A molecular dynamics simulation of the (dG)6 . (dC)6 minihelix including counterions and water

The results of a 60 ps molecular dynamics (MD) simulation of (dG)6.(dC)6 including 10 Na+ counterions and 292 water molecules are presented. All backbone angles and helix parameters for the hexamer are reported in this paper along with trajectory plots of selected angles. Hydrogen bonding between the bases along the helical axis was observed to fluctuate with time, showing the dynamic nature of the base-pairing interaction. These fluctuations gave rise to unusual hydrogen-bonding patterns. Good intrastrand base stacking and no interstrand base stacking were also observed. The hexamer minihelix retains an essentially B-DNA conformation throughout the entire simulation even though some helix parameters and backbone angles do not have strict B-DNA values. The most striking feature obtained from the simulation was a high propeller twist, which resulted in a narrow minor groove for the minihelix. It is proposed that (dG)n.(dC)n sequences are resistant to DNAase I because of this narrow minor groove in dilute aqueous solution.     

Crystal structure of an RNA duplex r(G GCGC CC)2 with non-adjacent G*U base pairs

The crystal structure of a self-complementary RNA duplex r(GGGCGCUCC)2with non-adjacent G*U and U*G wobble pairs separated by four Watson-Crick base pairs has been determined to 2.5 A resolution. Crystals belong to the space group R3; a = 33.09 A,alpha = 87.30 degrees with a pseudodyad related duplex in the asymmetric unit. The structure was refined to a final Rworkof 17.5% and Rfreeof 24.0%. The duplexes stack head-to-tail forming infinite columns with virtually no twist at the junction steps. The 3'-terminal cytosine nucleosides are disordered and there are no electron densities, but the 3' penultimate phosphates are observed. As expected, the wobble pairs are displaced with guanine towards the minor groove and uracil towards the major groove. The largest twist angles (37.70 and 40.57 degrees ) are at steps G1*C17/G2*U16 and U7*G11/C8*G10, while the smallest twist angles (28.24 and 27.27 degrees ) are at G2*U16/G3*C15 and C6*G12/U7*G11 and conform to the pseudo-dyad symmetry of the duplex. The molecule has two unequal kinks (17 and 11 degrees ) at the wobble sites and a third kink at the central G5 site which may be attributed to trans alpha (O5'-P), trans gamma (C4'-C5') backbone conformations. The 2'-hydroxyl groups in the minor groove form inter-column hydrogen bonding, either directly or through water molecules.     

Structural features and hydration of a dodecamer duplex containing two C.A mispairs.

X-ray diffraction techniques have been used to characterise the crystal and molecular structure of the deoxyoligomer d(C-G-C-A-A-A-T-T-C-G-C-G) at 2.5A resolution. The final R factor is 0.19 with the location of 78 solvent molecules. The oligomer crystallises in a B-DNA type conformation with two strands coiled about each other to produce a duplex. This double helix consists of four A.T and six G.C Watson-Crick base pairs and two C.A mispairs. The mismatched base pairs adopt a "wobble" type structure with the cytosine displaced laterally into the major groove, the adenine into the minor groove. We have proposed that the two close contacts observed in the C.A pairing represent two hydrogen bonds one of which results from protonation of adenine. The mispairs are accommodated in the double helix with small adjustments in the conformation of the sugar-phosphate backbone. Details of the backbone conformation, base stacking interactions, thermal parameters and the hydration are now presented and compared with those of the native oligomer d(C-G-C-G-A-A-T-T-C-G-C-G) and with variations of this sequence containing G.T and G.A mispairs.     

