Hydration of oligonucleotides in crystals

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

Hydration and recognition of methylated CpG steps in DNA.

The analysis of the hydration pattern around methylated CpG steps in three high resolution (1.7, 2.15 and 2.2 A) crystal structures of A-DNA decamers reveals that the methyl groups of cytosine residues are well hydrated. In comparing the native structure with two structurally distinct forms of the decamer d(CCGCCGGCGG) fully methylated at its CpG steps, this study shows also that in certain structural and sequence contexts, the methylated cytosine base can be more hydrated that the unmodified one. These water molecules seem to be stabilized in front of the methyl group through the formation C-H...O interactions. In addition, these structures provide the first observation of magnesium cations bound to the major groove of A-DNA and reveal two distinct modes of metal binding in methylated and native duplexes. These findings suggest that methylated cytosine bases could be recognized by protein or DNA polar residues through their tightly bound water molecules.     

Structure and recognition of sheared tandem G x A base pairs associated with human centromere DNA sequence at atomic resolution

G x A mismatched base pairs are frequently found in nucleic acids. Human centromere DNA sequences contain unusual repeating motifs, e.g. , (GAATG)n x (CATTC)n found in the human chromosome. The purine-rich strand of this repeating pentamer sequence forms duplex and hairpin structures with unusual stability. The high stability of these structures is contributed by the "sheared" G x A base pairs which present a novel recognition surface for ligands and proteins. We have solved the crystal structure, by the multiple-wavelength anomalous diffraction (MAD) method of d(CCGAATGAGG) in which the centromere core sequence motif GAATG is embedded. Three crystal forms were refined to near-atomic resolution. The structures reveal the detailed conformation of tandem G x A base pairs whose unique hydrogen-bonding surface has interesting interactions with bases, hydrated magnesium ions, cobalt(III)hexaammine, spermine, and water molecules. The results are relevant in understanding the structure associated with human centromere sequence in particular and G x A base pairs in nucleic acids (including RNA, like ribozyme) in general.     

Radiation-induced DNA damage as a function of hydration. I. Release of unaltered bases.

The release of unaltered bases from irradiated DNA, hydrated between 2.5 and 32.7 mol of water per mole of nucleotide (gamma), was investigated using HPLC. The objective of this study was to elucidate the yield of the four DNA bases as a function of dose, extent of hydration, and the presence or absence of oxygen. The increase in the yield of radiation-induced free bases was linear with dose up to 90 kGy, except for the DNA with gamma = 2.5, for which the increase was linear only to 10 kGy. The yield of free bases as a function of gamma was not constant in either the absence or the presence of oxygen over the range of hydration examined. For DNA with gamma between 2.5 and 15, the yield of free bases was nearly constant under nitrogen, but decreased under oxygen. However, for DNA with gamma greater than 15, the yield increased rapidly under both nitrogen and oxygen. The yield of free bases was described by a model that depended on two factors: 1) a change in the DNA conformation from a mixture of the A and C conformers in vacuum-dried DNA to predominantly the B conformer in the fully hydrated DNA, and 2) the proximity of the water molecules to the DNA. Irradiation of the inner water molecules (gamma less than 15) was less efficient than irradiation of the outer water molecules (gamma greater than 15), by a factor of approximately 3.3, in forming DNA lesions that resulted in the release of an unaltered base. This factor is similar to the previously published relative efficiency of 2.8 with which hydroxyl radicals and base cations induce DNA strand breaks. Our irradiation results are consistent with the hypothesis that the G value for the first 12-15 water molecules of the DNA hydration layer is the same as the G value for the form of DNA to which it is bound (i.e., the pseudo-C or the B form). Thus we suggest that the release of bases originating from irradiation of the hydration water is obtained predominantly: (1) by charge transfer from the direct ionization of the first 12-15 water molecules of the primary hydration layer and (2) by the attack of hydroxyl radicals generated in the outer, more loosely bound water molecules.     

