BI Conformation Research Articles

Substituting CF2 for O4' in Components of Nucleic Acids: Towards Systems with Reduced Propensity to Form Abasic Lesions.

Intrinsic structural features and energetics of nucleotides containing variously fluorinated sugars as potential building blocks of DNA duplexes and quadruplexes are explored systematically using the modern methods of density functional theory (DFT) and quantum chemical topology (QCT). Our results suggest that fluorination at the 2'-β or 2'-α,β positions somewhat stabilizes in vacuo the AI relative to the BI conformations. In contrast, substitution of the CF2 group for the O4' atom (O4'-CF2 modification) leads to a preference of the BI relative to AI DNA-like conformers. All the studied modifications result in a noticeable increase in the stability of the glycosidic bond [estimated by the relaxed force constants (RFC) approach], with particularly encouraging results for the O4'-CF2 derivative. Consequently, the O4'-CF2 modified systems are suggested and explored as promising scaffolds for the development of duplex and quadruplex structures with reduced propensity to form abasic lesions and to undergo DNA damage.

The Sequence Context and Methylation Dependence of DNA Drug Binding

The ability of molecules to bind DNA plays an important role in many biological processes including, transcription, regulation, replication, and repair. Understanding the factors that influence binding affinity is therefore essential to our understanding of DNA. In this study the effects of sequence context and binding-site methylation on the binding affinity and conformation of DNA dodecamers are explored. The Cre binding site (ACGT) was studied in its methylated and native forms with a number of different flanking sequences chosen to test the predictive power of a previously published dinucleotide scale, wherein certain dinucleotides were said to have an affinity for either the BI or BII DNA conformation. 31P-NMR was used to test the true conformational state of each phosphorous in the dodecamers, and it was found that the scale could not accurately predict the real conformation of DNA. Importantly, the conformation of the binding site, the central tetrad which was not changed, varied significantly based on the sequence context. Thus more complex relationships than dinucleotides, such as longer-range translational conformation effects, are shown to affect the DNA conformation. Fluorescence titration with the DNA intercalator 7-Aminoactinomycin D was performed to determine the KD of each of the dodecamers and provide biological context to our conformation results. Excellent binding affinities were seen in the ACAC, ACAG, and GGCC sequences, while weaker binding was observed for GGAG sequences. The effects of methylation on binding affinity and conformation demonstrated that methylation effects are modulated by sequence context.

Phosphorus Chemical Shifts in a Nucleic Acid Backbone from Combined Molecular Dynamics and Density Functional Calculations

A comprehensive quantum chemical analysis of the influence of backbone torsion angles on (31)P chemical shifts in DNAs has been carried out. An extensive DFT study employed snapshots obtained from the molecular dynamics simulation of [d(CGCGAATTCGCG)]2 to construct geometries of a hydrated dimethyl phosphate, which was used as a model for the phosphodiester linkage. Our calculations provided differences of 2.1 ± 0.3 and 1.6 ± 0.3 ppm between the B(I) and B(II) chemical shifts in two B-DNA residues of interest, which is in a very good agreement with the difference of 1.6 ppm inferred from experimental data. A more negative (31)P chemical shift for a residue in pure BI conformation compared to residues in mixed B(I)/B(II) conformation states is provided by DFT, in agreement with the NMR experiment. Statistical analysis of the MD/DFT data revealed a large dispersion of chemical shifts in both B(I) and B(II) regions of DNA structures. δP ranges within 3.5 ± 0.8 ppm in the B(I) region and within 4.5 ± 1.5 ppm in the B(II) region. While the (31)P chemical shift becomes more negative with increasing α in B(I)-DNA, it has the opposite trend in B(II)-DNA when both α and ζ increase simultaneously. The (31)P chemical shift is dominated by the torsion angles α and ζ, while an implicit treatment of β and ε is sufficient. The presence of an explicit solvent leads to a damping and a 2-3 ppm upfield shift of the torsion angle dependences.

