DNA Dodecamer Research Articles

Site-directed spin labeling measurements of nanometer distances in nucleic acids using a sequence-independent nitroxide probe.

In site-directed spin labeling (SDSL), local structural and dynamic information is obtained via electron paramagnetic resonance (EPR) spectroscopy of a stable nitroxide radical attached site-specifically to a macromolecule. Analysis of electron spin dipolar interactions between pairs of nitroxides yields the inter-nitroxide distance, which provides quantitative structural information. The development of pulse EPR methods has enabled such distance measurements up to 70 Å in bio-molecules, thus opening up the possibility of SDSL global structural mapping. This study evaluates SDSL distance measurement using a nitroxide (designated as R5) that can be attached, in an efficient and cost-effective manner, to a phosphorothioate backbone position at arbitrary DNA or RNA sequences. R5 pairs were attached to selected positions of a dodecamer DNA duplex with a known NMR structure, and eight distances, ranging from 20 to 40 Å, were measured using double electron-electron resonance (DEER). The measured distances correlated strongly (R2 = 0.98) with the predicted values calculated based on a search of sterically allowable R5 conformations in the NMR structure, thus demonstrating accurate distance measurements using R5. Furthermore, distance measurement in a 42 kD DNA was demonstrated. The results establish R5 as a sequence-independent probe for global structural mapping of DNA and DNA–protein complexes.

Structure and energy of a DNA dodecamer under tensile load

In the last decade, methods to study single DNA molecules under tensile load have been developed. These experiments measure the force required to stretch and melt the double helix and provide insights into the structural stability of DNA. However, it is not easy to directly relate the shape of the force curve to the structural changes that occur in the double helix under tensile load. Here, state-of-the-art computer simulations of short DNA sequences are preformed to provide an atomistic description of the stretching of the DNA double helix. These calculations show that for extensions larger that ∼25% the DNA undergoes a structural transformation and a few base pairs are lost from both the terminal and central part of the helix. This locally melted DNA duplex is stable and can be extended up to ∼50–60% of the equilibrium length at a constant force. It is concluded that melting under tension cannot be modeled as a simple two-state process. Finally, the important role of the cantilever stiffness in determining the shape of the force–extension curve and the most probable rupture force is discussed.

Base Flipping in a GCGC Containing DNA Dodecamer: A Comparative Study of the Performance of the Nucleic Acid Force Fields, CHARMM, AMBER, and BMS.

The improving quality of empirical force field parameters along with other methodological improvements and ever increasing computational resources have lead to more reliable computations on biological macromolecules. In the case of oligonucleotides, three force fields, namely CHARMM27, AMBER4.1, and BMS, have been developed and are widely used by the simulation community. Testing of these force fields to date has primarily focused on their treatment of the canonical forms of DNA and RNA. However, many biological functions of oligonucleotides involve significant variation of their structures from the canonical forms. In the present work, the three force fields are evaluated via computation of potentials of mean force (PMF) of the base flipping process in a DNA dodecamer, 5'-GTCAGCGCATGG-3'. Results are compared with available experimental data on the equilibrium between the opened and closed (i.e. Watson-Crick base paired) state of the underlined C and its WC partner G. Quantitative analysis shows CHARMM to be in the best agreement with experiment, closely followed by AMBER with BMS in the poorest agreement. Various components contributing to the change in the free energy such as base pair interactions, stacking interactions, solvation effects, and intrinsic potential energy changes were evaluated and compared. The results indicate that while all three force fields reasonably represent the canonical structures, the balance of forces contributing to their structural and dynamic properties differ significantly.

Sequence-Dependent Conformational Energy of DNA Derived from Molecular Dynamics Simulations: Toward Understanding the Indirect Readout Mechanism in Protein−DNA Recognition

