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.Web of Science6372146213

Hairpins participating in folding of human telomeric sequence quadruplexes studied by standard and T-REMD simulations

DNA G-hairpins are potential key structures participating in folding of human telomeric guanine quadruplexes (GQ). We examined their properties by standard MD simulations starting from the folded state and long T-REMD starting from the unfolded state, accumulating ~130 \u3bcs of atomistic simulations. Antiparallel G-hairpins should spontaneously form in all stages of the folding to support lateral and diagonal loops, with sub-\u3bcs scale rearrangements between them. We found no clear predisposition for direct folding into specific GQ topologies with specific syn/anti patterns. Our key prediction stemming from the T-REMD is that an ideal unfolded ensemble of the full GQ sequence populates all 4096 syn/anti combinations of its four G-stretches. The simulations can propose idealized folding pathways but we explain that such few-state pathways may be misleading. In the context of the available experimental data, the simulations strongly suggest that the GQ folding could be best understood by the kinetic partitioning mechanism with a set of deep competing minima on the folding landscape, with only a small fraction of molecules directly folding to the native fold. The landscape should further include nonspecific collapse processes where the molecules move via diffusion and consecutive random rare transitions, which could, e.g., structure the propeller loops

Click and detect: Versatile ampicillin aptasensor enabled by click chemistry on a graphene-alkyne derivative

Tackling the current problem of antimicrobial resistance (AMR) requires fast, inexpensive, and effective methods for controlling and detecting antibiotics in diverse samples at the point of interest. Cost-effective, disposable, point-of care electrochemical biosensors are a particularly attractive option. However, there is a need for conductive and versatile carbon-based materials and inks that enable effective bioconjugation under mild conditions for the develop ment of robust, sensitive, and selective devices. This work describes a simple and fast methodology to construct an aptasensor based on a novel graphene derivative equipped with alkyne groups prepared via fluorographene chem istry. Using click chemistry, an aptamer is immobilized and used as a suc cessful platform for the selective determination of ampicillin in real samples in the presence of interfering molecules. The electrochemical aptasensor displayed a detection limit of 1.36 nM, high selectivity among other antibi otics, the storage stability of 4 weeks, and is effective in real samples. Addi tionally, structural and docking simulations of the aptamer shed light on the ampicillin binding mechanism. The versatility of this platform opens up wide possibilities for constructing a new class of aptasensor based on disposable screen-printed carbon electrodes usable in point-of-care devices.Web of Scienc

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.Web of Science184

Computer Folding of RNA Tetraloops: Identification of Key Force Field Deficiencies

The computer-aided folding of biomolecules, particularly RNAs, is one of the most difficult challenges in computational structural biology. RNA tetraloops are fundamental RNA motifs playing key roles in RNA folding and RNA\u2013RNA and RNA\u2013protein interactions. Although state-of-the-art Molecular Dynamics (MD) force fields correctly describe the native state of these tetraloops as a stable free-energy basin on the microsecond time scale, enhanced sampling techniques reveal that the native state is not the global free energy minimum, suggesting yet unidentified significant imbalances in the force fields. Here, we tested our ability to fold the RNA tetraloops in various force fields and simulation settings. We employed three different enhanced sampling techniques, namely, temperature replica exchange MD (T-REMD), replica exchange with solute tempering (REST2), and well-tempered metadynamics (WT-MetaD). We aimed to separate problems caused by limited sampling from those due to force-field inaccuracies. We found that none of the contemporary force fields is able to correctly describe folding of the 5\u2032-GAGA-3\u2032 tetraloop over a range of simulation conditions. We thus aimed to identify which terms of the force field are responsible for this poor description of TL folding. We showed that at least two different imbalances contribute to this behavior, namely, overstabilization of base\u2013phosphate and/or sugar\u2013phosphate interactions and underestimated stability of the hydrogen bonding interaction in base pairing. The first artifact stabilizes the unfolded ensemble, while the second one destabilizes the folded state. The former problem might be partially alleviated by reparametrization of the van der Waals parameters of the phosphate oxygens suggested by Case et al., while in order to overcome the latter effect we suggest local potentials to better capture hydrogen bonding interactions

Toward convergence in folding simulations of RNA tetraloops: Comparison of enhanced sampling techniques and effects of force field modifications

