed, earlier molecular dynamics simulations of bound states indicated visible dynamics at the RNA-RRM interfaces. Here, we report the first atomistic simulation study of spontaneous binding of short RNA sequences to RRM domains of HuR and SRSF1 proteins. Using a millisecond-scale aggregate ensemble of unbiased simulations, we were able to observe a few dozen binding events. HuR RRM3 utilizes a pre-binding state to navigate the RNA sequence to its partially disordered bound state and then to dynamically scan its different binding registers. SRSF1 RRM2 binding is more straightforward but still multiple-pathway. The present study necessitated development of a goal-specific force field modification, scaling down the intramolecular van der Waals interactions of the RNA which also improves description of the RNA-RRM bound state. Our study opens up a new avenue for large-scale atomistic investigations of binding landscapes of protein-RNA complexes, and future perspectives of such research are discussed.

DNA Quadruplex Structure with a Unique Cation Dependency.

DNA quadruplex structures provide an additional layer of regulatory control in genome maintenance and gene expression and are widely used in nanotechnology. We report the discovery of an unprecedented tetrastranded structure formed from a native G-rich DNA sequence originating from the telomeric region of Caenorhabditis elegans. The structure is defined by multiple properties that distinguish it from all other known DNA quadruplexes. Most notably, the formation of a stable so-called KNa-quadruplex (KNaQ) requires concurrent coordination of K+ and Na+ ions at two distinct binding sites. This structure provides novel insight into G-rich DNA folding under ionic conditions relevant to eukaryotic cell physiology and the structural evolution of telomeric DNA. It highlights the differences between the structural organization of human and nematode telomeric DNA, which should be considered when using C. elegans as a model in telomere biology, particularly in drug screening applications. Additionally, the absence/presence of KNaQ motifs in the host/parasite introduces an intriguing possibility of exploiting the KNaQ fold as a plausible antiparasitic drug target. The structure's unique shape and ion dependency and the possibility of controlling its folding by using low-molecular-weight ligands can be used for the design or discovery of novel recognition DNA elements and sensors.

Prebiotic Synthesis of 3',5'-Cyclic Adenosine and Guanosine Monophosphates through Carbodiimide-Assisted Cyclization.

3',5'-Cyclic nucleotides play a fundamental role in modern biochemical processes and have been suggested to have played a central role at the origin of terrestrial life. In this work, we suggest that a formamide-based systems chemistry might account for their availability on the early Earth. In particular, we demonstrate that in a liquid formamide environment at elevated temperatures 3',5'-cyclic nucleotides are obtained in good yield and selectivity upon intramolecular cyclization of 5'-phosphorylated nucleosides in the presence of carbodiimides.

Complexity of Guanine Quadruplex Unfolding Pathways Revealed by Atomistic Pulling Simulations.

Guanine quadruplexes (GQs) are non-canonical nucleic acid structures involved in many biological processes. GQs formed in single-stranded regions often need to be unwound by cellular machinery, so their mechanochemical properties are important. Here, we performed steered molecular dynamics simulations of human telomeric GQs to study their unfolding. We examined four pulling regimes, including a very slow setup with pulling velocity and force load accessible to high-speed atomic force microscopy. We identified multiple factors affecting the unfolding mechanism, i.e.,: (i) the more the direction of force was perpendicular to the GQ channel axis (determined by GQ topology), the more the base unzipping mechanism happened, (ii) the more parallel the direction of force was, GQ opening and cross-like GQs were more likely to occur, (iii) strand slippage mechanism was possible for GQs with an all-anti pattern in a strand, and (iv) slower pulling velocity led to richer structural dynamics with sampling of more intermediates and partial refolding events. We also identified that a GQ may eventually unfold after a force drop under forces smaller than those that the GQ withstood before the drop. Finally, we found out that different unfolding intermediates could have very similar chain end-to-end distances, which reveals some limitations of structural interpretations of single-molecule spectroscopic data.

Sensitivity of the RNA Structure to Ion Conditions as Probed by Molecular Dynamics Simulations of Common Canonical RNA Duplexes.

