Environmental Effects on Guanine-Thymine Mispair Tautomerization Explored with Quantum Mechanical/Molecular Mechanical Free Energy Simulations.

DNA bases can adopt energetically unfavorable tautomeric forms that enable the formation of Watson-Crick-like (WC-like) mispairs, which have been proposed to give rise to spontaneous mutations in DNA and misincorporation errors in DNA replication and translation. Previous NMR and computational studies have indicated that the population of WC-like guanine-thymine (G-T) mispairs depends on the environment, such as the local nucleic acid sequence and solvation. To investigate these environmental effects, herein G-T mispair tautomerization processes are studied computationally in aqueous solution, in A-form and B-form DNA duplexes, and within the active site of a DNA polymerase λ variant. The wobble G-T (wG-T), WC-like G-T*, and WC-like G*-T forms are considered, where * indicates the enol tautomer of the base. The minimum free energy paths for the tautomerization from the wG-T to the WC-like G-T* and from the WC-like G-T* to the WC-like G*-T are computed with mixed quantum mechanical/molecular mechanical (QM/MM) free energy simulations. The reaction free energies and free energy barriers are found to be significantly influenced by the environment. The wG-T→G-T* tautomerization is predicted to be endoergic in aqueous solution and the DNA duplexes but slightly exoergic in the polymerase, with Arg517 and Asn513 providing electrostatic stabilization of G-T*. The G-T*→G*-T tautomerization is also predicted to be slightly more thermodynamically favorable in the polymerase relative to these DNA duplexes. These simulations are consistent with an experimentally driven kinetic misincorporation model suggesting that G-T mispair tautomerization occurs in the ajar polymerase conformation or concertedly with the transition from the ajar to the closed polymerase conformation. Furthermore, the order of the associated two proton transfer reactions is predicted to be different in the polymerase than in aqueous solution and the DNA duplexes. These studies highlight the impact of the environment on the thermodynamics, kinetics, and fundamental mechanisms of G-T mispair tautomerization, which plays a role in a wide range of biochemically important processes.

Modeling DNA Flexibility: Comparison of Force Fields from Atomistic to Multiscale Levels.

Accurate parametrization of force fields (FFs) is of ultimate importance for computer simulations to be reliable and to possess a predictive power. In this work, we analyzed, in multi-microsecond simulations of a 40-base-pair DNA fragment, the performance of four force fields, namely, the two recent major updates of CHARMM and two from the AMBER family. We focused on a description of double-helix DNA flexibility and dynamics both at atomistic and at mesoscale level in coarse-grained (CG) simulations. In addition to the traditional analysis of different base-pair and base-step parameters, we extended our analysis to investigate the ability of the force field to parametrize a CG DNA model by structure-based bottom-up coarse-graining, computing DNA persistence length as a function of ionic strength. Our simulations unambiguously showed that the CHARMM36 force field is unable to preserve DNA's structural stability at over-microsecond time scale. Both versions of the AMBER FF, parmbsc0 and parmbsc1, showed good agreement with experiment, with some bias of parmbsc0 parameters for intermediate A/B form DNA structures. The CHARMM27 force field provides stable atomistic trajectories and overall (among the considered force fields) the best fit to experimentally determined DNA flexibility parameters both at atomistic and at mesoscale level.

Structure of a P element transposase-DNA complex reveals unusual DNA structures and GTP-DNA contacts.

P element transposase catalyzes the mobility of P element DNA transposons within the Drosophila genome. P element transposase exhibits several unique properties, including the requirement for a guanosine triphosphate cofactor and the generation of long staggered DNA breaks during transposition. To gain insights into these features, we determined the atomic structure of the Drosophila P element transposase strand transfer complex using cryo-EM. The structure of this post-transposition nucleoprotein complex reveals that the terminal single-stranded transposon DNA adopts unusual A-form and distorted B-form helical geometries that are stabilized by extensive protein-DNA interactions. Additionally, we infer that the bound guanosine triphosphate cofactor interacts with the terminal base of the transposon DNA, apparently to position the P element DNA for catalysis. Our structure provides the first view of the P element transposase superfamily, offers new insights into P element transposition and implies a transposition pathway fundamentally distinct from other cut-and-paste DNA transposases.

