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Hydrated cytosine-Guanine base pair from DNA


Cytosine-Guanine base pair from DNA

Nucleic Acid Hydration

The hydration of the nucleic acids controls their structure and mechanism of action.


V Hydrogen bonds in the nucleic acids
V DNA hydration
V DNA processing



'The structure is an open one, and its water content is rather high.

At lower water contents we would expect the bases to tilt so that the structure could become more compact.'

James Watson and Francis Crick, 1953 [828]   


Pairing between single nucleic acid bases upon hydrogen bond formation in bulk water does not occur (although often shown for simplicity's sake, see below) unless there is a string of hydrogen-bonded bases. Nucleic acid hydration is crucially important for their conformation and utility [1093], as noted by Watson and Crick [828]. The organized hydration extends to several nanometers from the surface. The strength of these aqueous interactions is far greater than those for proteins due to their highly ionic character [542b]. The DNA double helix can take up several conformations (for example, right-handed A-DNA pitch 28.2 Å 11 bp, B-DNA pitch 34 Å 10 bp, C-DNA pitch 31 Å 9.33 bp, D-DNA pitch 24.2 Å 8 bp, and the left-handed Z-DNA pitch 43Å 12 bp) with differing hydration. The predominant natural DNA, B-DNA, has a wide and deep major groove and a narrow and deep minor groove and requires the greatest hydration. Lowering the hydration (for example, by adding ethanol) may cause transitions from B-DNA to A-DNA [2784] to Z-DNA.


The charged surface of A-T and C-G base pairs

Calculations using the Restricted Hartree-Fock wave function (RHF) using the 6-31G** basis set

The charged surface of AT and CG; Calculations using the Restricted Hartree-Fock wave function (RHF) using the 6-31G** basis set

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Hydrogen bonds in the nucleic acids

Hydrogen bonds between the chains of DNA


Hydrogen bonds between the chains of DNA

The double-helical chains of DNA and some RNA strands are held together by hydrogen bonds (see left). In themselves, the strengths of each of these hydrogen bonds are very similar to each other, and to the hydrogen bond between water molecules, except for the much weaker C-H···O=C bonds. Thus the guanine-cytosine base pair, with three medium-strong hydrogen bonds is held with about twice the strength as the adenine-thymine base pair [2493],e which only has two medium-strong hydrogen bonds. All these hydrogen bonds are strengthened and protected from solvent hydration by the hydrophobic stacking of the bases,f which is a major contributor to the helix stability. Without such protective stacking, the base pairs would rapidly separate and hydrate with solvent water. When forming the base pairs, hydrogen bonds to water molecules are broken [3454]. Each base-pair hydrogen bond costs at least two base-water hydrogen bonds less the water-water interaction of the released water molecules.


The form of the stacking and the resultant helix conformation is controlled by the external hydration of the helices.


Nuclear magnetic shieldings of the DNA nucleic acids, adenine, cytosine, guanine, and thymine in aqueous solutions have been calculated. The first solvation shell causes a marked deshielding of the protonated and amino nitrogens and for the hydrogens of the protonated nitrogen [3899].

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DNA hydration

The change in the free energy of the surrounding water aids the conversion of single-stranded DNA (ssDNA) into double-stranded DNA (dsDNA) as the water molecules are more stable around dsDNA than around ssDNA, even out to about 0.65 nm (3 hydration layers) [2693].


X-ray diffraction patterns for A-DNA and B-DNA, from


X-ray diffraction patterns for A-DNA and B-DNA from http://undsci.berkeley.edu/article/0_0_0/dna_06

B-DNA (see X-ray diffraction patterns right) needs about 30%, by weight, water to maintain its native conformation in the crystalline state. The transformation depends on various factors such as the sequence, ion composition, concentration, and water activity [2912]. Partial dehydration converts it to A-DNA (see X-ray diffraction patterns right, with a narrower and deeper major groove and very wide but shallow minor groove). The transition for this transformation occurs at about 20 water molecules per base pair, with its midpoint at about 15 water molecules per base pair [1343] (at about 85% relative humidity [3127]). The B-DNA possesses a spanning water network, and it is the loss of its continuity [1343], together with the competition between hydration and direct cation coupling to the free oxygen atoms in the phosphate groups [1394], that give rise to the transition to A-DNA. This dehydration-induced structural transition decreases the free energy required for A-DNA deformation and twisting, which is usefully employed by encouraging supercoiling but eventually leads to denaturation [441]. Further dehydration results in the least hydrated D-DNA (favored by excess counterions that shield the DNA phosphate charges), which has a very narrow minor groove with a string of alternating water and counterions distributed along its edge [816].