DFT study of minimal fragments of nucleic acid single chain for explication of sequence dependence of DNA duplex conformation

DFT studies of desoxydinucleoside monophosphate complexes with Na-ions (dDMPs), using DGAUSS and ADF software reveal that the characteristic B-type conformation of DNA duplexes is initially predisposed in single strand in the form of a local minimum. Such predisposition may be important for biological functioning of DNA by facilitating an addition of new nucleotide in the processes of DNA duplication and restoration of double helix after unwinding. The computational results demonstrate the important contribution of intra-strand interactions to conformational stability and sequence-dependent variability of B-DNA duplexes. The main characteristics of the “canonical” BI conformations, such as the regions of torsion angles of sugar-phosphate backbone, C2′-endo or very close to C2′-endo sugar puckerings, and nearly parallel base-ring arrangements are already present in the single strand and are universal for all 16 dDMPs. The dihedral angle between the bases does not depend on the extent of their overlap, which suggests the major contribution of sugar-phosphate backbone to formation of these minima. The study reveals characteristic sequence dependence of the base arrangements. Namely, an extensive base-ring overlap takes place for all purine–purine and purine–pyrimidine sequences, and negligible ring overlap is observed in all pyrimidine–purine and pyrimidine–pyrimidine sequences. These arrangements closely resemble the geometrical characteristics of BI DNA in duplex crystals. Preliminary results of search for other energy minima related to helical DNA structures revealed the existence of BII-like minima for some dDMPs.

Sequence Preference for BI/BII Conformations in DNA: MD and Crystal Structure Data Analysis

Deciphering sequence information from sugar-phosphate backbone is finely tuned through the conformational substates of DNA. BII conformation, one of the conformational substates of B-DNA, is known to play a key role in DNA-protein recognition. BI and BII are identified by the ϵ-ζ difference, which is negative in BI and positive in BII. Our analysis of MD and ciystal structures shows that BII conformation is sequence specific and dinucleotides GC, CG, CA, TG, TA show high preference to take up BII conformation, while TT, TC, CT, CC dinucleotides rarely take up this conformation. Significant changes were observed in the dinucleotide parameters viz. twist, roll, and slide for the steps having BII conformation. Interestingly, the magnitude of variation in the dinucleotide parameters is seen to depend mainly on two factors, the magnitude of ϵ-ζ difference and the presence or absence of BII conformation in the second strand, across the WC base-paired dinucleotide step. Based on these two factors, the conformational substate of a dinucleotide step can be further classified as BI.BI (BI conformation in both strands), BI.BII (BI conformation in one strand and BII conformation in the other), and BII.BII (BII conformation in both strands). The occurrence of BII in both strands was found to be quite rare and thus, it can be concluded that BI.BI and BI.BII hybrid steps are more favorable than a BII.BII step. In conformity with the sequence preference seen for dinucleotides in each strand, BII.BII combination of backbone conformation was observed only for GC, CG, CA, and TG containing dinucleotide steps. We further classified BII.BII step as strong BII and weak BII depending on the magnitude of the average ϵ-ζ difference. The dinucleotide steps which belong to the categoiy of strong BII, have large twist, high positive slide and negative roll values, while those in the weak BII group have roll, twist, and slide values similar to that of hybrid BI.BII steps. This conformational property could be contributing to the groove opening/closing and thus can modulate protein-DNA interaction.

Polyamine-nucleic acid interactions and the effects on structure in oriented DNA fibers.

Fibrous oriented calf thymus DNA containing the natural polyamines spermidine (Spd) and putrescine (Put), and the degradation polyamines cadaverine (Cad) and 1,3-diaminopropane (DAP), have been investigated at different water contents using nuclear magnetic resonance (NMR) methods, fiber X-ray diffraction and gravimetric measurements. When judged by X-ray only the DAP and Spd samples seem to undergo a B-A-form transition at reduced water activity. Solid-state two-dimensional rotor-synchronized magic angle spinning (2D-syncMAS) 31P-NMR, however, shows the A-form to be present also in the Put sample, and it appears that the separation between the amine units of diamines is correlated with the amount of A-form present. In addition, the solid-state NMR data show the polyamine-bound DNA samples to have a significant deviation from the ordinary B-form DNA structure, displaying similar amounts of BI and BII nucleotide conformations. The low water content of the samples suggest that the polyamines themselves act as hydrators of DNA. Water 2H-NMR results are in agreement with this observation. The quadrupolar splittings of the polyamine 2H signals for samples at low water content indicate some preferential spatial orientations of the polyamines in the ordered DNA environment. The polyamines show relatively fast macroscopic diffusion as detected by NMR self-diffusion measurements.

BII Nucleotides in the B and C Forms of Natural-sequence Polymeric DNA: A New Model for the C Form of DNA

A combination of solid-state 31P and 13C NMR, X-ray diffraction, and model building is used to show that the B and C forms of fibrous macromolecular DNA consist of two distinct nucleotide conformations, which correspond closely to the BI and BII nucleotide conformations known from oligonucleotide crystals. The proportion of the BII conformation is higher in the C form than in the B form. We show structural models for a 10 1 double helix involving BI nucleotides and a 9 1 double helix involving BII nucleotides. The 10 1 BI model is similar to a previous model of B-form DNA, while the 9 1 BII model is novel. The BII model has a very deep and narrow minor groove, a shallow and wide major groove, and highly inclined bases. This work shows that the B to C transition in fibers corresponds to BI to BII conformational changes of the individual nucleotides.