Concerted dynamics of metallo-base pairs in an A/B-form helical transition.

Metal-mediated base pairs expand the repertoire of nucleic acid structures and dynamics. Here we report solution structures and dynamics of duplex DNA containing two all-natural C-HgII-T metallo base pairs separated by six canonical base pairs. NMR experiments reveal a 3:1 ratio of well-resolved structures in dynamic equilibrium. The major species contains two (N3)T-HgII-(N3)C base pairs in a predominantly B-form helix. The minor species contains (N3)T-HgII-(N4)C base pairs and greater A-form characteristics. Ten-fold different 1J coupling constants (15N,199Hg) are observed for (N3)C-HgII (114 Hz) versus (N4)C-HgII (1052 Hz) connectivities, reflecting differences in cytosine ionization and metal-bonding strengths. Dynamic interconversion between the two types of C-HgII-T base pairs are coupled to a global conformational exchange between the helices. These observations inspired the design of a repetitive DNA sequence capable of undergoing a global B-to-A-form helical transition upon adding HgII, demonstrating that C-HgII-T has unique switching potential in DNA-based materials and devices.

Real-time monitoring of protein-induced DNA conformational changes using single-molecule FRET.

Apart from being storage devices for genetic information, nucleic acids can provide regulatory structures through evolutionarily optimized sequences. The interaction of proteins binding specifically to such sequences and resulting secondary structures, or the exposure of single-stranded DNA add a versatile regulatory framework for cells. Biochemical and structural biology experiments have revealed important underlying concepts of protein-DNA interactions but are often limited by ensemble averaging or static information. To decipher the dynamics of conformations adopted by protein-DNA complexes, single-molecule approaches have become a powerful resource over the past two decades. In particular single-molecule FRET (smFRET), which allows a read-out of DNA or protein conformations, became widely used. Here, we illustrate how to implement the technique and exemplarily describe how smFRET yields insights into conformational changes of DNA secondary structures induced by the single-stranded DNA binding protein SSB. We further explain how we use smFRET to study mechanisms of the replication initiator DnaA and the competition of DnaA and SSB for single-stranded DNA. We anticipate that smFRET will further develop into a particularly useful technique to study dynamic competitions of proteins for the same DNA substrate.

Changes in Microenvironment Modulate the B- to A-DNA Transition.

B- to A-DNA transition is known to be sensitive to the macroscopic properties of the solution, such as salt and ethanol concentrations. Microenvironmental effects on DNA conformational transition have been broadly studied. Providing an intuitive picture of how DNA responds to environmental changes is, however, still needed. Analyzing the chemical equilibrium of B-to-A DNA transition at critical concentrations, employing explicit-solvent simulations, is envisioned to help understand such microenvironmental effects. In the present study, free-energy calculations characterizing the B- to A-DNA transition and the distribution of cations were carried out in solvents with different ethanol concentrations. With the addition of ethanol, the most stable structure of DNA changes from the B- to A-form, in agreement with previous experimental observation. In 60% ethanol, a chemical equilibrium is found, showing reversible transition between B- and A-DNA. Analysis of the microenvironment around DNA suggests that with the increase of ethanol concentration, the cations exhibit a significant tendency to move toward the backbone, and mobility of water molecules around the major groove and backbone decreases gradually, leading eventually to a B-to-A transition. The present results provide a free-energy view of DNA microenvironment and of the role of cation motion in the conformational transition.

Extensive CRISPR RNA modification reveals chemical compatibility and structure-activity relationships for Cas9 biochemical activity.