Nucleic acid hydration, using dimethyl

phosphate, similar to [4393]


nucleic acid hydration, from [4393]


There is a significant difference in the hydration of (monomers of) RNA and DNA due to the presence of the 2'-OH groups in RNA. Somewhat surprisingly, their presence reduces the ability and strength of RNAs' hydrogen-bonding to water and influences the base-pair pattern and conformation of single nucleoside molecules [4241]. The reason for this is the formation of strong intramolecular hydrogen bonds between the 2'-OH and 3'-OH of the ribose residue (2'-O-H····3'-O-H) so reducing their ability to hydrogen bond to neighboring water molecules. As found in the loop domains of transfer RNA cloverleaf structures, short inter-phosphate distances need positively charged ions (e.g., Mg+2) and water molecules to maintain their bioactive folded structure [4393].


Hydration is greater and more strongly held around the phosphate groups that run along the inner edges of the DNA major grooves. However, the water molecules are not permanently situated due to the rather diffuse electron distribution of the phosphate groups. Hydration is more ordered and persistent around the bases with their more directional hydrogen-bonding ability and restricted space. Water molecules are held relatively strongly with residence times for the first hydration shell 0.5 - 1 ns. Because of the regular structure of DNA, hydrating water is held cooperatively along the double helix in both the major and minor grooves. The cooperative nature of this hydration aids both the zipping (annealing) and unzipping (unwinding) of the double helix. Water motion within the grooves is slowed down compared with the bulk water, with the most significant reduction within the more restricting minor groove [930]. It has been shown that the conformational fluctuations of the DNA facilitate these restricted water motions and accelerate the hydration dynamics within the groove's confinement [2776]. Molecular dynamics simulations indicate a high correlation of water's hydrogen bonds within the minor grooves, suggesting that this is the mechanism for the slow dynamics [4344]. On melting, about four water molecules per base pair are released despite extra hydration sites being released by the previously hydrogen-bonded base pairing [707], thus confirming the importance of this cooperative nature of the water-binding within the grooves. Using terahertz time-domain spectroscopy, a higher number of less well-bound water molecules are found arranged along the sugar-phosphate backbone beyond the first layer of those firmly bound to phosphate [4380].


Nucleic acids have several groups that can hydrogen bond to water, with RNA having a greater extent of hydration than DNA due to its extra oxygen atoms (that is, ribose O2') and unpaired base sites. These extra hydroxyl groups also create additional hydration in duplex RNA as they provide a scaffold for the minor groove hydration network [708]. Also, double-stranded RNA forms A-helical structures with shorter intra-strand phosphate-phosphatedistances (4.5 Å rather than 6.6 Å for B-DNA) and higher charge density.


DNA base pairs showing hydration sites


DNA base pairs showing hydration sites; in particular the G=C pair having three hydration sites in the minor groove compared with two in A=T

In DNA, the bases are hydrogen-bonded pairings, close to the 0.28 nm bond length found between hydrogen-bonded water molecules in liquid water. The aqueous environment causes a slight lengthening (≈ 1%) of the DNA hydrogen bonds and weakens them significantly (≈ 50%) [1867].d All these groups, except for the hydrogen-bonded ring nitrogen atoms (pyrimidine N3 and purine N1), are capable of one further hydrogen-bonding link to water within the major or minor grooves in B-DNA. A molecular dynamics simulation indicated that both grooves were equally hydrated with hydration roughly CN4/GN2/TO2 > AN6/CO2/GO6 > AN3/GN3/GN7/TO4 >> AN7 [1249].


Thus, in B-DNA, guanine will hydrogen-bond to a water molecule from the minor groove 2-amino- and major groove 6-keto-groups with further single hydration on the free ring nitrogen atoms (minor groove N3 and major groove N7). Cytosine will hydrogen-bond to a water molecule from both the major groove 4-amino- and minor groove 2-keto-groups. Adenine will hydrogen-bond to a water molecule from the major groove 6-amino-group with further single hydration on the free ring nitrogen atoms (minor groove N3 and major groove N7). Thymine (and uracil, if base-paired in RNA) will hydrogen-bond to a water molecule from both the minor groove 2-keto- and major groove 4-keto-groups. Phosphate hydration in the major groove is thermodynamically stronger but exchanges faster. There are six (from crystal structures, [143]) or seven (from molecular dynamics, [144]) hydration sites per phosphate a, not including hydration of the linking oxygen atoms to the deoxyribose or ribose residues. The deoxyribose oxygen atoms (O3' phosphodiester, ring O4' and O5' phosphodiester) all hydrogen-bond to one water molecule, whereas the free 2'-OH in ribose is much more capable of hydration and may hold on to about 2.5 water molecules. b The total for all these hydrations, in a G3 H-bondsC duplex, would be about 26-27, but about 14 of these water molecules are shared. There are many ways these water molecules can be arranged, with B-DNA possessing 22 possible primary hydration sites per base pair in a G3 H-bondsC duplex but only occupying 19 of them [144]. The DNA structure depends on how these sites are occupied; water providing the zip, holding the two strands together. It should be noted that cations may transiently replace about 2% of the hydrating water molecule sites.


