Solid-state NMR studies of nucleic acid components

The full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-pro t purposes provided that: • a full bibliographic reference is made to the original source • a link is made to the metadata record in DRO • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders. Please consult the full DRO policy for further details.


Introduction
Nucleotides, oligonucleotides and nucleic acids are fascinating molecules that are responsible for many cellular processes such as the storage of genetic information, catalysis, metabolic regulation and energy supply. 1 Furthermore, chemical modications of the components of nucleic acids open up new ways of ghting many diseases, such as AIDS or cancer. 2,3t rst sight, using NMR to study solid nucleic acid components may seem somewhat perverse.Lack of motional averaging in the solid state means that NMR spectra of solids generally exhibit signicantly poorer resolution than their solution-state counterparts, and it is generally the behaviour of nucleic acids in in vivo conditions rather than as solids that is of most biological interest.However, the overall re-orientational dynamics of molecules in solution signicantly complicates the interpretation of solution-state NMR results.Moreover, the polyanionic nature of nuclei acids means that their solid forms are usually well hydrated and the local environments (which NMR probes) are, as a result, not too dissimilar from in vivo conditions.Solid-state NMR (SS-NMR) is now widely used as complement to classical diffraction methods of characterising solid materials, with Bragg diffraction providing the overall long-range structure and NMR providing information on aspects, such as hydrogen atom positioning and dynamics, that are difficult to characterise by diffraction.This eld is commonly termed "NMR crystallography", although it is important to note that NMR techniques are not limited to crystalline solids.Solid-state NMR has the great virtue that chemical shis can be determined for solid materials of known structure, permitting the correlation of the NMR parameters directly with structural features. 4,5Solid-state NMR is thus a Martin Dračínský obtained his PhD in organic chemistry from Charles University in Prague in 2006.He is currently working as a researcher at Czech Academy of Sciences.His research deals with experimental solution and solid-state NMR spectroscopy applied mostly to modied nucleic acid components, theoretical prediction of NMR parameters (including vibrational and solvent effects) and classical and DFT molecular dynamics simulations.
Paul Hodgkinson obtained his PhD in physical chemistry from Oxford University in 1995, and, following postdoc periods as UC Berkeley and the ENS de Lyon, has been working at Durham University's Department of Chemistry since 1998, where he is now a Reader in Magnetic Resonance.His research group works in both on the fundamental principles of solid-state NMR as well as applications to chemical problems, focussing on the use of quantum calculation to enable the "NMR crystallography" of molecular organic solids.
valuable tool for nucleoside and nucleotide structural studies since it provides the means to acquire spectra corresponding to single conformations, in contrast to solution NMR methods.
The fundamental concepts of solid-state NMR have been extensively described, [6][7][8] and only summarised very briey here.The various interactions of NMR are fundamentally anisotropic with respect to the strong magnetic eld applied during the NMR experiment.In the solution state, however, fast molecular tumbling means that only the isotropic average of the interactions is observed via the NMR spectrum.Interactions with no isotropic component, such as the direct dipolar interaction between nuclear magnetic moments, and the quadrupolar interaction (due to coupling between nuclear electric quadrupole moments and local electric eld gradients) have no effect on the spectrum, leaving just the isotropic components of the chemical shi and indirect (or J) coupling.This leads to sharp well-resolved solution-state spectra for spin-1/2 nuclei, such as 1 H and 13 C.This same molecular tumbling is an efficient driver of relaxation processes.This can be an asset, returning the spin states quickly to equilibrium and allowing the NMR experiment to be repeated quickly, but it can be problematic for nuclei with a signicant electric quadrupole moment; rapid quadrupolar relaxation will typically lead to broad and unresolved lines for potentially useful nuclei such as 23 Na.In contrast, the lack of extensive dynamics in the solid state means that NMR spectra of solid materials are strongly broadened by the anisotropic interactions.This broadening can be signicantly reduced by the use of "magic-angle spinning" (MAS), which averages out, at least to rst order, the anisotropic components of the NMR interactions.The extensive nature of dipolar coupling between 1 H nuclei means that 1 H NMR spectra of solids are still relatively poorly resolved, even at the fastest MAS spinning rates currently available (about 100 kHz), and so the initial nucleus of choice for organic systems is generally 13 C.In contrast, specialised NMR techniques, such as the Multiple Quantum MAS (MQMAS) experiment, oen allow better site resolution for nuclei such as 23 Na to be obtained in the solid compared to the solution state.
Similarly 2 H NMR is a powerful technique for studying dynamics since the deuterium quadrupolar coupling is both small enough to be readily measured and highly sensitive to local dynamic processes.Dynamics of nucleic acids has been studied by 2 H NMR for more than 30 years; early studies were reviewed in 1991. 9In contrast to other techniques (solution NMR, X-ray), solid-state NMR can probe motions with a broad range of time scales.Other specialised experiments, most notably REDOR 10 experiments as discussed below, have been developed that allow dipolar couplings between pairs of nuclear spins to be measured directly.These provide more robust estimates of internuclear distances than the indirect estimation of dipolar couplings from "nuclear Overhauser effects" in the solution state.