Conformational features of benzo‐homologated yDNA duplexes by molecular dynamics simulation

yDNA is a base‐modified nucleic acid duplex containing size‐expanded nucleobases. Base‐modified nucleic acids could expand the genetic alphabet and thereby enhance the functional potential of DNA. Unrestrained 100 ns MD simulations were performed in explicit solvent on the yDNA NMR sequence [5′(yA T yA yA T yA T T yA T)₂] and two modeled yDNA duplexes, [5′(yC yC G yC yC G G yC G G)₂] and [(yT5′ G yT A yC yG C yA yG T3′)?(yA5′ C T C yG C G yT A yC A3′)]. The force field parameters for the yDNA bases were derived in consistent with the well‐established AMBER force field. Our results show that DNA backbone can withstand the stretched size of the bases retaining the Watson‐Crick base pairing in the duplexes. The duplexes retained their double helical structure throughout the simulations accommodating the strain due to expanded bases in the backbone torsion angles, sugar pucker and helical parameters. The effect of the benzo‐expansion is clearly reflected in the extended C1′‐C1′ distances and enlarged groove widths. The size expanded base modification leads to reduction in base pair twist resulting in larger overlapping area between the stacked bases, enhancing inter and intra strand stacking interactions in yDNA in comparison with BDNA. This geometry could favour enhanced interactions with the groove binders and DNA binding proteins., 2016. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 55–64, 2016     

Critical effect of the N2 amino group on structure, dynamics, and elasticity of DNA polypurine tracts

Unrestrained 5-20-ns explicit-solvent molecular dynamics simulations using the Cornell et al. force field have been carried out for d[GCG(N)11GCG]2 (N, purine base) considering guanine*cytosine (G*C), adenine*thymine (A*T), inosine*5-methyl-cytosine (I*mC), and 2-amino-adenine*thymine (D*T) basepairs. The simulations unambiguously show that the structure and elasticity of N-tracts is primarily determined by the presence of the amino group in the minor groove. Simulated A-, I-, and AI-tracts show almost identical structures, with high propeller twist and minor groove narrowing. G- and D-tracts have small propeller twisting and are partly shifted toward the A-form. The elastic properties also differ between the two groups. The sequence-dependent electrostatic component of base stacking seems to play a minor role. Our conclusions are entirely consistent with available experimental data. Nevertheless, the propeller twist and helical twist in the simulated A-tract appear to be underestimated compared to crystallographic studies. To obtain further insight into the possible force field deficiencies, additional multiple simulations have been made for d(A)10, systematically comparing four major force fields currently used in DNA simulations and utilizing B and A-DNA forms as the starting structure. This comparison shows that the conclusions of the present work are not influenced by the force field choice.     

Crystal structure of 2'-O-Me(CGCGCG)2, an RNA duplex at 1.30 A resolution. Hydration pattern of 2'-O-methylated RNA.

The molecular and crystal structure of 2'-O-Me (CGCGCG)2 has been determined using synchrotron radiation at near-atomic resolution (1.30 A), the highest resolution to date in the RNA field. The crystal structure is a half-turn A-type helix with some helical parameters deviating from canonical A-RNA, such as low base pair rise, elevated helical twist and inclination angles. In CG steps, inter-strand guanines are parallel while cytosines are not parallel. In steps GC this motif is reversed. This type of regularity is not seen in other RNA crystal structures. The structure includes 44 water molecules and two hydrated Mg2+ions one of which lies exactly on the crystallographic 2-fold axis. There are distinct patterns of hydration in the major and the minor grooves. The major groove is stabilised by water clusters consisting of fused five- and six-membered rings. Minor groove contains only a single row of water molecules; each water bridges either two self-parallel cytosines or two self-parallel guanines by a pair of hydrogen bonds. The structure provides the first view of the hydration scheme of 2'-O-methylated RNA duplex.     

Interactions of DNA binding ligands with PNA-DNA hybrids.

The interactions of two representative mixed-sequence (one with an AT-stretch) PNA-DNA duplexes (10 or 15 base-pairs) and a PNA2/DNA triplex with the DNA binding reagents distamycin A, 4',6-diamidino-2-phenylindole (DAPI), ethidium bromide, 8-methoxy-psoralen and the delta and lambda enantiomers of Ru(phen)2-dppz2+ have been investigated using optical spectroscopic methods. The behaviour of these reagents versus two PNA-PNA duplexes has also been investigated. With triple helical poly(dA)/(H-T10-Lys-NH2)2 no significant intercalative binding was detected for any of the DNA intercalators, whereas DAPI, a DNA minor groove binder, was found to exhibit a circular dichroism with a positive sign and amplitude consistent with minor groove binding. Similarly, a PNA-DNA duplex containing a central AATA motif, a typical minor groove binding site for the DNA minor groove binders distamycin A and DAPI, showed binding for both of these drugs, though with strongly reduced affinity. No important interactions were found for any of the ligands with a PNA-DNA duplex consisting of a ten base-pair mixed purine-pyrimidine sequence with only two AT base-pairs in the centre. Nor did any of the ligands show any detectable binding to the PNA-PNA duplexes (one containing an AATT motif). Various PNA derivatives with extentions of the backbone, believed to increase the flexibility of the duplex to opening of an intercalation slot, were tested for intercalation of ethidium bromide or 8-methoxypsoralen into the mixed sequence PNA-DNA duplex, however, without any observation of improved binding. The importance of the ionic contribution of the deoxyribose phosphate backbone, versus interactions with the nucleobases, for drug binding to DNA is discussed in the light of these findings.     

