Molecular dynamics simulation of nucleic acids: successes, limitations, and promise.

In the last five years we have witnessed a significant increase in the number publications describing accurate and reliable all-atom molecular dynamics simulations of nucleic acids. This increase has been facilitated by the development of fast and efficient methods for treating the long-range electrostatic interactions, the availability of faster parallel computers, and the development of well-validated empirical molecular mechanical force fields. With these technologies, it has been demonstrated that simulation is not only capable of consistently reproducing experimental observations of sequence specific fine structure of DNA, but also can give detailed insight into prevalent problems in nucleic acid structure, ion association and specific hydration of nucleic acids, polyadenine tract bending, and the subtle environmental dependence of the A-DNA-B-DNA duplex equilibrium. Despite the advances, there are still issues with the methods that need to be resolved through rigorous controlled testing. In general, these relate to deficiencies of the underlying molecular mechanical potentials or applied methods (such as the imposition of true periodicity in Ewald simulations and the need for energy conservation), and significant limits in effective conformational sampling. In this perspective, we provide an overview of our experiences, provide some cautionary notes, and provide recommendations for further study in molecular dynamics simulation of nucleic acids.     

NMR studies of dynamics in RNA and DNA by 13C relaxation

RNA and DNA molecules experience motions on a wide range of time scales, ranging from rapid localized motions to much slower collective motions of entire helical domains. The many functions of RNA in biology very often require this molecule to change its conformation in response to biological signals in the form of small molecules, proteins or other nucleic acids, whereas local motions in DNA may facilitate protein recognition and allow enzymes acting on DNA to access functional groups on the bases that would otherwise be buried in Watson-Crick base pairs. Although these statements make a compelling case to study the sequence dependent dynamics in nucleic acids, there are few residue-specific studies of nucleic acid dynamics. Fortunately, NMR studies of dynamics of nucleic acids and nucleic acids-protein complexes are gaining increased attention. The aim of this review is to provide an update of the recent progress in studies of nucleic acid dynamics by NMR based on the application of solution relaxation techniques.     

Molecular dynamics simulations of G-DNA and perspectives on the simulation of nucleic acid structures

The article reviews the application of biomolecular simulation methods to understand the structure, dynamics and interactions of nucleic acids with a focus on explicit solvent molecular dynamics simulations of guanine quadruplex (G-DNA and G-RNA) molecules. While primarily dealing with these exciting and highly relevant four-stranded systems, where recent and past simulations have provided several interesting results and novel insight into G-DNA structure, the review provides some general perspectives on the applicability of the simulation techniques to nucleic acids.     

DNA polymorphism: a comparison of force fields for nucleic acids

The improvements of the force fields and the more accurate treatment of long-range interactions are providing more reliable molecular dynamics simulations of nucleic acids. The abilities of certain nucleic acid force fields to represent the structural and conformational properties of nucleic acids in solution are compared. The force fields are AMBER 4.1, BMS, CHARMM22, and CHARMM27; the comparison of the latter two is the primary focus of this paper. The performance of each force field is evaluated first on its ability to reproduce the B-DNA decamer d(CGATTAATCG)(2) in solution with simulations in which the long-range electrostatics were treated by the particle mesh Ewald method; the crystal structure determined by Quintana et al. (1992) is used as the starting point for all simulations. A detailed analysis of the structural and solvation properties shows how well the different force fields can reproduce sequence-specific features. The results are compared with data from experimental and previous theoretical studies.     

Computer Simulation of Macromolecules

Computer simulation techniques are now an essential part of modern structural molecular biology. They are used in many different ways in order to study the conformation, dynamics and interactions of proteins and nucleic acids. In this paper, I shall review several of these applications and then focus on three specific areas, namely the conformation and dynamics of proteins including the use of free energy perturbation methods to study mutant proteins, the conformation and dynamics of DNA and DNA-drug complexes, and the use of computers with parallel architectures. Although simulation of molecules as large and complex as proteins and nucleic acids may be considered a grand challenge in itself, there are even greater challenges for the future.     