Atomistic molecular dynamics simulations represent an established technique for investigation of RNA structural dynamics. Despite continuous development, contemporary RNA simulations still suffer from suboptimal accuracy of empirical potentials (force fields, ffs) and sampling limitations. Development of efficient enhanced sampling techniques is important for two reasons. First, they allow us to overcome the sampling limitations, and second, they can be used to quantify ff imbalances provided they reach a sufficient convergence. Here, we study two RNA tetraloops (TLs), namely the GAGA and UUCG motifs. We perform extensive folding simulations and calculate folding free energies (ΔGfold°) with the aim to compare different enhanced sampling techniques and to test several modifications of the nonbonded terms extending the AMBER OL3 RNA ff. We demonstrate that replica-exchange solute tempering (REST2) simulations with 12–16 replicas do not show any sign of convergence even when extended to a timescale of 120 μs per replica. However, the combination of REST2 with well-tempered metadynamics (ST-MetaD) achieves good convergence on a timescale of 5–10 μs per replica, improving the sampling efficiency by at least 2 orders of magnitude. Effects of ff modifications on ΔGfold° energies were initially explored by the reweighting approach and then validated by new simulations. We tested several manually prepared variants of the gHBfix potential which improve stability of the native state of both TLs by ∼2 kcal/mol. This is sufficient to conveniently stabilize the folded GAGA TL while the UUCG TL still remains under-stabilized. Appropriate adjustment of van der Waals parameters for C–H···O5′ base-phosphate interaction may further stabilize the native states of both TLs by ∼0.6 kcal/mol.Web of Science1842656264

Automatic learning of hydrogen-bond fixes in an AMBER RNA force field

The capability of current force fields to reproduce RNA structural dynamics is limited. Several methods have been developed to take advantage of experimental data in order to enforce agreement with experiments. We herein extend an existing framework, which allows arbitrarily chosen force-field correction terms to be fitted by quantification of the discrepancy between observables back-calculated from simulation and corresponding experiments. We apply a robust regularization protocol to avoid overfitting, and additionally introduce and compare a number of different regularization strategies, namely L1-, L2-, Kish Size-, Relative Kish Size- and Relative Entropy-penalties. The training set includes a GACC tetramer as well as more challenging systems, namely gcGAGAgc and gcUUCGgc RNA tetraloops. Specific intramolecular hydrogen bonds in the AMBER RNA force field are corrected with automatically determined parameters that we call gHBfix$_{opt}$. A validation involving a separate simulation of a system present in the training set (gcUUCGgc) and new systems not seen during training (CAAU and UUUU tetramers) displays improvements regarding native population of the tetraloop as well as good agreement with NMR-experiments for tetramers when using the new parameters. Then we simulate folded RNAs (a kink-turn and L1 stalk rRNA) including hydrogen bond types not sufficiently present in the training set. This allows a final modification of the parameter set which is named gHBfix21 and is suggested to be applicable to a wider range of RNA systems.Comment: Supporting information included in ancillary file

Folding of guanine quadruplex molecules \u2013 funnel-like mechanism or kinetic partitioning? An overview from MD simulation studies

Background Guanine quadruplexes (GQs) play vital roles in many cellular processes and are of much interest as drug targets. In contrast to the availability of many structural studies, there is still limited knowledge on GQ folding. Scope of review We review recent molecular dynamics (MD) simulation studies of the folding of GQs, with an emphasis paid to the human telomeric DNA GQ. We explain the basic principles and limitations of all types of MD methods used to study unfolding and folding in a way accessible to non-specialists. We discuss the potential role of G-hairpin, G-triplex and alternative GQ intermediates in the folding process. We argue that, in general, folding of GQs is fundamentally different from funneled folding of small fast-folding proteins, and can be best described by a kinetic partitioning (KP) mechanism. KP is a competition between at least two (but often many) well-separated and structurally different conformational ensembles. Major conclusions The KP mechanism is the only plausible way to explain experiments reporting long time-scales of GQ folding and the existence of long-lived sub-states. A significant part of the natural partitioning of the free energy landscape of GQs comes from the ability of the GQ-forming sequences to populate a large number of anti-syn patterns in their G-tracts. The extreme complexity of the KP of GQs typically prevents an appropriate description of the folding landscape using just a few order parameters or collective variables. General significance We reconcile available computational and experimental studies of GQ folding and formulate basic principles characterizing GQ folding landscapes