Solution structure of a nitrous acid induced DNA interstrand cross-link

Nitrous acid is a mutagenic agent. It can induce interstrand cross-links in duplex DNA, preferentially at d(CpG) steps: two guanines on opposite strands are linked via a single shared exocyclic imino group. Recent synthetic advances have led to the production of large quantities of such structurally homogenous cross-linked duplex DNA. Here we present the high resolution solution structure of the cross-linked dodecamer [d(GCATCCGGATGC)]2 (the cross-linked guanines are underlined), determined by 2D NMR spectroscopy, distance geometry, restrained molecular dynamics and iterative NOE refinement. The cross-linked guanines form a nearly planar covalently linked 'G:G base pair' with only minor propeller twisting, while the cytidine bases of their normal base pairing partners have been flipped out of the helix and adopt well defined extrahelical positions in the minor groove. On the 5'-side of the cross-link, the minor groove is widened to accommodate these extrahelical bases, and the major groove becomes quite narrow at the cross-link. The cross-linked 'G:G base pair' is well stacked on the spatially adjacent C:G base pairs, particularly on the 3'-side guanines. In addition to providing the first structure of a nitrous acid cross-link in DNA, these studies could be of major importance to the understanding of the mechanisms of nitrous acid cross-linking and mutagenicity, as well as the mechanisms responsible for its repair in intracellular environments. It is also the shortest DNA cross-link structure to be described.     

Sequence context effect on the structure of nitrous acid induced DNA interstrand cross-links

In the preceding paper in this journal, we described the solution structure of the nitrous acid cross-linked dodecamer duplex [d(GCATCCGGATGC)]2 (the cross-linked guanines are underlined). The structure revealed that the cross-linked guanines form a nearly planar covalently linked 'G:G base pair', with the complementary partner cytidines flipped out of the helix. Here we explore the flanking sequence context effect on the structure of nitrous acid cross-links in [d(CG)]2 and the factors allowing the extrahelical cytidines to adopt such fixed positions in the minor groove. We have used NMR spectroscopy to determine the solution structure of a second cross-linked dodecamer duplex, [d(CGCTACGTAGCG)]2, which shows that the identity of the flanking base pairs significantly alters the stacking patterns and phosphate backbone conformations. The cross-linked guanines are now stacked well on adenines preceding the extrahelical cytidines, illustrating the importance of purine- purine base stacking. Observation of an imino proton resonance at 15.6 p.p.m. provides evidence for hydrogen bonding between the two cross-linked guanines. Preliminary structural studies on the cross-linked duplex [d(CGCGACGTCGCG)]2 show that the extrahelical cytidines are very mobile in this sequence context. We suggest that favorable van der Waals interactions between the cytidine and the adenine 2 bp away from the cross-link localize the cytidines in the previous cross-linked structures.     