Sequence dependence of DNA conformation plays a crucial role in its recognition by proteins and ligands. To clarify the relationship between sequence and conformation, it is necessary to quantify the conformational energy and specificity of DNA. Here, we make a systematic analysis of dodecamer DNA structures including all the 136 unique tetranucleotide sequences at the center by molecular dynamics simulations. Using a simplified conformational model with six parameters to describe the geometry of adjacent base pairs and harmonic potentials along these coordinates, we estimated the equilibrium conformational parameters and the harmonic potentials of mean force for the central base-pair steps from many trajectories of the simulations. This enabled us to estimate the conformational energy and the specificity for any given DNA sequence and structure. We tested our method by using sequence-structure threading to estimate the conformational energy and the Z-score as a measure of specificity for many B-DNA and A-DNA crystal structures. The average Z-scores were negative for both kinds of structures, indicating that the potential of mean force from the simulation is capable of predicting sequence specificity for the crystal structures and that it may be used to study the sequence specificity of both types of DNA. We also estimated the positional distribution of conformational energy and Z-score within DNA and showed that they are strongly position dependent. This analysis enabled us to identify particular conformations responsible for the specificity. The presented results will provide an insight into the mechanisms of DNA sequence recognition by proteins and ligands.

Using solid-state 31P{ 19F} REDOR NMR to measure distances between a trifluoromethyl group and a phosphodiester in nucleic acids

REDOR is a solid-state NMR technique frequently applied to biological structure problems. Through incorporation of phosphorothioate groups in the nucleic acid backbone and mono-fluorinated nucleotides, 31P{ 19F} REDOR has been used to study the binding of DNA to drugs and RNA to proteins through the detection of internuclear distances as large as 13–14 Å. In this work, 31P{ 19F} REDOR is further refined for use in nucleic acids by the combined use of selective placement of phosphorothioate groups and the introduction of nucleotides containing trifluoromethyl (–CF 3) groups. To ascertain the REDOR-detectable distance limit between an unique phosphorous spin and a trifluoromethyl group and to assess interference from intermolecular couplings, a series of model compounds and DNA dodecamers were synthesized each containing a unique phosphorous label and trifluoromethyl group or a single 19F nucleus. The dipolar coupling constants of the various 31P and 19F or –CF 3 containing compounds were compared using experimental and theoretical dephasing curves involving several models for intermolecular interactions.

Force field validation for nucleic acid simulations: Comparing energies and dynamics of a DNA dodecamer

Important questions exist regarding the quality of force fields used in molecular dynamics (MD) simulations and their interoperable use with other available MD implementations. NAMD is one of the most efficient and scalable parallel molecular dynamics codes for large-scale biomolecular simulations in the open source domain. It is the aim of this article to analyze and compare the dynamics of a benchmark DNA dodecamer d(CTTTTGCAAAAG)2 system, including its binding to a specific drug molecule arising from the use of various simulation protocols in NAMD using Amber98, with the dynamics arising from simulations of the same dodecamer using Amber98 in the AMBER package, one of the most well-established simulation codes for nucleic acids. Based upon a set of validation benchmarks, the details of which are discussed, we find that nucleic acid simulations using NAMD give meaningful results and that the essential features of the resulting dynamics are similar to those arising from the AMBER package. This sets the stage for reliable large-scale simulations of nucleic acids using NAMD.

X-ray analyses of DNA dodecamers containing 2'-deoxy-5-formyluridine

X-ray analyses of DNA dodecamers containing 2'-deoxy-5-formyluridine

Validation of the 53A6 GROMOS force field

The quality of biomolecular dynamics simulations relies critically on the force field that is used to describe the interactions between particles in the system. Force fields, which are generally parameterized using experimental data on small molecules, can only prove themselves in realistic simulations of relevant biomolecular systems. In this work, we begin the validation of the new 53A6 GROMOS parameter set by examining three test cases. Simulations of the well-studied 129 residue protein hen egg-white lysozyme, of the DNA dodecamer d(CGCGAATTCGCG)(2), and a proteinogenic beta(3)-dodecapeptide were performed and analysed. It was found that the new parameter set performs as well as the previous parameter sets in terms of protein (45A3) and DNA (45A4) stability and that it is better at describing the folding-unfolding balance of the peptide. The latter is a property that is directly associated with the free enthalpy of hydration, to which the 53A6 parameter set was parameterized.

ENGINEERING DNA TOPOLOGY WITH LOCKED NUCLEOSIDES: A STRUCTURAL STUDY

DNA dodecamers modified with nucleotide building blocks based on a bicyclo[3.1.0]hexane system that effectively “locks” the ribose template into an RNA-like or “North” (N) conformation were analyzed by various biophysical techniques including high field nuclear magnetic resonance (NMR). Replacement of either one or both of the center thymidines in the Dickerson Drew dodecamer (CGCGAAT*T*CGCG) caused a progressive shift in the bending propensity of the double helix as shown by a newly developed rapid technique that compares the residual dipolar coupling (RDC) values of the modified duplexes with those previously determined for the native DNA.

