Additive CHARMM force field for naturally occurring modified ribonucleotides

International audienceMore than 100 naturally occurring modified nucleotides have been found in RNA molecules, in particular in tRNAs. We have determined molecular mechanics force field parameters compatible with the CHARMM36 all-atom additive force field for all these modifications using the CHARMM force field parametrization strategy. Emphasis was placed on fine tuning of the partial atomic charges and torsion angle parameters. Quantum mechanics calculations on model compounds provided the initial set of target data, and extensive molecular dynamics simulations of nucleotides and oligonucleotides in aqueous solutions were used for further refinement against experimental data. The presented parameters will allow for computational studies of a wide range of RNAs containing modified nucleotides, including the ribosome and transfer RNAs. (C) 2016 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc

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

Molecular modeling and thermodynamics simulation of nucleic acids

Nucleic acids participate in many cellular processes. DNA is responsible for gene heredity and its structure is mainly in double helix, whereas RNA has wide functions in gene transcription and regulation so its structures are varied among species. RNA modifications which are known for their abundance and chemical diversity further increase the conformational variability. Functions of some RNAs closely tie to modifications. For example, modified nucleotides maintain correct tRNA structure so that enzyme and ribosome can recognize the tRNA in protein translation. Few epigenetic modifications are also found in DNA, such as 5-methyl cytidine. More often artificially modified DNA, like locked nucleic acid (LNA), is applied to alter the binding affinity of DNA duplex and triplex. Starting from the structures solved by experiments or modeled by programs, molecular dynamics (MD) simulations are employed to mimic the dynamic process and compute the thermodynamic properties, so that the structure and function of nucleic acids can be better understood. This thesis covers computational studies of both RNA and DNA structures. In paper I, the naturally modified ribonucleotides are parameterized in an additive CHARMM force field. The parameters are targeted on quantum chemistry data. The charge and dihedral parameters are fine-tuned for some molecules to reproduce the experimental conformation. This force field allows wider computational studies on modifications involved RNA molecules. In paper II, the new force field is used in the simulations of four tRNAs. The results show with modifications the structural stability, nucleotide conformation and base pair maintenance are almost better than those without modifications, especially in dihydrouridine loop and anticodon loop. The enhanced stability by magnesium ions is also observed. In paper III, MD simulations combined with electrophoretic mobility shift assay illustrate the LNA effects in DNA helical structures. The results show LNA substitutions in duplex strand or the third strand improve the triplex formation, because LNA pre-organizes the DNA strands to reduce their structural adaption required upon triplex forming. In paper IV, a method is developed to calculate free energy for LNA. The angle energies are transformed to convert the locked ribose to deoxyribose. The protocol can be in one-step or three-step by transforming bonded and nonbonded energies separately. Both protocols solve the reasonable solvation free energy and are expected to be applied in larger systems

Molecular Docking and Dynamics of SARS-CoV-2 Programmed Ribosomal Frameshifting RNA and Ligands for RNA-Targeting Alkaloids Prospecting

RNA-ligand docking is a part of computational biology, which is currently lowly recognized compared to the protein-ligand docking procedure commonly applied for drug discovery. This in silico study aims to create a simplified protocol for RNA-ligand docking, which is applicable to RNA-targeting small molecular drug screening. Four alkaloids (berberine, colchicine, nicotine, and tomatine) were subjected to this study and contended against the SARS-CoV-2 genomic RNA -1 PRF component targeting control drug, merafloxacin, including two known intercalator berberine and colchicine, a small alkaloid nicotine and a large alkaloid tomatine. The alkaloids were screened for drug-likeness properties (Lipinski’s Rules of 5 or LRo5), bioavailability indexes, and synthetic accessibility values using SwissADME before docking. The docking used PyRx – Autodock Vina and re-scored for RNA-ligand scoring using AnnapuRNA. The docking results have the interactions mapped using fingeRNAt and visualized using Discovery Studio. Molecular dynamics using CHARMM36 and AMBER forcefields were simulated in NAMD. The molecular dynamics 1 ns simulation results showed that the ligand interaction over time did not cause much interference with the RNA, indicated by the low number of RMSD changes between RNA itself and the RNA-ligand complex. Additionally, CHARMM36 forcefield provided more stable fluctuation compared to AMBER. The results indicated that tomatine disobeyed LRo5 and had a low bioavailability index and bad synthetic accessibility value, while the rest alkaloids passed. In the end, berberine has an even higher docking score than the control drug. The study also shows that this protocol can be useful for future RNA-ligand computational studies