CRISPR (clustered regularly interspaced short palindromic repeat) endonucleases are at the forefront of biotechnology, synthetic biology and gene editing. Methods for controlling enzyme properties promise to improve existing applications and enable new technologies. CRISPR enzymes rely on RNA cofactors to guide catalysis. Therefore, chemical modification of the guide RNA can be used to characterize structure-activity relationships within CRISPR ribonucleoprotein (RNP) enzymes and identify compatible chemistries for controlling activity. Here, we introduce chemical modifications to the sugar-phosphate backbone of Streptococcus pyogenes Cas9 CRISPR RNA (crRNA) to probe chemical and structural requirements. Ribose sugars that promoted or accommodated A-form helical architecture in and around the crRNA 'seed' region were tolerated best. A wider range of modifications were acceptable outside of the seed, especially D-2'-deoxyribose, and we exploited this property to facilitate exploration of greater chemical diversity within the seed. 2'-fluoro was the most compatible modification whereas bulkier O-methyl sugar modifications were less tolerated. Activity trends could be rationalized for selected crRNAs using RNP stability and DNA target binding experiments. Cas9 activity in vitro tolerated most chemical modifications at predicted 2'-hydroxyl contact positions, whereas editing activity in cells was much less tolerant. The biochemical principles of chemical modification identified here will guide CRISPR-Cas9 engineering and enable new or improved applications.

Shedding Light on the Hydrolysis Mechanism of cis, trans-[Ru(dmso)4Cl2] Complexes and Their Interactions with DNA-A Computational Perspective.

Using several computational tools such as density functional theory analysis, docking, and MD simulations, we performed a study on cis, trans-[Ru(II)(dmso)4Cl2] complexes, which have therapeutic potential as antimetastatic agents, and their association with DNA. Kohn-Sham energy decomposition analysis reveals that dmso ligands have much smaller interaction energies compared to the chlorido ligands, and their substitution by aquo ligands induces an extra stabilization of the other metal-ligand bonds. Once the complex is hydrolyzed, the aquo ligands have the weakest interactions to the metallic center and therefore are more labile for substitution by a DNA atom. Molecular docking and molecular dynamics were employed to understand the complex preassociation to DNA, pointing to a higher affinity of the hydrolyzed complexes, as well as showing spontaneous binding events during the simulations. Our results are consistent with the experimentally available data that suggest a mechanism in which the complexes are quickly hydrolyzed in solution, before forming cross-links with the DNA molecule. We present a set of methods that could be used to optimize these complexes computationally, aiding in the development of new drugs based on transition metals.

Temperature-Induced Replacement of Phosphate Proton with Metal Ion Captured in Neutron Structures of A-DNA.

Nucleic acids can fold into well-defined 3D structures that help determine their function. Knowing precise nucleic acid structures can also be used for the design of nucleic acid-based therapeutics. However, locations of hydrogen atoms, which are key players of nucleic acid function, are normally not determined with X-ray crystallography. Accurate determination of hydrogen atom positions can provide indispensable information on protonation states, hydrogen bonding, and water architecture in nucleic acids. Here, we used neutron crystallography in combination with X-ray diffraction to obtain joint X-ray/neutron structures at both room and cryo temperatures of a self-complementary A-DNA oligonucleotide d[GTGG(CSe)CAC]2 containing 2'-SeCH3 modification on Cyt5 (CSe) at pH 5.6. We directly observed protonation of a backbone phosphate oxygen of Ade7 at room temperature. The proton is replaced with hydrated Mg2+ upon cooling the crystal to 100 K, indicating that metal binding is favored at low temperature, whereas proton binding is dominant at room temperature.

Free-Energy Profiles for A-/B-DNA Conformational Transitions in Isolated and Aggregated States from All-Atom Molecular Dynamics Simulation.