Induced fit DNA recognition by a minor groove binding analogue of Hoechst 33258: fluctuations in DNA A tract structure investigated by NMR and molecular dynamics simulations

NMR analysis and molecular dynamics simulations of d(GGTAATTACC)₂ and its complex with a tetrahydropyrimidinium analogue of Hoechst 33258 suggest that DNA minor groove recognition in solution involves a combination of conformational selection and induced fit, rather than binding to a preorganised site. Analysis of structural fluctuations in the bound and unbound states suggests that the degree of induced fit observed is primarily a consequence of optimising van der Waals contacts with the walls of the minor groove resulting in groove narrowing through: (i) changes in base step parameters, including increased helical twist and propeller twist; (ii) changes to the sugar–phosphate backbone conformation to engulf the bound ligand; (iii) suppression of bending modes at the TpA steps. In contrast, the geometrical arrangement of hydrogen bond acceptors on the groove floor appears to be relatively insensitive to DNA conformation (helical twist and propeller twist). We suggest that effective recognition of DNA sequences (in this case an A tract structure) appears to depend to a significant extent on the sequence being flexible enough to be able to adopt the geometrically optimal conformation compatible with the various binding interactions, rather than involving ‘lock and key’ recognition.     

Crystal structure of a 14 bp RNA duplex with non-symmetrical tandem GxU wobble base pairs

Adjacent GxU wobble base pairs are frequently found in rRNA. Atomic structures of small RNA motifs help to provide a better understanding of the effects of various tandem mismatches on duplex structure and stability, thereby providing better rules for RNA structure prediction and validation. The crystal structure of an RNA duplex containing the sequence r(GGUAUUGC-GGUACC)2 has been solved at 2.1 A resolution using experimental phases. Novel refinement strategies were needed for building the correct solvent model. At present, this is the only short RNA duplex structure containing 5'-U-U-3'/3'-G-G-5' non-symmetric tandem GxU wobble base pairs. In the 14mer duplex, the six central base pairs are all displaced away from the helix axis, yielding significant changes in local backbone conformation, helix parameters and charge distribution that may provide specific recognition sites for biologically relevant ligand binding. The greatest deviations from A-form helix occur where the guanine of a wobble base pair stacks over a purine from the opposite strand. In this vicinity, the intra-strand phosphate distances increase significantly, and the major groove width increases up to 3 A. Structural comparisons with other short duplexes containing symmetrical tandem GxU or GxT wobble base pairs show that nearest-neighbor sequence dependencies govern helical twist and the occurrence of cross-strand purine stacks.     

Amide protein exchange and surface conformation of the basic pancreatic trypsin inhibitor in solution. Studies with two-dimensional nuclear magnetic resonance

Dickerson and his colleagues have described the structure of the DNA dodecamer C-G-C-G-A-A-T-T-C-G-C-G in the B form at a level that shows clearly several aspects of some base sequence-dependent departures from the ideal, regular helical structure of B-DNA. I argue that the detailed conformation is a consequence of simple steric repulsive forces between purine bases in consecutive base-pairs but on opposite backbones. These repulsions are a consequence of the “propeller twist” of the base-pairs, together with the larger size of the purine bases, and they may occur in either the major or the minor groove. The argument is conducted in terms of the structural mechanics of a deformable elastic system. These repulsive forces between the base-pairs are resisted by stresses in the helical backbones, which may be studied quantitatively via the variation in torsion angles δ along the backbones, at the points where the sugar rings are connected. There is also a correlation between the cross-chain purine repulsions and the perturbations in helical twist angle between successive base-pairs. The work suggests some comments on the proposed “alternating B” form, the Z form and the A form of DNA.     

Structures of the mismatched duplex d(GGGTGCCC) and one of its Watson-Crick analogues d(GGGCGCCC)

The mismatched duplex d(GGGTGCCC) (I) and its two Watson-Crick analogues (dGGGCGCCC) (II) and d(GGGTACCC) (III) were synthesized. The X-ray crystal structures of (I) and (II) were determined at resolutions of 2.5 and 1.7 A (1 A = 0.1 nm) and refined to R factors of 15 and 16%, respectively. (I) and (II) crystallize as A-DNA doublehelical octamers in space groups P61 and P4(3)2(1)2, respectively, and are stable at room temperature. The central two G.T mispairs of (I) adopt the wobble geometry as observed in other G.T mismatches. The two structures differ significantly in their local conformational features at the central helical regions as well as in some global ones. In particular, T-G adopts a large helical twist (44 degrees) whereas C-G adopts a small one (24 degrees). This difference can be rationalized on the basis of simple geometrical considerations. Base-pair stacking energies which were calculated for the two duplexes indicate that (I) is destabilized with respect to (II). Helix-coil transition measurements were performed for each of the three oligomers by means of ultraviolet absorbance spectrophotometry. The results indicate that the stability of the duplexes and the co-operativity of the transition are in the following order: (I) less than (III) less than (II). Such studies may help in understanding why certain regions of DNA are more likely to undergo spontaneous mutations than others.     