RNA molecules play a key role in countless biochemical processes. RNA interactions, which are of highly diverse nature, are determined by the fact that RNA is a highly negatively charged polyelectrolyte, which leads to intimate interactions with an ion atmosphere. Although RNA molecules are formally single-stranded, canonical (Watson-Crick) duplexes are key components of folded RNAs. A double-stranded (ds) RNA is also important for the design of RNA-based nanostructures and assemblies. Despite the fact that the description of canonical dsRNA is considered the least problematic part of RNA modeling, the imperfect shape and flexibility of dsRNA can lead to imbalances in the simulations of larger RNAs and RNA-containing assemblies. We present a comprehensive set of molecular dynamics (MD) simulations of four canonical A-RNA duplexes. Our focus was directed toward the characterization of the influence of varying ion concentrations and of the size of the solvation box. We compared several water models and four RNA force fields. The simulations showed that the A-RNA shape was most sensitive to the RNA force field, with some force fields leading to a reduced inclination of the A-RNA duplexes. The ions and water models played a minor role. The effect of the box size was negligible, and even boxes with a small fraction of the bulk solvent outside the RNA hydration sphere were sufficient for the simulation of the dsRNA.

Atomistic Picture of Opening-Closing Dynamics of DNA Holliday Junction Obtained by Molecular Simulations.

Holliday junction (HJ) is a noncanonical four-way DNA structure with a prominent role in DNA repair, recombination, and DNA nanotechnology. By rearranging its four arms, HJ can adopt either closed or open state. With enzymes typically recognizing only a single state, acquiring detailed knowledge of the rearrangement process is an important step toward fully understanding the biological function of HJs. Here, we carried out standard all-atom molecular dynamics (MD) simulations of the spontaneous opening-closing transitions, which revealed complex conformational transitions of HJs with an involvement of previously unconsidered "half-closed" intermediates. Detailed free-energy landscapes of the transitions were obtained by sophisticated enhanced sampling simulations. Because the force field overstabilizes the closed conformation of HJs, we developed a system-specific modification which for the first time allows the observation of spontaneous opening-closing HJ transitions in unbiased MD simulations and opens the possibilities for more accurate HJ computational studies of biological processes and nanomaterials.

Insight into formation propensity of pseudocircular DNA G-hairpins.

We recently showed that Saccharomyces cerevisiae telomeric DNA can fold into an unprecedented pseudocircular G-hairpin (PGH) structure. However, the formation of PGHs in the context of extended sequences, which is a prerequisite for their function in vivo and their applications in biotechnology, has not been elucidated. Here, we show that despite its 'circular' nature, PGHs tolerate single-stranded (ss) protrusions. High-resolution NMR structure of a novel member of PGH family reveals the atomistic details on a junction between ssDNA and PGH unit. Identification of new sequences capable of folding into one of the two forms of PGH helped in defining minimal sequence requirements for their formation. Our time-resolved NMR data indicate a possibility that PGHs fold via a complex kinetic partitioning mechanism and suggests the existence of K+ ion-dependent PGH folding intermediates. The data not only provide an explanation of cation-type-dependent formation of PGHs, but also explain the unusually large hysteresis between PGH melting and annealing noted in our previous study. Our findings have important implications for DNA biology and nanotechnology. Overrepresentation of sequences able to form PGHs in the evolutionary-conserved regions of the human genome implies their functionally important biological role(s).

The beginning and the end: flanking nucleotides induce a parallel G-quadruplex topology.

Genomic sequences susceptible to form G-quadruplexes (G4s) are always flanked by other nucleotides, but G4 formation in vitro is generally studied with short synthetic DNA or RNA oligonucleotides, for which bases adjacent to the G4 core are often omitted. Herein, we systematically studied the effects of flanking nucleotides on structural polymorphism of 371 different oligodeoxynucleotides that adopt intramolecular G4 structures. We found out that the addition of nucleotides favors the formation of a parallel fold, defined as the 'flanking effect' in this work. This 'flanking effect' was more pronounced when nucleotides were added at the 5'-end, and depended on loop arrangement. NMR experiments and molecular dynamics simulations revealed that flanking sequences at the 5'-end abolish a strong syn-specific hydrogen bond commonly found in non-parallel conformations, thus favoring a parallel topology. These analyses pave a new way for more accurate prediction of DNA G4 folding in a physiological context.

RNA kink-turns are highly anisotropic with respect to lateral displacement of the flanking stems.

Kink-turns are highly bent internal loop motifs commonly found in the ribosome and other RNA complexes. They frequently act as binding sites for proteins and mediate tertiary interactions in larger RNA structures. Kink-turns have been a topic of intense research, but their elastic properties in the folded state are still poorly understood. Here we use extensive all-atom molecular dynamics simulations to parameterize a model of kink-turn in which the two flanking helical stems are represented by effective rigid bodies. Time series of the full set of six interhelical coordinates enable us to extract minimum energy shapes and harmonic stiffness constants for kink-turns from different RNA functional classes. The analysis suggests that kink-turns exhibit isotropic bending stiffness but are highly anisotropic with respect to lateral displacement of the stems. The most flexible lateral displacement mode is perpendicular to the plane of the static bend. These results may help understand the structural adaptation and mechanical signal transmission by kink-turns in complex natural and artificial RNA structures.