Collective variable modelling of nucleic acids.

Collective variable models continue to contribute to our knowledge of nucleic acids. The past year has seen considerable progress both in modelling sequence-dependent effects on nucleic acid conformation and in understanding how proteins or external stresses influence nucleic acid structure. Algorithmic developments have also allowed collective models to be applied to studies of thermal fluctuations and dynamics. For larger systems, models with varying degrees of resolution are being refined and applied to nucleic acids containing hundreds or thousands of nucleotides.     

FRETmatrix: a general methodology for the simulation and analysis of FRET in nucleic acids

Förster resonance energy transfer (FRET) is a technique commonly used to unravel the structure and conformational changes of biomolecules being vital for all living organisms. Typically, FRET is performed using dyes attached externally to nucleic acids through a linker that complicates quantitative interpretation of experiments because of dye diffusion and reorientation. Here, we report a versatile, general methodology for the simulation and analysis of FRET in nucleic acids, and demonstrate its particular power for modelling FRET between probes possessing limited diffusional and rotational freedom, such as our recently developed nucleobase analogue FRET pairs (base–base FRET). These probes are positioned inside the DNA/RNA structures as a replacement for one of the natural bases, thus, providing unique control of their position and orientation and the advantage of reporting from inside sites of interest. In demonstration studies, not requiring molecular dynamics modelling, we obtain previously inaccessible insight into the orientation and nanosecond dynamics of the bases inside double-stranded DNA, and we reconstruct high resolution 3D structures of kinked DNA. The reported methodology is accompanied by a freely available software package, FRETmatrix, for the design and analysis of FRET in nucleic acid containing systems.     

DNA conformation and dynamics

Nucleotide conformation and dynamics are important for the study of radiation damage to DNA at the atomic level. It is necessary to study not only normal oligonucleotide structure but also those containing modified bases which result from interaction with OH-radicals. There are now over 8000 atomic coordinate entries in the Brookhaven Protein Data Bank, of which over 900 relate to experimentally determined structures of nucleic acids and nucleic acid/protein complexes. We review some of these data which have led to the elucidation of novel DNA conformations, insight into DNA sequence specificity and knowledge of protein/DNA interactions. Further understanding of the conformation, stability and dynamics of nucleic acids has come from molecular modelling. We have used such techniques to study chemical modifications to bases such as alkylation of thymine and guanine and the effects of curvature in longer sequences. Recent improvements in this area include the inclusions of explicit counter-ions and solvent molecules, the use of Particle Mesh Ewald methods to incorporate the long-range electrostatic interactions and the use of longer time scale simulations. We have employed these methods to analyse the effects of incorporation of 8-oxodeoxyguanosine into duplex DNA. This lesion is a common result of radiation damage and is known to have important effects in mutagenesis, cancer and ageing. Received: 7 October 1998 / Accepted in revised form: 18 January 1999     

Phosphorus-31 Transverse Relaxation Rate Measurements by NMR Spectroscopy: Insight into Conformational Exchange Along the Nucleic Acid Backbone

Phosphorus ((31)P) NMR spectroscopy can provide important information about the dynamics of nucleic acids. In this communication, we propose an inversely detected (31)P transverse relaxation rate ( R (2)) measurement experiment. This experiment enables fast measurement of accurate (31)P transverse relaxation rates and provides the possibility to detect slow motions mapped by the phosphorus nuclei along the nucleic acid backbone. Dispersion curves show some (31)P nuclei experiencing chemical exchange in the millisecond time scale. Under the assumption of a two-state exchange process, the reduced lifetimes of the exchanging sites (tau(ex)) obtained are in accordance with base pair lifetime estimates deduced from imino proton exchange measurements.     