In ordinary aqueous solution, B-DNA is the major structural form of DNA. After the addition of ethanol, DNA is thought to be aggregated/condensed in the A-form structure. However, there is uncertainty as to whether the B-to-A conformational change is connected to the aggregation/condensation steps. In this study, we performed all-atom molecular dynamics simulations and calculated the free-energy surface involved in the A/B conformational transition for isolated and aggregated Dickerson-Drew dodecamers (DDDs) in water and 85% ethanol environments. We found in the case of an isolated DDD, the overall free-energy profile is entirely downhill to give the B-DNA conformation in both water and 85% ethanol. However, in the aggregated state and 85% ethanol environment, there is a free-energy minimum associated with the A-DNA region in addition to the global B-DNA minimum, and there is a ∼3 kcal/mol free-energy barrier to the A-to-B conformational change. The molecular dynamics results suggest that aggregation of DNA is essential for forming A-DNA.

Tunable DNA Hybridization Enables Spatially and Temporally Controlled Surface-Anchoring of Biomolecular Cargo.

The controlled immobilization of biomolecules onto surfaces is relevant in biosensing and cell biological research. Spatial control is achieved by surface-tethering molecules in micro- or nanoscale patterns. Yet, there is an increasing demand for temporal control over how long biomolecular cargo stays immobilized until released into the medium. Here, we present a DNA hybridization-based approach to reversibly anchor biomolecular cargo onto micropatterned surfaces. Cargo is linked to a DNA oligonucleotide that hybridizes to a sequence-complementary, surface-tethered strand. The cargo is released from the substrate by the addition of an oligonucleotide that disrupts the duplex interaction via toehold-mediated strand displacement. The unbound tether strand can be reloaded. The generic strategy is implemented with small-molecule or protein cargo, varying DNA sequences, and multiple surface patterning routes. The approach may be used as a tool in biological research to switch membrane proteins from a locally fixed to a free state, or in biosensing to shed biomolecular receptors to regenerate the sensor surface.

Dehydrated DNA in B-form: ionic liquids in rescue.

The functional B-conformation of DNA succumbs to the A-form at low water activity. Methods for room temperature DNA storage that rely upon 'anhydrobiosis', thus, often encounter the loss of DNA activity due to the B→A-DNA transition. Here, we show that ionic liquids, an emerging class of green solvents, can induce conformational transitions in DNA and even rescue the dehydrated DNA in the functional B-form. CD spectroscopic analyses not only reveal rapid transition of A-DNA in 78% ethanol medium to B-conformation in presence of ILs, but also the high resistance of IL-bound B-form to transit to A-DNA under dehydration. Molecular dynamics simulations show the unique ability of ILs to disrupt Na+ ion condensation and form 'IL spine' in DNA minor groove to drive the A→B transition. Implications of these findings range from the plausible use of ILs as novel anhydrobiotic DNA storage medium to a switch for modulating DNA conformational transitions.

Kinetics of the B-A transition of DNA: analysis of potential contributions to a reaction barrier.

Because of open problems in the relation between results obtained by relaxation experiments and molecular dynamics simulations on the B-A transition of DNA, relaxation measurements of the B-A dynamics have been extended to a wider range of conditions. Field-induced reaction effects are measured selectively by the magic angle technique using a novel cell construction preventing perturbations from cell window anisotropy. The kinetics was recorded for the case of poly[d(AT)] up to the salt concentration limit of 4.4 mM, where aggregation does not yet interfere. Now experimental data on the B-A dynamics are available for poly[d(AT)] at salt concentrations of 0.18, 0.73, 2.44 and 4.4 mM. In all cases, a spectrum of time constants is found, ranging from ~ 10 µs up to components approaching ~ 1 ms. The relatively small dependence of these data on the salt concentration indicates that electrostatic effects on the kinetics are not as strong as may be expected. The ethanol content at the transition center is a linear function of the logarithm of the salt concentration, and the slope is close to that expected from polyelectrolyte theory. The B-A transition dynamics was also measured in D2O at a salt concentration of 2.4 mM: the center of the transition is found at 20.0 mol/l H2O and at 20.1 mol/l D2O with an estimated accuracy of ± 0.1 mol/l; the spectrum of time constants at the respective transition centers is very similar. The experimental results are discussed regarding the data obtained by molecular dynamics simulations.