Molecular dynamics study of solvation effect on diffusivity changes of DNA fragments

DNA sequence analyzing and base pair separation techniques have attracted much attention, such as denaturing gradient gel electrophoresis, temperature gradient gel electrophoresis, and capillary electrophoresis. However, details of sequence separation mechanisms in electrophoresis are not clarified enough. Understanding and controlling flow characteristics of DNA are important not only for fundamental research but also for further developments of bio-nano technologies. In the present study, we theoretically discuss the relationship between diffusivity and hydrated structures of DNA fragments in water solvent using molecular dynamics methods. In particular, influence of base pair substitutions on the diffusivity is investigated, focusing on an adenine-thymine (AT) rich B–DNA decamer 5’-dCGTATATATA-3’. Consequently, it is found that water molecules that concentrate on dissociated base pairs form hydrated structures and change the diffusivity of DNA decamers. The diffusion coefficients are affected by the substitution of GC for AT because of the different manner of interactions between the base molecules and water solvent. This result predicts a possibility of base pair separation according to differences in the diffusivity.     

Molecular dynamics simulations of DNA in solutions with different counter-ions

Molecular dynamics simulations of the [d(ATGCAGTCAG]2 fragment of DNA, in water and in the presence of three different counter-ions (Li+, Na+ and Cs+) are reported. Three-dimensional hydration structure and ion distribution have been calculated using spatial distribution functions for a detailed picture of local concentrations of ions and water molecules around DNA. According to the simulations, Cs+ ions bind directly to the bases in the minor groove, Na+ ions bind prevailing to the bases in the minor groove through one water molecule, whereas Li+ ions bind directly to the phosphate oxygens. The different behavior of the counter-ions is explained by specific hydration structures around the DNA and the ions. It is proposed how the observed differences in the ion binding to DNA may explain different conformational behavior of DNA. Calculated self-diffusion coefficients for the ions agree well with the available NMR data.     

Structures of building blocks in clusters of sweetpotato amylopectin

φ,β-Limit dextrins of domains and clusters of sweetpotato amylopectin were subjected to extensive hydrolysis by Bacillus amyloliquefaciens α-amylase to release building blocks and reveal the internal structures of clusters. The composition of building blocks was analyzed by size-fractionation, gel permeation chromatography, and high performance anion exchange chromatography. Different domains and clusters had structurally similar building blocks with around three chains per building block and internal chain length around 2.9. Singly branched and doubly branched building blocks were the largest and second largest groups in the clusters. Type A clusters had more large building blocks and contained 5–6 blocks per cluster with an inter-block chain length (IB-CL) of 7.0, whereas type B clusters had less large building blocks and contained 3–4 blocks per cluster with IB-CL 7.9. Models on how the building blocks could be organized into type A and type B clusters are discussed.     

Structure-based prediction of DNA target sites by regulatory proteins

Regulatory proteins play a critical role in controlling complex spatial and temporal patterns of gene expression in higher organism, by recognizing multiple DNA sequences and regulating multiple target genes. Increasing amounts of structural data on the protein-DNA complex provides clues for the mechanism of target recognition by regulatory proteins. The analyses of the propensities of base-amino acid interactions observed in those structural data show that there is no one-to-one correspondence in the interaction, but clear preferences exist. On the other hand, the analysis of spatial distribution of amino acids around bases shows that even those amino acids with strong base preference such as Arg with G are distributed in a wide space around bases. Thus, amino acids with many different geometries can form a similar type of interaction with bases. The redundancy and structural flexibility in the interaction suggest that there are no simple rules in the sequence recognition, and its prediction is not straightforward. However, the spatial distributions of amino acids around bases indicate a possibility that the structural data can be used to derive empirical interaction potentials between amino acids and bases. Such information extracted from structural databases has been successfully used to predict amino acid sequences that fold into particular protein structures. We surmised that the structures of protein-DNA complexes could be used to predict DNA target sites for regulatory proteins, because determining DNA sequences that bind to a particular protein structure should be similar to finding amino acid sequences that fold into a particular structure. Here we demonstrate that the structural data can be used to predict DNA target sequences for regulatory proteins. Pairwise potentials that determine the interaction between bases and amino acids were empirically derived from the structural data. These potentials were then used to examine the compatibility between DNA sequences and the protein-DNA complex structure in a combinatorial "threading" procedure. We applied this strategy to the structures of protein-DNA complexes to predict DNA binding sites recognized by regulatory proteins. To test the applicability of this method in target-site prediction, we examined the effects of cognate and noncognate binding, cooperative binding, and DNA deformation on the binding specificity, and predicted binding sites in real promoters and compared with experimental data. These results show that target binding sites for several regulatory proteins are successfully predicted, and our data suggest that this method can serve as a powerful tool for predicting multiple target sites and target genes for regulatory proteins.     