Unexpected BII Conformer Substate Population in Unoriented Hydrated Films of the d(CGCGAATTCGCG)2 Dodecamer and of Native B-DNA from Salmon Testes

Conformational substates of B-DNA had been observed so far in synthetic oligonucleotides but not in naturally occurring highly polymeric B-DNA. Our low-temperature experiments show that native B-DNA from salmon testes and the d(CGCGAATTCGCG)2 dodecamer have the same BI and BII substates. Nonequilibrium distribution of conformer population was generated by quenching hydrated unoriented films to 200K, and isothermal structural relaxation toward equilibrium by interconversion of substates was followed by Fourier transform infrared spectroscopy. BI interconverts into BII on isothermal relaxation at 200K, whereas on slow cooling from ambient temperature, BII interconverts into BI. Our estimation of the dodecamer's BI-to-BII conformer substate population by curve resolution of the symmetrical stretching vibration of the ionic phosphate is 2.4±0.5 to 1 at 200K, and it is 1.3±0.5 to 1 between 270 and 290K. Pronounced spectral changes upon BI-to-BII interconversion are consistent with base destacking coupled with migration of water from ionic phosphate toward the phosphodiester and sugar moieties. Nonspecific interaction of proteins with the DNA backbone could become specific by induced-fit-type interactions with either BI or BII backbone conformations. This suggests that the BI-to-BII substate interconversion could be a major contributor to the protein recognition process.

Measurement of3JC2‘PScalar Couplings in a 17 kDa Protein Complex with13C,15N-Labeled DNA Distinguishes the BIand BIIPhosphate Conformations of the DNA

Measurement of³<i>J</i>_C2_‘_PScalar Couplings in a 17 kDa Protein Complex with¹³C,¹⁵N-Labeled DNA Distinguishes the B_Iand B_IIPhosphate Conformations of the DNA

Solution structure of a GA mismatch DNA sequence, d(CCATGAATGG)2, determined by 2D NMR and structural refinement methods.

GA mismatches in DNA have drawn attention because of their special repair mechanisms, stability, and variety of conformations. A symmetric 10-base oligodeoxyribonucleotide duplex, d(CCATGAATGG)2, containing two GA mismatches has been investigated by one- and two-dimensional multinuclear NMR and molecular refinement procedures to ascertain the conformational details of the 5'-pyrimidine-GA-purine-3' sequence. A molecular model established from the NMR results has a B-type right-handed helix with each of the bases retaining the normal anti-glycosidic torsional angles. Type I mismatched base pairs have GNH2-AN7 and GN3-ANH2 (edge-to-edge) hydrogen bonds, while type II base pairs have GN1H-AN1 and GO6-ANH2 (face-to-face) bonds. The conformation at the GA mismatch site has type I GA base pairs and an unusual cross-strand stacking of the adjacent G5 and A6 bases, which causes significant overwinding of the helix at the mismatch site. Unusual shifts of the 31P resonances suggest that the phosphate linkage between G5 and A6 is no longer in the low-energy BI conformation. One-dimensional imino and phosphorus NMR studies were carried out on a number of DNA sequences containing adjacent 5'-GA-3' mismatched base pairs to investigate the sequence dependence of the conformations and base-pairing types. Type I and type II conformations have very different imino proton and 31P NMR spectral patterns that can be used to classify any sequence with adjacent GA mismatches by base-pairing and conformational type. The NMR results indicate that the conformation selected is dictated completely by the flanking sequence: 5'-pyrimidine-GA-purine-3' sequences adopt the type I conformation, while 5'-purine-GA-pyrimidine-3' sequences have the type II conformation.

