Molecular dynamics simulations of chemically modified ribonucleotides

Post-transcriptional modifications are crucial for RNA function, with roles ranging from the stabilization of functional RNA structures to modulation of RNA--protein interactions. Additionally, artificially modified RNAs have been suggested as optimal oligonucleotides for therapeutic purposes. The impact of chemical modifications on secondary structure has been rationalized for some of the most common modifications. However, the characterization of how the modifications affect the three-dimensional RNA structure and dynamics and its capability to bind proteins is still highly challenging. Molecular dynamics simulations, coupled with enhanced sampling methods and integration of experimental data, provide a direct access to RNA structural dynamics. In the context of RNA chemical modifications, alchemical simulations where a wild type nucleotide is converted to a modified one are particularly common. In this Chapter, we review recent molecular dynamics studies of modified ribonucleotides. We discuss the technical aspects of the reviewed works, including the employed force fields, enhanced sampling methods, and alchemical methods, in a way that is accessible to experimentalists. Finally, we provide our perspective on this quickly growing field of research. The goal of this Chapter is to provide a guide for experimentalists to understand molecular dynamics works and, at the same time, give to molecular dynamics experts a solid review of published articles that will be a useful starting point for new research.Comment: Submitted as a chapter for the book "RNA Structure and Function", series "RNA Technologies", published by Springe

Why Is a High Temperature Needed by Thermus thermophilus Argonaute During mRNA Silencing: A Theoretical Study

Thermus thermophiles Argonaute (TtAgo) is a complex, which is consisted of 5′-phosphorylated guide DNA and a series of target DNA with catalytic activities at high temperatures. To understand why high temperatures are needed for the catalytic activities, three molecular dynamics simulations and binding free energy calculations at 310, 324, and 338K were performed for the TtAgo-DNA complex to explore the conformational changes between 16-mer guide DNA/15-mer target DNA and TtAgo at different temperatures. The simulation results indicate that a collapse of a small β-strand (residues 507–509) at 310 K caused Glu512 to move away from the catalytic residues Asp546 and Asp478, resulting in a decrease in catalytic activity, which was not observed in the simulations at 324 and 338 K. The nucleic acid binding channel became enlarged at 324 and 338K, thereby facilitating the DNA to slide in. Binding free energy calculations and hydrogen bond occupancy indicated that the interaction between TtAgo and the DNA was more stable at 324K and 338K than at 310 K. The DNA binding pocket residues Lys575 and Asn590 became less solvent accessible at 324 and 338K than at 310 K to influence hydrophilic interaction with DNA. Our simulation studies shed some light on the mechanism of TtAgo and explained why a high temperature was needed by TtAgo during gene editing of CRISPR

RNA Structural Dynamics As Captured by Molecular Simulations: A Comprehensive Overview

With both catalytic and genetic functions, ribonucleic acid (RNA) is perhaps the most pluripotent chemical species in molecular biology, and its functions are intimately linked to its structure and dynamics. Computer simulations, and in particular atomistic molecular dynamics (MD), allow structural dynamics of biomolecular systems to be investigated with unprecedented temporal and spatial resolution. We here provide a comprehensive overview of the fast-developing field of MD simulations of RNA molecules. We begin with an in-depth, evaluatory coverage of the most fundamental methodological challenges that set the basis for the future development of the field, in particular, the current developments and inherent physical limitations of the atomistic force fields and the recent advances in a broad spectrum of enhanced sampling methods. We also survey the closely related field of coarse-grained modeling of RNA systems. After dealing with the methodological aspects, we provide an exhaustive overview of the available RNA simulation literature, ranging from studies of the smallest RNA oligonucleotides to investigations of the entire ribosome. Our review encompasses tetranucleotides, tetraloops, a number of small RNA motifs, A-helix RNA, kissing-loop complexes, the TAR RNA element, the decoding center and other important regions of the ribosome, as well as assorted others systems. Extended sections are devoted to RNA-ion interactions, ribozymes, riboswitches, and protein/RNA complexes. Our overview is written for as broad of an audience as possible, aiming to provide a much-needed interdisciplinary bridge between computation and experiment, together with a perspective on the future of the field