Detection of unusual nucleic acids with monoclonal antibodies specific for triplexes, poly(ADP-ribose), DNA-RNA hybrids, and Z-DNA

Monoclonal antibodies which are specific for several unusual nucleic acids are now available. These include Jel 318 which is specific for triplexes, ADP-1 specific for poly(ADP-ribose), Jel 99 specific for RNA-DNA duplexes, and Jel 150 specific for Z-DNA. With the aid of these antibodies and an immunoblotting procedure, unusual nucleic acids can be detected and the amount estimated from a variety of sources. The method involves binding the nucleic acid to either nitrocellulose or Zeta Probe (a cationic nylon membrane), probing with the appropriate monoclonal antibody, followed by addition of an 125I-labeled anti-mouse second antibody. The blot is then developed by autoradiography. The technique is extremely sensitive and can be used to estimate unusual nucleic acids from crude cell extracts.     

Hydration of oligonucleotides in crystals

The solvent molecules found around crystallized oligonucleotides after X-ray refinement are analysed in terms of interaction sites to bases, phosphates and sugars in the three main forms of nucleic acid structures, the A-form, the B-form and the Z-form. The average numbers of contacts to nucleic acid atoms made by solvent molecules are identical in the three forms, but it appears that the average number of contacts solvent molecules make with each other depends on the resolution of the structure. The phosphate anionic oxygen atoms are the most hydrated, while the O(3′) and O(5′) backbone atoms and the ring oxygen atom O(4′) are the least hydrated. Among the hydrophilic atoms of the bases, there is a modulation of the relative water affinities with the nucleic acid form. Numerous hydration sites are such that water molecules can bridge hydrophilic atoms of the same residue, of adjacent residues on the same strand, of distant residues on the two strands, or belonging to symmetry-related residues. Through the helical periodicity of the nucleic acid structure, those bridges can lead to regular and striking hydration networks involving several water molecules and characteristic of the nucleic acid form. Solvent dynamics, as seen by temperature factor versus occupancy plots, seems intimately related to nucleic acid structure and dynamics, since they depend on hydration sites around the nucleic acids.     

Monte Carlo simulations of nucleotide crystal hydrates and their counter-ions

A knowledge of structural and energetic aspects of water- and ion-nucleic acid interactions is essential for the understanding of the role of solvent and counterions in stabilising the various helical forms of nucleic acids. In this study, Monte Carlo computer simulation techniques have been used to predict structural properties of solvent networks in small nucleic acid crystal hydrates containing the ions sodium, ammonium and calcium. Appropriate parameters to describe the interaction potentials of the ions are added to those previously developed for water and nucleic acid atoms. A comparison is made between the predicted and experimental results and it is concluded that the potential functions used lead to simulated solvent structure in reasonable agreement with experimental data, at least in the cases of sodium and calcium. It is now feasible to use these functions in studies of hydration of larger helical fragments of nucleic acids of more direct biological interest.     

Site-specific incorporation of nitroxide spin-labels into 2'-positions of nucleic acids

A protocol is described for the incorporation of nitroxide spin-labels into specific 2'-sites within nucleic acids. This labeling strategy facilitates the investigation of nucleic acid structure and dynamics using electron paramagnetic resonance (EPR) spectroscopy and macromolecular complex formation using paramagnetic relaxation enhancement NMR spectroscopy. A spin-labeling reagent, 4-isocyanato TEMPO, which can be prepared in one facile step or obtained commercially, is used for postsynthetic modification of site-specifically 2'-amino-modified nucleic acids. This spin-labeling protocol has been applied primarily to RNA, but is also applicable to DNA. Subsequently, EPR spectroscopic analysis of the spin-labeled nucleic acids allows for the measurements of distances, solvent accessibilities and conformation dynamics. Using the spin-labeling strategy described here, spin-labeled samples can be prepared in 2-4 d.     

Nucleic acid solvation: from outside to insight

Nucleic acids are polyanionic molecules that were historically considered to be solely surrounded by a shell of water molecules and a neutralizing cloud of monovalent and divalent cations. In this respect, recent experimental and theoretical reports demonstrate that water molecules within complex nucleic acid structures can display very long residency times, and assist drug binding and catalytic reactions. Finally, anions can also bind to these polyanionic systems. Many of these recent insights are provided by state-of-the-art molecular dynamics simulations of nucleic acid systems, which will be described together with relevant methodological issues.     