Hydration of the minor groove of Β-DNA, from

hydration of the minor groove of DNA (dodecamer CGCAATTCGCG; NDB BD0008; http://ndbserver.rutgers.edu/atlas/xray/structures/B/bd0008/bd0008.html)


The hydration of the B-DNA minor groove is dependent on the DNA sequence with water-bridge lifetimes varying from 1 to 300 ps [1767], depending on the sequence. The hydration usually involves single water molecules connecting the strands. However, connection via pairs of water molecules, with varying interchange between these forms, may allow greater structural flexibility in the DNA and interactions with specific proteins [1605]. There is a spine of hydration running down the bottom of the B-DNA minor groove, particularly where there is the A=T duplex [145] (see right, where the water oxygen atoms are shown large green and red, where the red atoms are the primary hydration water and the green atoms are the secondary hydration water, [1136]), which is vital in stabilizing it [146]. Thus, A=T duplex sequences favor water binding in the minor groove, and protein binding driven by the significant entropy release on this low entropy water's release [1136]. Water molecules hydrogen-bond by donating two hydrogen bonds, so bridging between thymine 2-keto(s) and adenine ring N3(s) in sequential opposite strands (that is, not paired bases). This water has been called cross-strand bridging water (CSBW) and appears to be necessary for charge transfer (hole transport) between separated guanine bases down the DNA duplex [1221]). CSBW water is fully hydrogen-bonded by accepting two further hydrogen bonds from secondary hydration water, so fixing the primary hydration water more firmly in place such that they exchange slower (0.9 ns) than any other water hydrating the DNA. The primary hydration may occur regularly down the minor groove connecting the strands and is chiral even in the liquid state [2916]. Such water molecules shield the double helix and protect it from excess heat and UV photo damage [2916].


Transcription factor binding to the minor groove is accompanied by loss of this water [2921]. A further cooperative effect is through the secondary hydration. The minor groove has a complex hydration pattern, including water hexagons from the initial spine of hydration (above) through secondary hydration out to the 4th aqueous shell [797]. This hydration is more strongly held than in the G3 H-bondsC duplex giving rise to greater apparent hydration (about 44 water molecules per A=T duplex base pair [179]). The A=T base-pairing produces the narrower minor groove and more pronounced spine of hydration. In contrast, the G3 H-bondsC base pairing produces a wider minor groove with more extensive primary hydration, partly due to the 50% greater hydration sites.


Such solvent interactions are key to the hydration environment, and hence its recognition [1565], around the nucleic acids and directly contributes to the DNA conformation. They act together with the positively charged counterions, to give a complex sequence-dependent electrostatic environment and capable of specifically interacting with biologically important ligands [3400].


B-DNA possessing higher phosphate hydration, less exposed sugar residues, and a smaller hydrophobic surface, is stabilized at high water activity, whereas A-DNA, with its shared inter-phosphate water bridges, is more stable at low water activity. Thus, if the relative humidity is kept constant, there will be a transformation from B-DNA to A-DNA with increasing temperature [179]. The much greater loss in primary hydration of G3 H-bondsC base pairs (compared with A=T base pairs) on changing from B-DNA to A-DNA is apparently responsible for the tendency of G3 H-bondsC base pairs rather than A=T base pairs to form the A-DNA conformation. In contrast to B-DNA, A-DNA possesses a hollow core down its axis where water can create a hydrogen-bonded structure linking to the bases from the side of the major groove (as shown above) [553]. Any disruption of this core structure may lead to the A-DNAgoes to B-DNA transition.


Using cryo neutron crystallography, the H-bonding patterns of water molecules around the left-handed Z-DNA duplex
[d(CGCGCG)]2 has been investigated at 1.5 Å resolution[4271]. Although the bases form standard Watson-Crick pairs in Z-DNA, the cytosine and guanosine residues adopt different sugar puckers (C2'-endo and C3'-endo, respectively). This study showed that water is shared among guanine and cytosine keto and amino groups in the minor groove and on the convex surface.