Quadruplex Formation by a Guanine-Rich PNA Oligomer

A guanine-rich PNA dodecamer having the sequence H-G4T4G4-Lys-NH2 (G-PNA) hybridizes with a DNA dodecamer of homologous sequence to form a four-stranded quadruplex (Datta, B.; Schmitt, C.; Armitage, B. A. J. Am. Chem. Soc. 2003, 125, 4111-4118). This report describes quadruplex formation by the PNA alone. UV melting curves and fluorescence resonance energy transfer experiments reveal formation of a multistranded structure stabilized by guanine tetrads. The ion dependency of these structures is analogous to that reported for DNA quadruplexes. Electrospray ionization mass spectrometry indicates that both dimeric and tetrameric quadruplexes are formed by G4-PNA, with the dimeric form being preferred. These results have implications for the use of G-rich PNA for homologous hybridization to G-rich targets in chromosomal DNA and suggest additional applications in assembling quadruplex structures within lipid bilayer environments.

Structure and axis curvature in two dA6·dT6DNA oligonucleotides: Comparison of molecular dynamics simulations with results from crystallography and NMR spectroscopy

Molecular dynamics (MD) simulations have been performed on the A6 containing DNA dodecamers d(GGCAAAAAACGG) solved by NMR and d(CGCAAAAAAGCG) solved by crystallography. The experimental structures differ in the direction of axis bending and in other small but important aspects relevant to the DNA curvature problem. Five nanosecond MD simulations of each sequence have been performed, beginning with both the NMR and crystal forms as well as canonical B-form DNA. The results show that all simulations converge to a common form in close proximity to the observed NMR structure, indicating that the structure obtained in the crystal is likely a strained form due to packing effects. A-tracts in the MD model are essentially straight. The origin of axis curvature is found at pyrimidine-purine steps in the flanking sequences.

Structural significances of pseudouridination and methylation of uridine residues in tRNA: X-Ray analyses of DNA dodecamers containing uridine and pseudouridine

Pseudouridine (psi) and 5-methyluridine (T) residues found in tRNA are prepared through modification of the uridine residues by specific enzymes just after transcription. On the other hands, thymidine residues in DNA are incorporated using dTTP which is derived from UTP before replication. In order to investigate the necessity of such modification and the structural properties of these residues, we have determined two X-ray structures of DNA dodecamers containing dU or d psi, and compared with that containing dT. There is found remarkable difference in arrangement of water molecules hydrogen-bonded to these residues, which form a Watson-Crick type base pair. The psi residue stabilizes the phosphate-backbone conformation of the phosphate group via water mediated hydrogen bond networks, while dT residues seem to prevent from attacking of water molecules.

Sequence-Dependence of the Energetics of Opening of AT Basepairs in DNA

Proton exchange and nuclear magnetic resonance spectroscopy are being used to characterize the energetics of opening of AT/TA basepairs in the DNA dodecamer 5′-d(GCTATAAAAGGG)-3′/5′-d(CCCTTTTATAGC)-3′. The dodecamer contains the TATA box of the adenovirus major late promoter. The equilibrium constants for opening of each basepair are measured from the dependence of the exchange rates of imino protons on ammonia concentration. The enthalpy, entropy, and free energy changes in the opening reaction of each basepair are determined from the temperature dependence of the exchange rates. The results reveal that the opening enthalpy changes encompass a wide range of values, namely, from 17 to 29 kcal/mol. The largest values are observed for the AT basepairs in 7th and 8th positions. These values, and the exchange rates of the corresponding imino protons, suggest that these two basepairs open in a single concerted reaction. The enthalpy changes for opening of the central six basepairs are correlated to the opening entropy changes. This enthalpy-entropy compensation minimizes the variations in the opening free energies among these central basepairs. Deviations from the enthalpy-entropy compensation pattern are observed for basepairs located close to the ends of the duplex structure, suggesting a different mode of opening for these basepairs.