Loss of m(1)acp(3)Ψ ribosomal RNA modification is a major feature of cancer

The ribosome is an RNA-protein complex that is essential for translation in all domains of life. The structural and catalytic core of the ribosome is its ribosomal RNA (rRNA). While mutations in ribosomal protein (RP) genes are known drivers of oncogenesis, oncogenic rRNA variants have remained elusive. We identify a cancer-specific single-nucleotide variation in 18S rRNA at nucleotide 1248.U in up to 45.9% of patients with colorectal carcinoma (CRC) and present across >22 cancer types. This is the site of a unique hyper-modified base, 1-methyl-3-α-amino-α-carboxyl-propyl pseudouridine (m(1)acp(3)Ψ), a >1-billion-years-conserved RNA modification at the peptidyl decoding site of the ribosome. A subset of CRC tumors we call hypo-m(1)acp(3)Ψ shows sub-stoichiometric m(1)acp(3)Ψ modification, unlike normal control tissues. An m(1)acp(3)Ψ knockout model and hypo-m(1)acp(3)Ψ patient tumors share a translational signature characterized by highly abundant ribosomal proteins. Thus, m(1)acp(3)Ψ-deficient rRNA forms an uncharacterized class of "onco-ribosome" which may serve as a chemotherapeutic target for treating cancer patients

Understanding structural features of biomolecular interactions : from classical simulations to ab initio calculations

The structures of biomolecules and their interactions dictate their functions. In this thesis, five papers are presented to illustrate how the dynamics of biomolecules can be investigated and derivation of desired thermodynamic quantities obtained by utilising a diverse range of computational techniques, from simulations utilising classical mechanical descriptions to calculations employing quantum mechanical descriptions. Classical simulation, referring to molecular dynamics simulation with atomistic force fields, has been used in every paper in this thesis. In Paper I, classical simulation and homology modelling are used to investigate the dynamics of a protein as well as that of its homologues, which have a missing region. Protein purification and production of these homologues was also attempted. When state transitions like protonation and tautomerisation equilibria are central to the query, we employed lambda-dynamics, an extension to conventional simulation that can describe transitions between states by including coupling parameter lambda in the dynamics. In Papers II and III, protonation and tautomerisation equilibria respectively are central to the query. In Paper II, lambda-dynamics and multiple pH regime are both used to calculate the pK shifts of cytidine in triplex nucleic acid environments. In some of the triplex nucleic acid systems, sugar modification LNA is present. The force field parameters of LNA have been updated to provider better descriptions for pK calculations. In Paper III, lambda-dynamics is used to describe tautomerisation equilibrium between two tautomers of pseudoisocytidine in singlestranded and triple-stranded nucleic acids in order to observe how the equilibrium shifts in different environments. In vitro binding assay is used to corroborate the computational results. When greater accuracy for certain properties like electrostatics or energetics is required, we employed quantum mechanical calculations as well as hybrid methods which combine classical and quantum mechanical descriptions. In Paper IV, QM and QM/MM calculations were performed to calculate the energetic difference between two tautomers in the ribosome. In Paper V, protein-specific polarised charge, a charge update scheme that updates the atomic charges with QM and Poisson-Boltzmann calculations during classical simulation, is used for better electrostatics description of a peptide

Molecular Dynamics Study of Single Stranded Peptide Nucleic Acids

A PNA molecule is a DNA strand where the sugar-phosphate backbone has been replaced by a structurally homomorphous pseudopeptide chain consisting of N(2-aminoethyl)-glycine units. PNA binds strongly to both DNA and RNA. However, an analysis of the X-ray and NMR data show that the dihedral angles of PNA/DNA or PNA/DNA complexes are very different from those of DNA:DNA or RNA:RNA complexes. In addition, the PNA strand is very flexible. One way to improve the binding affinity of PNA for DNA/RNA is to design a more pre-organized PNA structure. An effective way to rigidify the PNA strand is to introduce ring structures into the backbone. In several experimental studies, the ethylenediamine portion of aminoethylglycine peptide nucleic acids (aegPNA) has been replaced with one or more (S,S)- trans cyclopentyl (cpPNA) units. This substitution has met with varied success in terms of DNA/RNA recognition. In the present work, molecular modeling studies were performed to investigate PNA and modified PNA analogs. Molecular dynamics (MD) simulations is a principal tool in the theoretical study of biological molecules. This computational method calculates the time dependent behavior of a molecular system and provides detailed information on the fluctuations and conformational changes. The MD simulation uses an empirical parameterized energy functions. These parameters play an important role in the quality of the simulations. Therefore, novel empirical force field parameters were developed for cyclopentane modified PNA analogs. We demonstrate that our parameterization can accurately reproduce high level quantum mechanical calculations. Detailed investigations on the conformational and dynamical properties of single stranded aegPNA and cpPNA were undertaken to determine how the cyclopentane ring will improve binding and to determine the contributions of both entropy and dihedral angle preference to the observed stronger binding. The effects of single and multiple modifications of the PNA backbone were also analyzed to understand changes in conformational and dynamical properties

QUANTUM COMPUTATIONS AND MOLECULAR DYNAMICS SIMULATIONS: FROM THE FUNDAMENTALS OF ANTIMICROBIAL RESISTANCE TO NEUROLOGICAL DISEASES