Effects of the minor groove pyrimidine nucleobase functional groups on the stability of duplex DNA: the impact of uncompensated minor groove amino groups

DNA sequences containing four types of analog nucleosides are described. All four are pyridine derivatives constructed as C-nucleosides so that they mimic the pyrimidine derivatives 2'-deoxyuridine, thymidine or 2'-deoxycytidine, but in all cases the analogs lack the corresponding O2-carbonyls that in duplex DNA are located in the minor groove. In place of the O2-carbonyl is a hydrogen atom, a polar fluorine atom, or a nonpolar methyl group. The described C-nucleosides have native-like bidentate Watson-Crick hydrogen-bonding faces and can form essentially normal W-C base pairs of varying stability with A or G. In each modified base pair, two inter-residue hydrogen bonds should be present. In spite of a common number of interstrand hydrogen bonds, the thermodynamic stabilities of the prepared duplexes, each containing two analog base pairs, vary dramatically. Most notably, base pairs containing uncompensated purine amino groups (those lacking a hydrogen-bonding partner) in the minor groove exhibit the most dramatic reductions in thermodynamic stability. Removal of such uncompensated amino groups results in increased duplex stability. Base pairs containing fluorine in the minor groove positioned adjacent to an amino group seem to enhance duplex stability marginally (relative to --H or --CH(3)), but there is little evidence to suggest that fluorine is an effective hydrogen-bonding partner in these systems. The presence of minor groove methyl groups results in the least stable duplexes in each series of sequences.     

Comparison of positively charged DNG with DNA duplexes: a computational approach

Molecular dynamics is used to investigate the structural properties of the cationic DNA analogue deoxynucleic guanidine (DNG), in which a guanidinium group replaces the phosphate moiety of DNA. This study examines the DNG duplex dodecamers d(Ag)(12).d(Tg)(12) and d(Gg)(12).d(Cg)(12), as well as their DNA counterparts. Watson-Crick base-pairing is maintained in the solvated DNG duplex models during the 5ns simulations. The idealized DNG dodecamers assume many parameters characteristic of the corresponding native DNA, assuming B-DNA conformations. Several helical parameters are rather unique to DNG, including buckle, slide, inclination, propeller, and X-displacement. Fewer transitions in backbone torsions occur in the DNG duplexes compared to those of the DNA, which may result from the greater rigidity of the sp(2) hybridized guanidinium group verses the flexible sp(3) phosphate group. The DNG helices have exceptionally shallow major grooves and very deep minor grooves. The major and minor groove widths of DNG are narrower than those of the respective DNA counterparts.     

C-C-A-G-G-C-m5C-T-G-G. Helical fine structure, hydration, and comparison with C-C-A-G-G-C-C-T-G-G.

The self-complementary DNA duplex C-C-A-G-G-C-m5C-T-G-G has been refined against 1.75-A x-ray diffraction data to an R value of 17.4%. In the crystal of space group P6, 10-base pair DNA fragments with characteristic sequence-related fine structure stack end to end to form long antiparallel B-type double helices. As shown by a structure analysis at lower resolution (Heinemann, U., and Alings, C. (1991) EMBO J. 10, 35-43), the overall geometry of C-C-A-G-G-C-m5C-T-G-G is similar to that of the unmethylated analog C-C-A-G-G-C-C-T-G-G despite a different crystal environment. The present high resolution structure analysis permits a detailed comparison of the two duplexes and their hydration spheres. Helical parameters are significantly correlated between both molecules, with the exception of the base pair propeller. Sugar pucker and backbone torsion angles alpha, gamma, delta, and chi show similar mean values, but their individual values deviate significantly between duplexes. In contrast, torsion angles beta, epsilon, and zeta change along the strands of both duplexes in much the same way. The effect of single-site methylation on DNA conformation appears to be small and limited to the base pairs directly involved. Methylation tends to push base pairs toward the minor groove of the helix. A regular minor groove hydration pattern involves dual hydrogen bonding of water molecules to O-4' and base atoms of C-C-A-G-G-C-m5C-T-G-G.     