Stabilizing or Destabilizing Oligodeoxynucleotide Duplexes Containing Single 2′‐Deoxyuridine Residues with 5‐Alkynyl Substituents

The 5-position of pyrimidines in DNA duplexes offers a site for introducing alkynyl substituents that protrude into the major groove and thus do not sterically interfere with helix formation. Substituents introduced at the 5-position of the deoxyuridine residue of dU:dA base pairs may stabilize duplexes and reinforce helices weakened by a low G/C content, which would otherwise lead to false negative results in DNA chip experiments. Here we report on a method for preparing oligonucleotides with a 5-alkynyl substituent at a 2'-deoxyuridine residue by on-support Sonogashira coupling involving the fully assembled oligonucleotide. A total of 25 oligonucleotides with 5-alkynyl substituents were prepared. The substituents either decrease the UV melting point of the duplex with the complementary strand or increase it by up to 7.1 degrees C, compared with that of the unmodified control duplex. The most duplex-stabilizing substituent, a pyrenylbutyramidopropyne moiety, is likely to intercalate but does not prevent sequence-specific base pairing of the modified deoxyuridine residue or the neighboring nucleotides. It also increases the signal for a target strand when employed on a small oligonucleotide microarray. The ability to tune the melting point of a DNA dodecamer duplex with a single side chain over a temperature range of >11 degrees C may prove useful when developing DNA sequences for biomedical applications.

Exploring the Counterion Atmosphere around DNA: What Can Be Learned from Molecular Dynamics Simulations?

The counterion distribution around a DNA dodecamer (5′-CGCGAATTCGCG-3′) is analyzed using both standard and novel techniques based on state of the art molecular dynamics simulations. Specifically, we have explored the population of Na + in the minor groove of DNA duplex, and whether or not a string of Na + can replace the spine of hydration in the narrow AATT minor groove. The results suggest that the insertion of Na + in the minor groove is a very rare event, but that when once the ion finds specific sites deep inside the groove it can reside there for very long periods of time. According to our simulation the presence of Na + inside the groove does not have a dramatic influence in the structure or dynamics of the duplex DNA. The ability of current MD simulations to obtain equilibrated pictures of the counterion atmosphere around DNA is critically discussed.

Large-scale molecular dynamics simulation of DNA: implementation and validation of the AMBER98 force field in LAMMPS

Molecular modelling played a central role in the discovery of the structure of DNA by Watson and Crick. Today, such modelling is done on computers: the more powerful these computers are, the more detailed and extensive can be the study of the dynamics of such biological macromolecules. To fully harness the power of modern massively parallel computers, however, we need to develop and deploy algorithms which can exploit the structure of such hardware. The Large-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS) is a scalable molecular dynamics code including long-range Coulomb interactions, which has been specifically designed to function efficiently on parallel platforms. Here we describe the implementation of the AMBER98 force field in LAMMPS and its validation for molecular dynamics investigations of DNA structure and flexibility against the benchmark of results obtained with the long-established code AMBER6 (Assisted Model Building with Energy Refinement, version 6). Extended molecular dynamics simulations on the hydrated DNA dodecamer d(CTTTTGCAAAAG)(2), which has previously been the subject of extensive dynamical analysis using AMBER6, show that it is possible to obtain excellent agreement in terms of static, dynamic and thermodynamic parameters between AMBER6 and LAMMPS. In comparison with AMBER6, LAMMPS shows greatly improved scalability in massively parallel environments, opening up the possibility of efficient simulations of order-of-magnitude larger systems and/or for order-of-magnitude greater simulation times.

NMR solution structure of an oxaliplatin 1,2-d(GG) intrastrand cross-link in a DNA dodecamer duplex.

We have determined, at high resolution, the NMR solution structure of an oxaliplatin-GG DNA dodecamer in the AGGC sequence context by 2D NMR studies. Homonuclear assignment strategies resulted in unambiguous assignment of 203 out of 249 protons, which corresponds to assignment of approximately 81% of the protons. Assignments of H5' and H5" protons were tentative due to resonance overlap. The structure of the oxaliplatin duplex was calculated using the program CNS with a simulated annealing protocol. A total of 510 experimental restraints were employed in the structure calculation. Of 20 calculated structures, the 15 with the lowest energy were accepted as a family. The RMSD of the 15 lowest energy structures was 0.68 A, indicating good structural convergence. The theoretical NOESY spectrum obtained by back-calculation from the final average structure showed excellent agreement with the experimental data, indicating that the final structure was in good agreement with the experimental NMR data. Significant conformational differences were observed between the oxaliplatin-GG 12-mer DNA we studied and all previous solution structures of cisplatin-GG DNA duplexes. For example, the oxaliplatin-GG adduct shows much less distortion at the AG base-pair step than the cisplatin-GG adducts. In addition, the oxaliplatin-GG structure also has a narrow minor groove and an overall axis bend of about 31 degrees, both of which are very different from the recent NMR structures for the cisplatin-GG adducts. These structural differences may explain some of the biological differences between oxaliplatin- and cisplatin-GG adducts.