In addition to methodological advances, the development of "NMR crystallography" has been driven by the availability of DFT calculations that allow NMR properties to be calculated efficiently. 11,12Such quantum calculations provide a direct link between structure and NMR observables; rather than the NMR spectrum simply being used as a ngerprint of a particular solid-form, it is now possible to relate structural and NMR parameters in a quantitative fashion.Several modelling and simulation techniques have been proposed to describe the inuence of intermolecular interactions in the solid state on chemical shis.In the cluster model, neighbouring molecules or fragments are considered explicitly during the chemicalshielding calculations.However, modelling a solid as a 'large molecule' or a cluster has many difficulties.The choice of the cluster, in particular its termination, is critical, as the calculations must be maintained at a manageable size.It is thus more efficient to exploit the translational repetition in crystals.In the last decade, the gauge-including projector-augmented wave (GIPAW) procedure has been developed for the prediction of the magnetic-resonance parameters in fully periodic solids. 13The wide applications of GIPAW-based calculations to "NMR crystallography" are now well documented. 12,14Note also that nucleic acid components have oen been used as model compounds for the development and testing of computational methods.For example, the GIPAW approach has been tested against cluster calculations for solid isocytosine, where the cluster modelling was clearly found to be inferior. 15n this paper, we review recent applications (mostly aer 2000) of solid-state NMR and NMR crystallography in studies of the structure of nucleic acid components, their intra-and intermolecular interactions, and dynamics.

Structure of nucleic acids components
One of the major conformational variables in nucleic acids is the pucker of the (deoxy)ribose ring.High-resolution X-ray studies have shown that the most common ring conformations in DNA are 3 0 -endo and 2 0 -endo.The pucker is inextricably linked to the helix geometry.For instance, the conformation is C2 0 -endo and C3 0 -endo in the B-form and the A-form of DNA respectively.The conformations of individual monomers in nucleic acids are thus important for their biological function.
In the determination of nucleic acid structure by solutionstate NMR, the backbone has a relatively low density of constraints because of the difficulty of obtaining conformational parameters from J couplings or NOE information within crowded spectral regions, particularly for large nucleic acids.For example, determining the backbone torsion angle g(O5 0 -C5 0 -C4 0 -C3 0 ) from 3 J HH through the measurement of J H4 0 -H5 0 and J H4 0 -H5 00 is oen impractical because of the severe spectral overlapping of H5 0 and H5 00 resonances, the difficulty in their stereo assignment, as well as poor detection because of the proximity of the water peak. 16Fig. 1 shows the conventional nucleotide atom numbering and torsion angle denitions.
In the solid, all of the major forms of DNA are accessible, either as bres or as crystalline oligomers.In addition, crystalline nucleosides and nucleotides with a variety of ring puckers are known.Chemical shis for a series of solid nucleosides and nucleotides with different deoxyribose ring conformations have been measured and the 13 C chemical shis were found to be related in a direct way to the ring pucker; 3 0 -endo conformers have signicantly lower C3 0 and C5 0 chemical shis (5-10 ppm) relative to comparable 3 0 -exo and 2 0 -endo conformers. 4The latter two conformers were distinguished by smaller, but still signicant, differences in the carbon chemical shis at the C2 0 and C4 0 positions.The same trends have also been observed for chemical shis calculated by DFT methods for isolated nucleosides. 17CP-MAS NMR spectroscopy has also been used to investigate the dependence of 13 C chemical shis on specic conformational parameters of a variety of RNA nucleosides and nucleotides.It was shown that 13 C chemical shis can be used to determine sugar pucker and glycosidic (c) and exocyclic (g) angles in these systems with a high degree of certainty. 18imilarly, the dependence of 13 C chemical shis of the sugar ring on backbone torsion angle g(O5 0 -C5 0 -C4 0 -C3 0 ) and d(C5 0 -C4 0 -C3 0 -O3 0 ) as well as the sugar pucker, has been determined using crystalline nucleosides and nucleotides.The experimental data agreed well with DFT-calculated chemical shis, implying that 13 C chemical shis are a useful tool for the determination of nucleic acid structure.The chemical shis of C3 0 , C4 0 , and C5 0 may be used for a reliable determination of the backbone torsion angles and the sugar pucker in most cases. 16n unusual DNA structure in Pf1 bacteriophage has been characterised by solid-state NMR.On the basis of experimental chemical shis, obtained with dynamic-nuclear-polarisationenhanced spectroscopy, it was concluded that Pf1 DNA exhibits a 2 0 -endo conformation because of its high C3 0 and C5 0 chemical shis.The 13 C and 15 N chemical shis of the DNA bases fall outside their typical regions in DNA, pointing to an absence of Watson-Crick hydrogen bonding. 19For example, adenine C4 and C5, and thymine C2 and C5 had unusually high chemical shis, falling 1-2 ppm above the range of chemical shis observed in B-DNA and cytosine C4, and guanosine C4 and C5 had unusually low chemical shis.These observations were consistent with the absence of hydrogen bonding previously observed for thermal melting of DNA duplexes. 