Hydration forces between aligned DNA helices undergoing B to A conformational change: In-situ X-ray fiber diffraction studies in a humidity and temperature controlled environment.

Hydration forces between DNA molecules in the A- and B-Form were studied using a newly developed technique enabling simultaneous in situ control of temperature and relative humidity. X-ray diffraction data were collected from oriented calf-thymus DNA fibers in the relative humidity range of 98%-70%, during which DNA undergoes the B- to A-form transition. Coexistence of both forms was observed over a finite humidity range at the transition. The change in DNA separation in response to variation in humidity, i.e. change of chemical potential, led to the derivation of a force-distance curve with a characteristic exponential decay constant of∼2Å for both A- and B-DNA. While previous osmotic stress measurements had yielded similar force-decay constants, they were limited to B-DNA with a surface separation (wall-to-wall distance) typically>5Å. The current investigation confirms that the hydration force remains dominant even in the dry A-DNA state and at surface separation down to∼1.5Å, within the first hydration shell. It is shown that the observed chemical potential difference between the A and B states could be attributed to the water layer inside the major and minor grooves of the A-DNA double helices, which can partially interpenetrate each other in the tightly packed A phase. The humidity-controlled X-ray diffraction method described here can be employed to perform direct force measurements on a broad range of biological structures such as membranes and filamentous protein networks.

Understanding B-DNA to A-DNA transition in the right-handed DNA helix: Perspective from a local to global transition.

The right-handed DNA helix exhibits two major conformations, A-DNA and B-DNA, depending on the environmental conditions. The B-DNA to A-DNA (B→A) transition is sequence specific, cooperative, and reversible. The reduced water activity due to the addition of solvents like ethanol or the presence of protein or drug molecules causes B→A transition. In several biological cases, B→A transition occurs at a local level where small fragments of a long DNA sequence undergoes B→A transition. In this review, we have discussed various aspects of B→A transition such as the role of water, sequence specificity, mechanism of B→A transition, etc. The review primarily focuses on the B→A mechanism involved at a local level, and finally its connection to the global transition in theoretical and experimental studies.

Crystal structure of d(CCGGGGTACCCCGG)2 at 1.4†Šresolution.

The X-ray crystal structure of the DNA tetradecamer sequence d(CCGGGGTACCCCGG)2 is reported at 1.4 Šresolution in the tetragonal space group P41212. The sequence was designed to fold as a four-way junction. However, it forms an A-type double helix in the presence of barium chloride. The metal ion could not be identified in the electron-density map. The crystallographic asymmetric unit consists of one A-type double helix with 12 base pairs per turn, in contrast to 11 base pairs per turn for canonical A-DNA. A large number of solvent molecules have been identified in both the grooves of the duplex and around the backbone phosphate groups.

Polarizable Force Field for DNA Based on the Classical Drude Oscillator: I. Refinement Using Quantum Mechanical Base Stacking and Conformational Energetics.

Empirical force fields seek to relate the configuration of a set of atoms to its energy, thus yielding the forces governing its dynamics, using classical physics rather than more expensive quantum mechanical calculations that are computationally intractable for large systems. Most force fields used to simulate biomolecular systems use fixed atomic partial charges, neglecting the influence of electronic polarization, instead making use of a mean-field approximation that may not be transferable across environments. Recent hardware and software developments make polarizable simulations feasible, and to this end, polarizable force fields represent the next generation of molecular dynamics simulation technology. In this work, we describe the refinement of a polarizable force field for DNA based on the classical Drude oscillator model by targeting quantum mechanical interaction energies and conformational energy profiles of model compounds necessary to build a complete DNA force field. The parametrization strategy employed in the present work seeks to correct weak base stacking in A- and B-DNA and the unwinding of Z-DNA observed in the previous version of the force field, called Drude-2013. Refinement of base nonbonded terms and reparametrization of dihedral terms in the glycosidic linkage, deoxyribofuranose rings, and important backbone torsions resulted in improved agreement with quantum mechanical potential energy surfaces. Notably, we expand on previous efforts by explicitly including Z-DNA conformational energetics in the refinement.