Structural characterization of the RNase E S1 domain and identification of its oligonucleotide-binding and dimerization interfaces.

S1 domains occur in four of the major enzymes of mRNA decay in Escherichia coli: RNase E, PNPase, RNase II, and RNase G. Here, we report the structure of the S1 domain of RNase E, determined by both X-ray crystallography and NMR spectroscopy. The RNase E S1 domain adopts an OB-fold, very similar to that found with PNPase and the major cold shock proteins, in which flexible loops are appended to a well-ordered five-stranded beta-barrel core. Within the crystal lattice, the protein forms a dimer stabilized primarily by intermolecular hydrophobic packing. Consistent with this observation, light-scattering, chemical crosslinking, and NMR spectroscopic measurements confirm that the isolated RNase E S1 domain undergoes a specific monomer-dimer equilibrium in solution with a K(D) value in the millimolar range. The substitution of glycine 66 with serine dramatically destabilizes the folded structure of this domain, thereby providing an explanation for the temperature-sensitive phenotype associated with this mutation in full-length RNase E. Based on amide chemical shift perturbation mapping, the binding surface for a single-stranded DNA dodecamer (K(D)=160(+/-40)microM) was identified as a groove of positive electrostatic potential containing several exposed aromatic side-chains. This surface, which corresponds to the conserved ligand-binding cleft found in numerous OB-fold proteins, lies distal to the dimerization interface, such that two independent oligonucleotide-binding sites can exist in the dimeric form of the RNase E S1 domain. Based on these data, we propose that the S1 domain serves a dual role of dimerization to aid in the formation of the tetrameric quaternary structure of RNase E as described by Callaghan et al. in 2003 and of substrate binding to facilitate RNA hydrolysis by the adjacent catalytic domains within this multimeric enzyme.

Molecular Dynamics Simulations of DNA Using the Generalized Born Solvation Model: Quantitative Comparisons with Explicit Solvation Results

It is now well established that, with the correct treatment of hydration and long-range electrostatic effects, simulations of nucleic acid structure, dynamics, and recognition can yield remarkably reliable results and have genuine predictive power. While the “gold standard” remains modeling studies with the explicit treatment of water molecules and counterions, new implicit hydration models are being advanced as suitable choices for simulations where a shorter time-to-solution is required. In this study, we have evaluated the reliability of one such model, the generalized Born/surface area (GB/SA) continuum model by using it to study the interaction between the minor groove binding drug Hoechst 33258 and the DNA dodecamer d(CTTTTGCAAAAG)2. There is extensive NMR and explicit hydrated modeling data on this system, and it shows the interesting property of highly cooperative binding of two molecules of the drug, such that the 1:1 complex is never observed. We find that with a suitable choice of parameters, the GB/SA model can perform adequately, reproducing the structural and dynamical characteristics observed in the previous explicitly solvated simulations in approximately a quarter of the computational time. However, limitations of this GB/SA method become apparent when the thermodynamic properties are evaluated.

Comparison of the solution structures of a DNA dodecamer using NOE and residual dipolar coupling data

The solution structure of a DNA dodecamer is determined by combining dipolar cross-relaxation and residual dipolar coupling (RDCs) data. The 13C– 1H RDCs result from the partial alignment of the molecule after its dissolution in different oriented dilute liquid crystals: polyethylene glycols, bicelles and phages. It clearly appears that thanks to the use of RDCs, the global curvature of the duplex solution structures is reduced. This is shown to result from the use of long-range RDC restraints and from the symmetry properties of a self-complementary dodecamer. To cite this article: F. Alvarez-Salgado, et al., C.R. Chimie 7 (2004).