Molecular structure of the netropsin-d(CGCGATATCGCG) complex: DNA conformation in an alternating AT segment

The molecular structure of the complex between a minor groove binding drug (netropsin) and the DNA dodecamer d(CGCGATATCGCG) has been solved and refined by single-crystal X-ray diffraction analysis to a final R factor of 20.0% to 2.4-A resolution. The crystal is similar to that of the other related dodecamers with unit cell dimensions of a = 25.48 A, b = 41.26 A, and c = 66.88 A in the space group P2(1)2(1)2(1). In the complex, netropsin binds to the central ATAT tetranucleotide segment in the narrow minor groove of the dodecamer B-DNA double helix as expected. However, in the structural refinement the drug is found to fit the electron density in two orientations equally well, suggesting the disordered model. This agrees with the results from solution studies (chemical footprinting and NMR) of the interactions between minor groove binding drugs (e.g., netropsin and distamycin A) and DNA. The stabilizing forces between drug and DNA are provided by a combination of ionic, van der Waals, and hydrogen-bonding interactions. No bifurcated hydrogen bond is found between netropsin and DNA in this complex due to the unique dispositions of the hydrogen-bond acceptors (N3 of adenine and O2 of thymine) on the floor of the DNA minor groove. Two of the four AT base pairs in the ATAT stretch have low propeller twist angles, even though the DNA has a narrow minor groove. Alternating helical twist angles are observed in the ATAT stretch with lower twist in the ApT steps than in the TpA step.     

Solution structure of RNA duplexes containing alternating CG base pairs: NMR study of r(CGCGCG)2 and 2'-O-Me(CGCGCG)2 under low salt conditions.

Structures of r(CGCGCG)2 and 2'-O-Me(CGCGCG)2 have been determined by NMR spectroscopy under low salt conditions. All protons and phosphorus nuclei resonances have been assigned. Signals of H5'/5" have been assigned stereospecifically. All 3JH,H and 3JP,H coupling constants have been measured. The structures were determined and refined using an iterative relaxation matrix procedure (IRMA) and the restrained MD simulation. Both duplexes form half-turn, right-handed helices with several conformational features which deviate significantly from a canonical A-RNA structure. Duplexes are characterised as having C3'-endo sugar pucker, very low base-pair rise and high helical twist and inclination angles. Helices are overwound with <10 bp per turn. There is limited inter-strand guanine stacking for CG steps. Within CG steps of both duplexes, the planes of the inter-strand cytosines are not parallel while guanines are almost parallel. For the GC steps this pattern is reversed. The 2'-O-methyl groups are spatially close to the 5'-hydrogens of neighbouring residues from the 3'-side and are directed towards the minor groove of 2'-O-Me(CGCGCG)2 forming a hydrophobic layer. Solution structures of both duplexes are similar; the effect of 2'-O-methylation on the parent RNA structure is small. This suggests that intrinsic properties imposed by alternating CG base pairs govern the overall conformation of both duplexes.     

Molecular dynamics simulation of the DNA triplex d(TC)5.d(GA)5.d(C+T)5.

A molecular dynamics simulation of the DNA triple helix d(TC)5.d(GA)5.d(C+T)5 is described (C+ represents a protonated cytosine residue). The simulation has been performed using the program AMBER 3.1 and includes counterions and explicit solvent under periodic boundary conditions. Both the dynamic and time-averaged behaviour of the system has been analysed. Considerable deviations from the fibre-diffraction model for DNA triple helix structure are observed, including the repuckering of the purine strand sugars that has been identified in some nuclear magnetic resonance (n.m.r.) studies. The simulation suggests that this conformational change may be driven by the possibility of improved interactions between the phosphate groups of this strand and both the solvent and counterions. Several examples of a particular conformational transition are observed, involving correlated changes in the backbone angles alpha and gamma. These transitions provide a possible explanation for some unusual n.m.r. data that have been reported. The structure of the triple helix major groove also suggests an explanation for the observed stabilization of DNA triplexes by polyvalent cations, and their ability to interact with drugs that bind in the minor groove of DNA duplexes.