MD simulations reveal the basis for dynamic assembly of Hfq-RNA complexes.

The conserved protein Hfq is a key factor in the RNA-mediated control of gene expression in most known bacteria. The transient intermediates Hfq forms with RNA support intricate and robust regulatory networks. In Pseudomonas, Hfq recognizes repeats of adenine-purine-any nucleotide (ARN) in target mRNAs via its distal binding side, and together with the catabolite repression control (Crc) protein, assembles into a translation-repression complex. Earlier experiments yielded static, ensemble-averaged structures of the complex, but details of its interface dynamics and assembly pathway remained elusive. Using explicit solvent atomistic molecular dynamics simulations, we modeled the extensive dynamics of the Hfq-RNA interface and found implications for the assembly of the complex. We predict that syn/anti flips of the adenine nucleotides in each ARN repeat contribute to a dynamic recognition mechanism between the Hfq distal side and mRNA targets. We identify a previously unknown binding pocket that can accept any nucleotide and propose that it may serve as a 'status quo' staging point, providing nonspecific binding affinity, until Crc engages the Hfq-RNA binary complex. The dynamical components of the Hfq-RNA recognition can speed up screening of the pool of the surrounding RNAs, participate in rapid accommodation of the RNA on the protein surface, and facilitate competition among different RNAs. The register of Crc in the ternary assembly could be defined by the recognition of a guanine-specific base-phosphate interaction between the first and last ARN repeats of the bound RNA. This dynamic substrate recognition provides structural rationale for the stepwise assembly of multicomponent ribonucleoprotein complexes nucleated by Hfq-RNA binding.

Computer Aided Development of Nucleic Acid Applications in Nanotechnologies.

Utilization of nucleic acids (NAs) in nanotechnologies and nanotechnology-related applications is a growing field with broad application potential, ranging from biosensing up to targeted cell delivery. Computer simulations are useful techniques that can aid design and speed up development in this field. This review focuses on computer simulations of hybrid nanomaterials composed of NAs and other components. Current state-of-the-art molecular dynamics simulations, empirical force fields (FFs), and coarse-grained approaches for the description of deoxyribonucleic acid and ribonucleic acid are critically discussed. Challenges in combining biomacromolecular and nanomaterial FFs are emphasized. Recent applications of simulations for modeling NAs and their interactions with nano- and biomaterials are overviewed in the fields of sensing applications, targeted delivery, and NA templated materials. Future perspectives of development are also highlighted.

Multiscale Modeling of Phosphate···π Contacts in RNA U-Turns Exposes Differences between Quantum-Chemical and AMBER Force Field Descriptions.

Phosphate···π, also called anion···π, contacts occur between nucleobases and anionic phosphate oxygens (OP2) in r(GNRA) and r(UNNN) U-turn motifs (N = A,G,C,U; R = A,G). These contacts were investigated using state-of-the-art quantum-chemical methods (QM) to characterize their physicochemical properties and to serve as a reference to evaluate AMBER force field (AFF) performance. We found that phosphate···π interaction energies calculated with the AFF for dimethyl phosphate···nucleobase model systems are less stabilizing in comparison with double-hybrid DFT and that minimum contact distances are larger for all nucleobases. These distance stretches are also observed in large-scale AFF vs QM/MM computations and classical molecular dynamics (MD) simulations on several r(gcGNRAgc) tetraloop hairpins when compared to experimental data extracted from X-ray/cryo-EM structures (res. ≤ 2.5 Å) using the WebFR3D bioinformatic tool. MD simulations further revealed shifted OP2/nucleobase positions. We propose that discrepancies between the QM and AFF result from a combination of missing polarization in the AFF combined with too large AFF Lennard-Jones (LJ) radii of nucleobase carbon atoms in addition to an exaggerated short-range repulsion of the r-12 LJ repulsive term. We compared these results with earlier data gathered on lone pair···π contacts in CpG Z-steps occurring in r(UNCG) tetraloops. In both instances, charge transfer calculations do not support any significant n → π* donation effects. We also investigated thiophosphate···π contacts that showed reduced stabilizing interaction energies when compared to phosphate···π contacts. Thus, we challenge suggestions that the experimentally observed enhanced thermodynamic stability of phosphorothioated r(GNRA) tetraloops can be explained by larger London dispersion.

Photoinduced water-chromophore electron transfer causes formation of guanosine photodamage.