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DNA processing

The processing of the genetic information within DNA is facilitated by highly discriminatory and strong protein binding. It has been shown that the interfacial water molecules can serve as 'hydration fingerprints' of a given DNA sequence [889]. The usual 'hydration fingerprint' of the DNA is disrupted by DNA damage, facilitateing repair protein attachment. The hydration spine (see above) is capable of carrying messages, as facilitated proton movement down the water wire, between binding sites in a similar, if complementary, manner to the electron transfer through the DNA residues [2258] and so coordinate the repair process.


The primary driving force for the specificity of protein binding is the entropy increase due to the release of bound water molecules (estimated at 3.6 kJ ˣ mol−1 for minor groove water and 2.3 kJ ˣ mol−1 for major groove water, both at 300 K [1096]),c with the DNA sequence determining the hydration pattern in the major and minor grooves (see above). Less perfect (that is, weaker) binding involves mainly secondary hydration water loss. It would allow sliding of the protein along the DNA [1176], facilitated by the remaining primary hydration water molecules [889]. For example, about 110 water molecules are released on binding the restriction endonuclease EcoRI to its site GAATTC, leaving an essentially dry interface and firmly bound complex with binding constant ≈ 10,000 times that for nonspecific binding. However, changing just one base out of the recognition sequence leaves those water molecules mostly unaffected and only little different from EcoRI non-specifically binding to DNA [1176b]. Thus, the key to forming specific links between proteins and DNA is that the interfacial water molecules allow the protein facile movement along the binding cleft while retaining contact information [1443]. Final binding makes use of both direct and water-mediated hydrogen bonds; for example, the restriction endonuclease MspI makes specific connections with all eight bases in the four base pair recognition sequence (5'-CCGG-3' and complementary 3'-GGCC-5'), by six direct and five water-mediated hydrogen bonds and thirteen water-mediated links to the phosphates [1444].


Water screening the charges between DNA and protein


water screening the charges between DNA and protein

Protein sliding along the DNA is assisted by uniform complementary electrostatic interactions between the positive protein and negative DNA as moderated by the intervening water, whereby the protein follows the helical pathway of the groove rather than jumping between the major groove and the more negative minor groove [1176c]. Where negative charges exist on the protein that create unfavorable binding electrostatics, similar charges may be screened, as shown right. It is essential that a balance of positive and negative charges exist to ensure that the binding is generally not too strong, avoiding excessive binding friction except where required.


The organization of the hydration close to the DNA results in weaker hydrogen bonding further out at 5–15 Å that allows unusually rapid movement of these water molecules. The ease of displacement of this translationally mobile water by approaching proteins or ligands will be energetically favored and reduce the activation barrier for DNA surface. interactions [2448],


It has been (independently) proposed that the separation of DNA double helices is enabled by forming clathrate-like water structuring using its screening dipoles [1222], an idea that ties this basic life process to the ESreversible arrowCS equilibrium and its icosahedral water clusters.


Highly structured water molecules, with lengthy residence times, have been found to be essential for the structural dynamics and function of ribozymes [1106] (catalytically competent highly structured non-coding RNAs) where water analogously communicates structural rearrangements to its action around many proteins.

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a    Both methods have drawbacks. X-ray crystallography gives better resolution at lower hydration, but the lower hydration will change the structure. Another disadvantage is its inability to define the orientation of water molecules. The results of molecular dynamics calculations are highly dependent on the methodology and force field used. [Back]


b    The ribose residue is a furan possessing a flexible (five-membered) ring with low energy barriers between several conformations. This encourages localized weak hydrogen-bonding around its free 2'-hydroxyl group (for more detail, see the polysaccharide hydration section). [Back]


c    The entropy 3.6 kJ ˣ mol−1 for minor groove water may be compared with that of ice (6.0 kJ ˣ mol−; 0 °C), indicating that the minor groove water tends towards being ice-like. [Back]


d    Raman spectroscopy has shown that the hydrogen bonds in the primary (inner) hydration shell are very strongly bound to the DNA and appear to be about 0.3 Å shorter than the hydrogen bonds in bulk water [2487]. [Back]


e    The commonly-found assumption that the two A=C hydrogen bonds and the three CºG hydrogen bonds are all approximately equal has been challenged with the two base pairs being given about equal bonding strength [3468]. [Back]


f    In the π-stacking of aromatic compounds, such as the formation of benzene dimers, there is an entropic stabilization associated with the shrinkage of the solvent-excluded volume as the two surfaces come together with the elimination of the H2O-Π-benzene hydrogen bonds from each surface. This entropic stabilization is nearly canceled out due to the enthalpic cost required for dewetting the internal space. Such an enthalpy-entropy compensation leads the association-free energy to be predominantly dictated by the solute-solute interaction enthalpy. [3511]. [Back]



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