Insights into nucleic acid conformational dynamics from massively parallel stochastic simulations

The helical hairpin is one of the most ubiquitous and elementary secondary structural motifs in nucleic acids, capable of serving functional roles and participating in long-range tertiary contacts. Yet the self-assembly of these structures has not been well-characterized at the atomic level. With this in mind, the dynamics of nucleic acid hairpin formation and disruption have been studied using a novel computational tool: large-scale, parallel, atomistic molecular dynamics simulation employing an inhomogeneous distributed computer consisting of more than 40,000 processors. Using multiple methodologies, over 500 micro s of atomistic simulation time has been collected for a large ensemble of hairpins (sequence 5'-GGGC[GCAA]GCCU-3'), allowing characterization of rare events not previously observable in simulation. From uncoupled ensemble dynamics simulations in unperturbed folding conditions, we report on 1), competing pathways between the folded and unfolded regions of the conformational space; 2), observed nonnative stacking and basepairing traps; and 3), a helix unwinding-rewinding mode that is differentiated from the unfolding and folding dynamics. A heterogeneous transition state ensemble is characterized structurally through calculations of conformer-specific folding probabilities and a multiplexed replica exchange stochastic dynamics algorithm is used to derive an approximate folding landscape. A comparison between the observed folding mechanism and that of a peptide beta-hairpin analog suggests that although native topology defines the character of the folding landscape, the statistical weighting of potential folding pathways is determined by the chemical nature of the polymer.     

Molecular dynamics in protein-single stranded DNA complexes. Two distinct nucleoside mobilities, in poly(deoxythymidylic acid)-poly-L-lysine complexes

Stoichiometric amounts of poly-L-lysine were added to site-specifically spin labeled single stranded nucleic acids and the resulting complexes analyzed by electron spin resonance spectroscopy (ESR). The nucleic acids were spin labeled to different extents and with labels of varying tether length. The ESR data are used to determine nucleoside dynamics and some structural features in these complexes. It is concluded that two distinct base mobilities exist in the complexes; one set is characterized by a mean correlation time tau -R = 2 ns, and the other one by a tau -R greater than or equal to 50 ns. A model is proposed which suggests that a poly-L-lys single stranded nucleic acid complex consists of low mobility segments flanked by more mobile bases. An interesting feature of the proposed model is its applicability to explain ESR data of single strand binding protein-spin labeled nucleic acid complexes, which can also be interpreted in terms of two distinct nucleoside mobility states. It is hypothesized that this phenomenon could be of biological significance for the release of protein ligands from a protein-nucleic acid complex.     

Fluorescence energy transfer monitored competitive equilibria of nucleic acids: applications in thermodynamics and screening

Precise thermodynamic characterization of nucleic acid complex stability is required to understand a variety of biologically significant events as well as to exploit the specific recognition capabilities of nucleic acids in biotechnology, diagnostics, and therapeutics. The development of a database of nucleic acid thermodynamics with sufficient precision to foster further developments in these areas requires new and improved measurement techniques. The combination of a competitive equilibrium titration with fluorescence energy transfer based detection provides a method for precise measurement of differences in free energy values for nucleic acid duplexes that far exceeds in precision those accessible via conventional methods. The method can be applied to detect and to characterize any deviation in a nucleic acid that alters duplex stability. Such deviations include, but are not limited to, mismatches; single nucleotide polymorphisms (SNP); chemically modified nucleotide bases, sugars or phosphates; and conformational anomalies or folding motifs, such as, loops or hairpins.     

Proteins involved in binding and cellular uptake of nucleic acids

The study of mechanisms of nucleic acid transport across the cell membrane is valuable both for understanding the biological function of extracellular nucleic acids and the practical use of nucleic acids in gene therapy. It has been clearly demonstrated that cell surface proteins are necessary for transport of nucleic acids into cells. A large amount of data has now been accumulated about the proteins that participate in nucleic acid transport. The methods for revealing and identification of these proteins, possible mechanisms of protein-mediated transport of nucleic acids, and cellular functions of these proteins are described.