Hydration of protein-protein interfaces

We present an analysis of the water molecules immobilized at the protein-protein interfaces of 115 homodimeric proteins and 46 protein-protein complexes, and compare them with 173 large crystal packing interfaces representing nonspecific interactions. With an average of 15 waters per 1000 A2 of interface area, the crystal packing interfaces are more hydrated than the specific interfaces of homodimers and complexes, which have 10-11 waters per 1000 A2, reflecting the more hydrophilic composition of crystal packing interfaces. Very different patterns of hydration are observed: Water molecules may form a ring around interfaces that remain "dry," or they may permeate "wet" interfaces. A majority of the specific interfaces are dry and most of the crystal packing interfaces are wet, but counterexamples exist in both categories. Water molecules at interfaces form hydrogen bonds with protein groups, with a preference for the main-chain carbonyl and the charged side-chains of Glu, Asp, and Arg. These interactions are essentially the same in specific and nonspecific interfaces, and very similar to those observed elsewhere on the protein surface. Water-mediated polar interactions are as abundant at the interfaces as direct protein-protein hydrogen bonds, and they may contribute to the stability of the assembly.     

G . T base-pairs in a DNA helix: the crystal structure of d(G-G-G-G-T-C-C-C)

The synthetic deoxyoctanucleotide d(G-G-G-G-T-C-C-C) crystallizes as an A-type DNA double helix containing two adjacent G . T base-pair mismatches. The structure has been refined to an R-factor of 14% at 2.1 A resolution with 104 solvent molecules located. The two G . T mismatches adopt the "wobble" form of base-pairing. The mismatched bases are linked by a network of water molecules interacting with the exposed functional groups in both the major and minor grooves. The presence of two mispaired bases in the octamer has surprisingly little effect on the global structure of the helix or the backbone and glycosidic torsional angles. Base stacking around the mismatch is perturbed, but the central G-T step shows particularly good base overlap, which may contribute to the relatively high stability of this oligomer.     

G.T wobble base-pairing in Z-DNA at 1.0 A atomic resolution: the crystal structure of d(CGCGTG).

The DNA oligomer d(CGCGTG) crystallizes as a Z-DNA double helix containing two guanine-thymine base pair mismatches of the wobble type. The crystal diffracts to 1 A resolution and the structure has been solved and refined. At this resolution, a large amount of information is revealed about the organization of the water molecules in the lattice generally and more specifically around the wobble base pairs. By comparing this structure with the analogous high resolution structure of d(CGCGCG) we can visualize the structural changes as well as the reorganization of the solvent molecules associated with wobble base pairing. There is only a small distortion of the Z-DNA backbone resulting from introduction of the GT mismatched base pairs. The water molecules cluster around the wobble base pair taking up all of the hydrogen bonding capabilities of the bases due to wobble pairing. These bridging water molecules serve to stabilize the base-base interaction and, thus, may be generally important for base mispairing either in DNA or in RNA molecules.     