DNA bending studied byMD and 2DNMR NOESY simulations: Role of the junction sequence between the A/T tracts

We have performed all-atom constant temperature (T = 285 K) molecular dynamics (MD) simulations on d(GA4T4C)2 and d(GT4A4C)2 for 200 ps using the NMR-derived structures as the starting points. The major purpose of MD simulation is to search the conformational space so that we can construct an ensemble of conformations that will reproduce the experimentally observed 2D NMR NOESY spectra. Analyses of the ensemble of duplex structures reveal that in both oligomers constituent nucleotides predominantly display BI conformations. A simple two-parameter analysis shows that the nature of the long-range collective bending motion is different in the two duplexes. The conformations obtained by use of molecular dynamics are very similar to each other for the same molecule, the root mean square deviation (RMSD) being 1.6 A for d(GA4T4C)2 and 1.1 A for d(GT4A4C)2. We have computed theoretical 2D NOESY spectra for an ensemble of a large library of 1200 structures in the MD trajectory for d(GA4T4C)2. Theoretical MD-derived 2D NOESY spectra, averaged through an automated loop computation procedure over the 1200 timewise additivity NOESY simulation spectra, are compared with the corresponding experimental spectra. The agreement between the theoretical and experimental NOESY spectra is good. The results suggest that there is no longer any need to interpret NMR data on the basis of a “nonexistent” time-average structure and that worthwhile information about the realistic composition of the ensemble of conformations that are present in solution can be obtained by interconnecting MD simulations and NMR data.

Determination of the conformation of d(GGAAATTTCC)2 in solution by use of proton NMR and restrained molecular dynamics

The conformation of the putative bent DNA d(GGAAATTTCC)2 in solution was studied by use of 1H NMR and restrained molecular dynamics. Most of the resonances were assigned sequentially. A total of 182 interproton distance restraints were determined from two-dimensional nuclear Overhauser effect spectra with short mixing times. Torsion angle restraints for each sugar moiety were determined by qualitative analysis of a two-dimensional correlated spectrum. Restrained molecular dynamics was carried out with the interproton distances and torsion angles incorporated into the total energy function of the system in the form of effective potential terms. As initial conformations for restrained molecular dynamics, classical A-DNA and B-DNA were adopted. The root mean square deviation (rmsd) between these two conformations is 5.5 A. The conformations obtained by use of restrained molecular dynamics are very similar to each other, the rmsd being 0.8 A. On the other hand, the conformations obtained by use of molecular dynamics without experimental restraints or restrained energy minimization depended heavily on the initial conformations, and convergence to a similar conformation was not attained. The conformation obtained by use of restrained molecular dynamics exhibits a few remarkable features. The second G residue takes on the BII conformation [Fratini, A. V., Kopka, M. L., Drew, H. R., & Dickerson, R. E. (1982) J. Biol. Chem. 257, 14686-14707] rather than the standard BI conformation. There is discontinuity of the sugar puckering between the eighth T and ninth C. The minor groove of the oligo(dA) tract is rather compressed. As a result, d(GGAAATTTCC)2 is bent.

Chiral phosphorothioate analogues of B-DNA : The crystal structure of Rp-d¦Gp(S)CpGp(S)CpGp(S)C¦

The compound Rp-d¦Gp(S)CpGp(S)CpGp(S)C¦, an analogue of the deoxyoligomer d(G-C) 3, crystallizes in space group P2 12 12 1 with a = 34.90 A ̊ , b = 39.15 A ̊ and c = 20.64 A ̊ . The structure, which is not isomorphous with any previously determined deoxyoligonucleotide, was refined to an R factor of 14.5% at a resolution of 2.17 Å, with 72 solvent molecules located. The two strands of the asymmetric unit form a right-handed double helix, which is a new example of a B-DNA conformation and brings to light an important and overlooked component of flexibility of the double helix. This flexibility is manifest in the alternation of the backbone conformation between two states, defined by the adjacent torsion angles ε and ζ, trans.gauche—( BI) and gauche—.trans ( BII). BI is characteristic of classical of B-DNA and has an average C (1′) to C (1′) separation of 4.5 Å. The corresponding separation for BII is 5.3 Å. Each state is associated with a distinct phosphate orientation where the plane of the PO2 (or POS) group is alternately near horizontal or vertical with respect to the helix axis. The BI and BII conformations are out of phase on the two strands. As a consequence, on one strand purine-pyrimidine stacking is better than pyrimidine-purine, while the converse holds for the other strand. At each base-pair step, good and bad stacking alternate across the helix axis. The pattern of alternation is regular in the context of a fundamental dinucleotide repeat. Re-examination of the B-DNA dodecamer d(C-G-C-G-A-A-T-T-C-G-C-G) shows that the C-G-C-G regions contain the BI and BII conformations, and the associated dual phosphate orientation and asymmetric base stacking. Different mechanisms are used in the two structures to avoid clashes between guanine residues on opposite strands, a combination of lateral slide, tilt and helical twist in the present structure, and base roll, tilt and longitudinal slide (Calladine rules) in the dodecamer. The flexibility of the phosphate orientations demonstrated in this structure is important, since it offers a structural basis for protein-nucleic acid recognition.