UV-induced photolysis of aqueous guanine nucleosides produces 8-oxo-guanine and Fapy-guanine, which can induce various types of cellular malfunction. The mechanistic rationale underlying photodestructive processes of guanine nucleosides is still largely obscure. Here, we employ accurate quantum chemical calculations and demonstrate that an excited-state non-bonding interaction of guanosine and a water molecule facilitates the electron-driven proton transfer process from water to the chromophore fragment. This subsequently allows for the formation of a crucial intermediate, namely guanosine photohydrate. Further (photo)chemical reactions of this intermediate lead to the known products of guanine photodamage.

A Cr2O3-doped graphene sensor for early diagnosis of liver cirrhosis: a first-principles study.

Liver cirrhosis is among the leading causes of death worldwide. Because of its asymptomatic evolution, timely diagnosis of liver cirrhosis via non-invasive techniques is currently under investigation. Among the diagnostic methods employing volatile organic compounds directly detectable from breath, sensing of limonene (C10H16) represents one of the most promising strategies for diagnosing alcohol liver diseases, including cirrhosis. In the present work, by means of state-of-the-art Density Functional Theory calculations including the U correction, we present an investigation on the sensing capabilities of a chromium-oxide-doped graphene (i.e., Cr2O3-graphene) structure toward limonene detection. In contrast with other structures such as g-triazobenzol (g-C6N6) monolayers and germanane, which revealed their usefulness in detecting limonene via physisorption, the proposed Cr2O3-graphene heterostructure is capable of undergoing chemisorption upon molecular approaching of limonene over its surface. In fact, a high adsorption energy is recorded (∼-1.6 eV). Besides, a positive Moss-Burstein effect is observed upon adsorption of limomene on the Cr2O3-graphene heterostructure, resulting in a net increase of the bandgap (∼50%), along with a sizeable shift of the Fermi level toward the conduction band. These findings pave the way toward the experimental validation of such predictions and the employment of Cr2O3-graphene heterostructures as sensors of key liver cirrhosis biomarkers.

Molecular basis for AU-rich element recognition and dimerization by the HuR C-terminal RRM.

Human antigen R (HuR) is a key regulator of cellular mRNAs containing adenylate/uridylate-rich elements (AU-rich elements; AREs). These are a major class of cis elements within 3' untranslated regions, targeting these mRNAs for rapid degradation. HuR contains three RNA recognition motifs (RRMs): a tandem RRM1 and 2, followed by a flexible linker and a C-terminal RRM3. While RRM1 and 2 are structurally characterized, little is known about RRM3. Here we present a 1.9-Å-resolution crystal structure of RRM3 bound to different ARE motifs. This structure together with biophysical methods and cell-culture assays revealed the mechanism of RRM3 ARE recognition and dimerization. While multiple RNA motifs can be bound, recognition of the canonical AUUUA pentameric motif is possible by binding to two registers. Additionally, RRM3 forms homodimers to increase its RNA binding affinity. Finally, although HuR stabilizes ARE-containing RNAs, we found that RRM3 counteracts this effect, as shown in a cell-based ARE reporter assay and by qPCR with native HuR mRNA targets containing multiple AUUUA motifs, possibly by competing with RRM12.

A Computational Quantum-Based Perspective on the Molecular Origins of Life's Building Blocks.

The search for the chemical origins of life represents a long-standing and continuously debated enigma. Despite its exceptional complexity, in the last decades the field has experienced a revival, also owing to the exponential growth of the computing power allowing for efficiently simulating the behavior of matter-including its quantum nature-under disparate conditions found, e.g., on the primordial Earth and on Earth-like planetary systems (i.e., exoplanets). In this minireview, we focus on some advanced computational methods capable of efficiently solving the Schro¨dinger equation at different levels of approximation (i.e., density functional theory)-such as ab initio molecular dynamics-and which are capable to realistically simulate the behavior of matter under the action of energy sources available in prebiotic contexts. In addition, recently developed metadynamics methods coupled with first-principles simulations are here reviewed and exploited to answer to old enigmas and to propose novel scenarios in the exponentially growing research field embedding the study of the chemical origins of life.

Acid-Catalyzed RNA-Oligomerization from 3',5'-cGMP.

The assembly of ancient informational polymers from nucleotide precursors is the central challenge of life's origin on our planet. Among the possible solutions, dry polymerization of 3',5'-cyclic guanosine monophosphate (3',5'-cGMP) has been proposed as a candidate to create oligonucleotides of 15-20 units in length. However, the reported sensitivity of the reaction to the presence of cations raised questions of whether this chemistry could be relevant in a geological context. The experiments in this study show that the presence of cations is not restrictive as long as the reaction is conducted in an acidic environment, in contrast to previous reports that suggested optimal conditions at pH 9.