Assignment of the non-exchangeable proton resonances of d(C-G-C-G-A-A-T-T-C-G-C-G) using two-dimensional nuclear magnetic resonance methods

A general method of assigning the non-exchangeable protons in the nuclear magnetic resonance spectra of small DNA molecules has been developed based upon two-dimensional autocorrelated (COSY) and nuclear Overhauser (NOESY) spectra in 2H2O solutions. Groups of protons in specific sugars or bases are identified by their scalar couplings (COSY), then connected spatially in a sequential fashion using the Overhauser effect (NOESY). The method appears to be generally applicable to moderate-sized DNA duplexes with structures close to B DNA. The self-complementary DNA sequence d(C-G-C-G-A-A-T-T-C-G-C-G) has been synthesized by the solid-phase phosphite triester technique and studied by this method. Analysis of the COSY spectrum and the NOESY spectrum leads to the unambiguous assignment of all protons in the molecule except the poorly resolved H5' and H5" resonances. The observed NOEs indicate qualitatively that, in solution, the d(C-G-C-G-A-A-T-T-C-G-C-G) helix is right-handed and close to the B DNA form with a structure similar to that determined by crystallography.     

A 3D building blocks approach to analyzing and predicting structure of proteins

A new approach is introduced for analyzing and ultimately predicting protein structures, defined at the level of C alpha coordinates. We analyze hexamers (oligopeptides of six amino acid residues) and show that their structure tends to concentrate in specific clusters rather than vary continuously. Thus, we can use a limited set of standard structural building blocks taken from these clusters as representatives of the repertoire of observed hexamers. We demonstrate that protein structures can be approximated by concatenating such building blocks. We have identified about 100 building blocks by applying clustering algorithms, and have shown that they can "replace" about 76% of all hexamers in well-refined known proteins with an error of less than 1 A, and can be joined together to cover 99% of the residues. After replacing each hexamer by a standard building block with similar conformation, we can approximately reconstruct the actual structure by smoothly joining the overlapping building blocks into a full protein. The reconstructed structures show, in most cases, high resemblance to the original structure, although using a limited number of building blocks and local criteria of concatenating them is not likely to produce a very precise global match. Since these building blocks reflect, in many cases, some sequence dependency, it may be possible to use the results of this study as a basis for a protein structure prediction procedure.     

Intercalation of water molecules between nucleic acid bases in the crystal structures of 6-azathymine hemihydrate and 5-amino-2-thiocytosine dihydrochloride dihydrate

The crystal structure of 6-azathymine hemihydrate (6AzTH) exhibits a novel intercalation of water molecules interposed half-way between the modified bases 6.3 to 6.7 A apart. The crystal contains four molecules of 6-azathymine (6AzT) and two water molecules as the independent repeating unit. These two water molecules together with the four bases form two separate water sandwiches. In the crystal structure these sandwiches form two sets of local clusters. The anhydrous crystalline form of 6AzT, on the other hand, is stabilized by base stacking interactions. Both the water molecules in 6AzTH that are involved in sandwich formation have trigonal coordination around them. A reexamination of the crystal structure of 5-amino-2-thiocytosine (5A2TC) revealed that one of the water molecules in this structure also forms a water sandwich and has trigonal coordination whereas the other water molecule with tetrahedral coordination does not form a sandwich. The environment and the characteristics of the intercalated water molecule in these structures suggest a possible role for such water intercalations in the dynamics of DNA. Crystals of 6AzTH are monoclinic, space group P21/n, with unit cell parameters a = 8.861 (1), b = 13.177 (3), c = 20.662 (2) A, beta = 93.35 (1) degrees, and Z = 16. From diffractometer data (2503 reflections, greater than or equal to 3 sigma), the crystal structure was solved and refined to an R of 0.056.     