Biophysical phenomena are modeled using a combination of quantum and classical methods to interpret and supplement three distinct and diverse problems in this dissertation. In the first project, decarboxylation reactions are ubiquitous across chemical and biological disciplines, yet the origin of non-catalytic solvent effects remains elusive. Specific solvent structure and energetics have not been well described for the monoanion of malonate, nor corrected from the gas-phase charge-assisted intramolecular hydrogen bond model known as “pseudochair”. In the aqueous phase, a low-lying energy conformer known as the “orthogonal conformation” is computed to be preferred by a three-water cluster of hydrogen bonding over the pseudochair intramolecular hydrogen bond accounting for 87% of the experimental activation enthalpy. The orthogonal conformation is further stabilized by water clusters that satisfy the ten theoretical donor/acceptor hydrogen bonding sites on malonate, reproducing the enthalpy of activation with errors under 0.6 kcal/mol, underscoring the relationship between solvent effects, conformation, and activation parameters for decarboxylation. In the second project, enzyme allostery can be induced by exogenous ligands, thereby impacting enzyme kinetics. Yet, a hypothesized allosteric effect of dichlorodiphenyldichloroethylene (p,p’-DDE) that inhibits androgen receptor’s (AR) activity, by inducing release of the endogenous ligand dihydrotestosterone (DHT) has not been well studied or defined. Through enhanced molecular dynamics, a series of residues were computed to transmit an allosteric response from the binding factor 3 (BF-3) site to the active site, destabilizing DHT through conformational changes of AR. The most probable potential path for the allosteric response is transmitted through a series of residues connecting the BF-3 site to the active site, inclusive of: Phe673, Val715, Leu722, Phe725, Ile737, Trp741, Met742, Leu744, Met745, Leu812, Phe813, Tyr834, and Ile899. Each amino acid changes rotameric state upon the stabilization of Phe673 and Tyr834 at the BF-3 site by p,p’-DDE. Three egress directions were identified, and the dissociation free energy of DHT when p,p’-DDE is bound and unbound at the BF-3 site were compared. For the three paths, the dissociation energy was lowered, relative to simulations without p,p’-DDE, suggesting that DHT is destabilized within the active pocket when p,p’-DDE is bound at the BF-3 site underscoring how allostery triggers dissociation. In the final project, to interpret the phenomena of aggregation of complementary gamma-modified peptide nucleic acids (gPNAs) as a preliminary step to understanding differential G-quadruplexes (GQ) and hairpin (HP) RNA binding, torsional and electrostatic parameters for the miniature polyethylene glycol (miniPEG) modified gPNA backbone were parameterized for MD simulation. The relative energetics for the backbone were parameterized to be within 0.2 kcal/mol. Our MD simulation indicates that the miniPEG reduces base pair hydrogen bonds selectively at A-T base pairs, ultimately promoting duplex dissociation to gPNA single strands. GQ and HP structures were independently simulated as a preliminary step to investigate gPNA binding. The parameterization of modified gPNA and structural derivation of GQ and HP structures are necessary first steps in identifying and exploiting important gPNA/RNA interactions

Computational modeling of protein-ion binding and nucleic acids

Metal ions and nucleic acids are essential for a variety of biological functions. Metal ions play roles in enzyme catalysis, signal transduction and muscle contraction, and the stabilization of protein and nucleic acids structures. Nucleic acids are integral to gene expression and regulation. There are many unsolved questions regarding the function and thermodynamics of metal ions and nucleic acids. Molecular modeling has been an indispensable tool for microscopic understanding of biological processes. While tremendous success has been achieved for the modeling of proteins and organic molecules, it remains challenging to accurate model charged molecules, such as metal ions and nucleic acids. The difficulty mainly arises from the inadequate description of electrostatic interaction and polarization. In this work, accurate models for metal ions and nucleic acids based on AMOEBA polarizable force field were developed. These models along with advanced quantum mechanical methods were then used to study some practical problems. First, the principles underlying Ca²⁺/Mg²⁺ selectivity in ion-binding proteins were studied. It was shown that the Ca²⁺/Mg²⁺ selectivity can be explained by many-body polarization, which depends on the chemistry and geometry of the binding pocket. Second, an existing controversial question regarding the conduction mechanism of potassium channels was resolved by molecular dynamics (MD) simulations with AMOEBA. Contrary to previous beliefs, the conduction operates through nearly ion-saturated states. This mechanism is compatible with almost all existing experimental data. Third, free energy calculation with AMOEBA was used to predict the effect of chemical modifications on the stability of DNA-RNA hybrids, which has implications for the development of gene therapy. Overall, the AMOEBA polarizable force field significantly improves the accuracy for modeling of metal ions and nucleic acids. It is expected that application of polarizable force field will lead to more exciting findings on biological systems.Biomedical Engineerin