Molecular Simulation Studies of Dynamics and Interactions in Nucleic Acids

In my thesis work, I conducted molecular simulation studies to explore dynamics and interactions in nucleic acids. I began my work by applying conventional molecular dynamics (MD) simulations to study the local and global dynamics of the transactivation response (TAR) element from the type-1 human immunodeficiency virus (HIV-1) and the effect of binding of ligands on the dynamics of TAR RNA. I determined that the TAR RNA structure was stabilized on binding of ligands due to the decreased flexibility in helices that comprise TAR RNA. This rigidity of the TAR RNA structure was coupled with the decreased flipping of bulge nucleotides. I also observed that different initial conformations of TAR RNA converged to similar conformations in the course of MD simulations. Finally, I observed the formation of binding pockets in unliganded TAR structures that could accommodate ligands of various sizes. After comprehensively exploring the dynamics of TAR RNA with and without ligands, I conducted more specific studies on the interactions that were formed or broken during the (un)binding process of two ligands, a small molecule inhibitor and a helical peptide, from the viral RNA molecules using non-equilibrium simulations. Firstly, I observed that the dissociation of a small molecule is coupled with a base flipping event which I described using physical variables and thermodynamic properties. Secondly, I observed that the dissociation process of a helical peptide is facilitated by a network of hydrogen bonding and salt bridging interactions which are formed across four distinct dissociation pathways. I also resolved the free-energy profiles for each pathway which revealed metastable states and dissociation barriers. Based on the free-energy profiles, I proposed a preferred dissociation pathway and identified one arginine amino acid that plays an important role in the recognition of the peptide by the viral RNA. Next, I focused on studying a more complex reaction coordinate (RC) that could describe a base flipping mechanism in a double-stranded RNA (dsRNA) molecule using transition path sampling (TPS) methods. Additionally, I used the likelihood maximization method to determine a refined RC based on an ensemble of 1000 transition trajectories created by the path sampling algorithm. The refined RC consisted of two collective variables (CVs), a distance and a dihedral angle between the neighboring nucleotides and the flipping base. I also projected a free-energy profile along the refined RC which revealed three free-energy minima. I proposed that one of the free-energy minima represented a wobbled conformation of the flipping nucleobase. I also analyzed the reactive trajectories which showed that the base flipping is coupled with global conformational changes in a stem-loop of dsRNA. Outside of studies involving RNA, I conducted conventional MD simulations to study the dynamics of a porphyrin/DNA nanoassembly which revealed the overall left-handed orientation of the nanoassembly. I characterized the resulting porphyrin/DNA system using various physical variables. Overall, my thesis revealed the local and global dynamics of RNA as well as DNA systems, and perturbations to dynamics originating in binding of ligands of various sizes

Impedance-Based Real-Time Monitoring of Mammalian Cells upon Introduction of Xenobiotics into the Cytoplasm by In Situ Electroporation