Interfacial water as a "hydration fingerprint" in the noncognate complex of BamHI

The molecular code of specific DNA recognition by proteins as a paradigm in molecular biology remains an unsolved puzzle primarily because of the subtle interplay between direct protein-DNA interaction and the indirect contribution from water and ions. Transformation of the nonspecific, low affinity complex to a specific, high affinity complex is accompanied by the release of interfacial water molecules. To provide insight into the conversion from the loose to the tight form, we characterized the structure and energetics of water at the protein-DNA interface of the BamHI complex with a noncognate sequence and in the specific complex. The fully hydrated models were produced with Grand Canonical Monte Carlo simulations. Proximity analysis shows that water distributions exhibit sequence dependent variations in both complexes and, in particular, in the noncognate complex they discriminate between the correct and the star site. Variations in water distributions control the number of water molecules released from a given sequence upon transformation from the loose to the tight complex as well as the local entropy contribution to the binding free energy. We propose that interfacial waters can serve as a "hydration fingerprint" of a given DNA sequence.     

Hydration of nucleic acid fragments: comparison of theory and experiment for high-resolution crystal structures of RNA, DNA, and DNA-drug complexes.

A computationally efficient method to describe the organization of water around solvated biomolecules is presented. It is based on a statistical mechanical expression for the water-density distribution in terms of particle correlation functions. The method is applied to analyze the hydration of small nucleic acid molecules in the crystal environment, for which high-resolution x-ray crystal structures have been reported. Results for RNA [r(ApU).r(ApU)] and DNA [d(CpG).d(CpG) in Z form and with parallel strand orientation] and for DNA-drug complexes [d(CpG).d(CpG) with the drug proflavine intercalated] are described. A detailed comparison of theoretical and experimental data shows positional agreement for the experimentally observed water sites. The presented method can be used for refinement of the water structure in x-ray crystallography, hydration analysis of nuclear magnetic resonance structures, and theoretical modeling of biological macromolecules such as molecular docking studies. The speed of the computations allows hydration analyses of molecules of almost arbitrary size (tRNA, protein-nucleic acid complexes, etc.) in the crystal environment and in aqueous solution.     

N6-methoxyadenine in damaged DNA has two faces in property of Watson-Crick base pairing.

In order to investigate mutation mechanism with oxyamine, two DNA dodecamers containing 2'-deoxy-N6-methoxyadenosine have been synthesized and their crystal structures have been determined. The dodecamers are associated in B form duplexes. The methoxy groups attached to the adenine bases do not affect the DNA conformation significantly. Electron densities clearly show that N6-methoxyadenine moiety forms Watson-Crick type pairing with both, thymine and cytosine bases. Such two faces in pairing are the origin of pyrimidine transition mutation.     

Synthesis and hybridization properties of inverse oligonucleotides.

The synthesis of adenine and thymine cyclopentylethyl nucleosides is presented. This novel constrained monomeric building block is very difficult to incorporate into oligonucleotides. It was introduced in 13mer oligodeoxynucleotide sequences at a single position using H-phosphonate chemistry. Phosphoramidite chemistry completely failed in this particular case. The H-phosphonate building blocks were obtained starting from the corresponding phosphoramidites. Stability of duplexes with RNA and DNA is significantly reduced.     

Ordered water structure in an A-DNA octamer at 1.7 A resolution

The crystal structure of the deoxyoctamer d(G-G-Br U-A-BrU-A-C-C) was refined to a resolution of 1.7 A using combined diffractometer and synchrotron data. The analysis was carried out independently in two laboratories using different procedures. Although the final results are identical the comparison of the two approaches highlights potential problems in the refinement of oligonucleotides when only limited data are available. As part of the analysis the positions of 84 solvent molecules in the asymmetric unit were established. The DNA molecule is highly solvated, particularly the phosphate-sugar back-bone and the functional groups of the bases. The major groove contains, in the central BrU-A-BrU-A region, a ribbon of water molecules forming closed pentagons with shared edges. These water molecules are linked to the base O and N atoms and to the solvent chains connecting the O-1 phosphate oxygen atoms on each strand. The minor groove is also extensively hydrated with a continuous network in the central region and other networks at each end. The pattern of hydration is briefly compared with that observed in the structure of a B-dodecamer.