Polarizable Force Field for DNA Based on the Classical Drude Oscillator: II. Microsecond Molecular Dynamics Simulations of Duplex DNA.

The structure and dynamics of DNA are governed by a sensitive balance between base stacking and pairing, hydration, and interactions with ions. Force-field models that include explicit representations of electronic polarization are capable of more accurately modeling the subtle details of these interactions versus commonly used additive force fields. In this work, we validate our recently refined polarizable force field for DNA based on the classical Drude oscillator model, in which electronic degrees of freedom are represented as negatively charged particles attached to their parent atoms via harmonic springs. The previous version of the force field, called Drude-2013, produced stable A- and B-DNA trajectories on the order of hundreds of nanoseconds, but deficiencies were identified that included weak base stacking ultimately leading to distortion of B-DNA duplexes and unstable Z-DNA. As a result of extensive refinement of base nonbonded terms and bonded parameters in the deoxyribofuranose sugar and phosphodiester backbone, we demonstrate that the new version of the Drude DNA force field is capable of simulating A- and B-forms of DNA on the microsecond time scale and the resulting conformational ensembles agree well with a broad set of experimental properties, including solution X-ray scattering profiles. In addition, simulations of Z-form duplex DNA in its crystal environment are stable on the order of 100 ns. The revised force field is to be called Drude-2017.

Comparison of X-ray crystal structures of a tetradecamer sequence d(CCCGGGTACCCGGG)2 at 1.7 Å resolution.

We present here a comparison of three different X-ray crystal structures of DNA tetradecamer sequence d(CCCGGGTACCCGGG)2 all at about 1.7 Å resolution. The sequence was designed as an attempt to form a DNA four-way junction with A-type helical arms. However, in the presence of zinc, magnesium, and in the absence of any metal ion, it does not take up the junction structure, but forms an A-type double helix. This allowed us to study possible conformational changes in the double helix due to the presence of metal ions. Upon addition of the zinc ion, there is a change in the space group from P41212 to P41. The overall conformation of the duplex remains the same. There are small changes in the interaction of the metal ions with the DNA. In the zinc-bound structure, there are two zinc ions that show direct interaction with the N7 atoms of terminal G13 bases at either end of the molecule. There are small changes in the interhelical contacts. The consequence of these differences is to break some of the symmetry and change the space group.

Consecutive non-natural PZ nucleobase pairs in DNA impact helical structure as seen in 50 µs molecular dynamics simulations.

Z: Little is known about the influence of multiple consecutive 'non-standard' ( , 6-amino-5-nitro-2(1H)-pyridone, and , 2-amino-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one) nucleobase pairs on the structural parameters of duplex DNA. nucleobase pairs follow standard rules for Watson-Crick base pairing but have rearranged hydrogen bonding donor and acceptor groups. Using the X-ray crystal structure as a starting point, we have modeled the motions of a DNA duplex built from a self-complementary oligonucleotide (5΄-CTTATPPPZZZATAAG-3΄) in water over a period of 50 µs and calculated DNA local parameters, step parameters, helix parameters, and major/minor groove widths to examine how the presence of multiple, consecutive nucleobase pairs might impact helical structure. In these simulations, the -containing DNA duplex exhibits a significantly wider major groove and greater average values of stagger, slide, rise, twist and h-rise than observed for a 'control' oligonucleotide in which nucleobase pairs are replaced by . The molecular origins of these structural changes are likely associated with at least two differences between and . First, the electrostatic properties of differ from in terms of density distribution and dipole moment. Second, differences are seen in the base stacking of pairs in dinucleotide steps, arising from energetically favorable stacking of the nitro group in with π-electrons of the adjacent base.