Whole-cell biosensors are irreplaceable tool for studies of cellular mechanisms and behavior of the cell as a smallest living unit. Their development have progressed rapidly over past decades and nowadays we have powerful tools to study cell-based assays and to examine behavior of the cells exposed to different kinds of stimuli and challenges. Limited and selective permeability of the plasma membrane prevents the introduction of hydrophilic xenomolecules into the cytoplasm of mammalian cells. However, it is essential for many fields of cell biology, biomedicine or biotechnology to allow transport of such molecules (e.g. nucleic acids, antibodies, peptides or drugs) across the cell membrane. An ultimate goal of this thesis was to establish proof-of-principle assays for delivery of various bioactive molecules into adherent cells by in situ electroporation, and to monitor how these compounds influence cellular behavior, once they are internalized within the cell cytosol. Studies of in situ electroporation (ISE) were conducted using different types of mammalian cells (BAEC, CHO-K1/CHO-GFP, HaCaT, NRK and NIH-3T3) grown to confluence on small planar gold film electrodes. For every cell line individually, electric pulse parameters were optimized to achieve maximal loading efficiency, while keeping the invasiveness of the operation as low as possible. Impedance monitoring of in situ electroporation conducted with high time resolution showed biphasic changes of impedance signal after pulse application, indicating (i) fast recovery of the cell membrane integrity and (ii) relatively slow process of cell recovery after changes induced by membrane permeabilization. For the first time, release of intracellular material from the cells by ISE was studied using ECIS setup. Direct time-resolved imaging of NRK cells showed measurable efflux of fluorescence-labeled probes upon multiply applied electric pulses. In situ electroporation allowed transfer of second messenger (8-OH-)cAMP in the cell monolayers. Subsequent changes in impedance signal were in agreement with those observed after stimulation of the cells with membrane-permeable compounds CPT-cAMP and forskolin, as a consequence of triggering of the corresponding signaling cascades. Transport of nucleic acids into cytoplasm and nuclei of NRK and CHO-GFP cells was conducted in a highly-efficient manner. Besides fluorescent DNA aptamers, various types of siRNA molecules were successfully delivered into cells by in situ electroporation and their long-term sequence-specific silencing effect on cells was demonstrated and quantified by using microscopy and/ or impedance analysis. Transfection performance of ISE was compared with conventional and widespread delivery transfection method. In conclusion, this thesis demonstrated that in situ electroporation allows for highly efficient delivery of emerging types of molecules into monolayers of various types of cells

Probing the Binding Interactions between Chemically Modified siRNAs and Human Argonaute 2 Using Microsecond Molecular Dynamics Simulations

The use of chemical modifications in small interfering RNAs (siRNAs) is warranted to impart drug-like properties. However, certain chemical modifications especially those on the sugar have deleterious effects on the RNA interference (RNAi) when they are placed at key positions in the seed region of an siRNA guide strand. In order to probe the effect of chemically modified siRNAs [(2′-<i>O</i>-methyl, 4′-<i>C</i>-aminomethyl-2′-<i>O</i>-methyl, 2′-<i>O</i>-(2-methoxyethyl), and 2′-<i>O</i>-benzyl] on human Argonaute 2 (hAGO2), the catalytic engine of RNAi, we have developed a model of its open conformation. Results from microsecond MD simulations of 15 different siRNA−hAGO2 complexes provide insights about how the key noncovalent interactions and conformational changes at the seed region are modulated, depending upon the nature and position of chemical modifications. Such modification induced structural changes can affect siRNA loading into hAGO2, which may influence RNAi activity. Our studies show that microsecond MD simulations can provide useful information for the design of therapeutically relevant siRNAs

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Influence of Connexin Expression/Co-Expression Levels on Electrical Impulse Propagation Investigated in the HL-1 Cell Model

In the myocardium action potentials are transmitted from cell-to-cell through gap junctions. These specialised junctions play a pivotal role in regulating the speed and safety of impulse propagation by controlling the amount of depolarised current that is passed from excited to non-excited regions of the heart. In mammalian hearts gap junction proteins connexin43, connexin40 and connexin45 are co-expressed in distinctive combinations and relative quantities in functionally specialised subsets of cardiac myocyte. The functional consequences of these connexin expression/co-expression patterns in modulating impulse propagation are poorly understood. To study the relative importance of membrane excitability and electrical coupling in relation to propagation velocities, clones of the HL-1 mouse atrial myocyte tumour line were used as an in vitro cell model. Five clones were characterised for expression of myocytic markers, calcium handling proteins and connexins, two of which (#2 and #6) displayed large differences in conduction velocities using microelectrode arrays. To ascertain which factor(s) were the main determinants of speed of conduction, the membrane excitability (voltage-gated channels) and electrical coupling (gap junctions) between the two clones were compared. Sodium, L- and T-type calcium channels were present in both clones but no significant differences were found in the current densities. However, large differences were seen in expression levels of connexin43, connexin40 and connexin45. RNA interference combined with microelectrode arrays was employed to establish the relative importance of each connexin in impulse propagation. The results indicate that electrical coupling by gap junctions is a major determinant of conduction velocities in HL-1 cell lines. Further experiments using RNA interference to suppress the expression of proteins thought to play a role in the action potential parameters should help in defining the part played by either the active or passive electrical properties in action potential propagation