20he chemical shi of the 31 P nucleus in the backbone of nucleic acids is inuenced by the torsion angle z, which, at least in the B-type of nucleic acids, is either in the gauche region (approximately À60 , BI class) or in the trans region (approximately 180 , BII class).A phosphorus isotropic chemical shi difference of 1.8 ppm between the two classes has been extracted from 31 P CP-MAS spectra of model solid oligonucleotides, the BII phosphorus atom having higher chemical shi than the BI. 21This study used macroscopically oriented samples, with bres parallel to the rotor axis, allowing the orientation of the phosphate group with respect to the bre axis to be determined.Although the BI 4 BII conformational exchange is always fast in solution, a range of 31 P shis is observed in solution which is consistent with some systems existing predominantly in one conformation and others in a distribution, with the average 31 P shi being determined by the BI/BII ratio. 22,23hemical shi information can, however, be difficult to interpret due to non-local effects.Torsion angles may be more directly estimated by solid-state NMR using experiments that are sensitive to the relative orientations of nuclear spin interaction tensors.For example, experiments exploiting the evolution of a double quantum coherence under the heteronuclear local elds of neighbouring spins have been used to measure the d torsion angles of two 2 0 -deoxynucleosides doubly 13 C-labelled at the C3 0 and C4 0 positions. 24Similarly, the H1 0 -C1 0 -C6-H6 projection torsion angle dening the relative orientation of the nucleoside pyrimidine and ribose rings in uniformly labelled [ 13 C, 15 N]uridine has been estimated by selective excitation of 13 C double-quantum coherences under MAS at rotational resonance. 25otational echo double resonance (REDOR) is a solid-state NMR technique used to measure dipolar couplings and hence distances between pairs of different nuclear spins, which is frequently applied to biological structure problems.The distance range accessible by REDOR generally exceeds that of NOE or residual dipolar coupling measurements in solution.For example, the high magnetogyric ratio of 31 P and 19 F nuclei means that 31 P, 19 F dipolar couplings are relatively strong, and 31 P- 19 F distances of up to 16 Å have been measured. 26The high number of the phosphodiesters in the backbone of nucleic acids results in poorly resolved 31 P NMR spectra.To enable site-specic detection of 31 P- 19 F distances, a single phosphate group has been replaced by a phsphorothioate group, and uorinated nucleotides have been placed in specic positions of model oligonucleotides. 27 31P-19 F REDOR has also been used to monitor changes in minor groove width of a DNA oligomer upon binding of the drug distamycin A at different stoichiometries (Fig. 2). 28requency-selective 31 P- 13 C REDOR has been used to determine Pa-C8, Pb-C8, and Pg-C8 distances in ATP within the Na, K-ATPase enzyme.These distances were then used to propose the ATP conformation in the enzyme. 29These distances were compatible with a previous 13 C-detected proton spin diffusion experiment, which was used to detect contacts between ATP and the binding site of the enzyme.The P-C distances followed the order C2 > C8 > ribose, which is consistent with the adenine ring of ATP being in contact with the binding site and the ribose ring being relatively exposed. 30olid-phase synthesis has become the method of choice for producing oligonucleotides of dened sequence. 31P CP-MAS experiments have been used to monitor the solid-phase oligonucleotide-elongation reactions.The technique readily distinguishes the oxidation state of the phosphorus atom (phosphate/phosphite), the presence or absence of a protecting Fig. 1 The structure of a (2 0 -deoxy)ribonucleotide fragment with atom numbering and selected torsion angles.
group attached to phosphorus, or phosphate vs. phosphorothioate groups. 31Similarly, 31 P CP-MAS experiments have been used to study the complexation of 2 0 -deoxyadenosine-5 0 -phosphate (dAMP) with the surface of a mesoporous aluminium oxide.A single 31 P resonance was observed upon complexation.However, 27 Al MAS spectra show both tetrahedral and octahedral aluminium environments expected for the mesoporous alumina. 27Al- 31 P REDOR experiments revealed that the phosphate group of dAMP interacts predominantly with the octahedrally coordinated aluminium species at the surface.A comparison of experimental 31 P CSA tensor parameters (obtained by the analysis of spinning side-bands) with those calculated for model clusters indicated that the binding was via a monodentate complex. 32

Hydrogen bonding
The potential of nucleobases to form well-dened hydrogenbonded base pairs is not only a major determinant of nucleic acid structure, but is also fundamental to important biological processes, such as replication and transcription.In addition, hydrogen-bond interactions between nucleobases and amino acid side chains are believed to play a crucial role in the recognition of specic nucleotide sequences by DNA-binding proteins. 33,34Detailed characterization of hydrogen bond interactions between biomolecular building blocks has, therefore, been the subject of numerous experimental and theoretical studies.NMR spectroscopy is one of the most powerful tools to study the strength and geometry of hydrogen bonds, although the study of hydrogen-bond interactions of small molecules by NMR is oen hampered by the fast exchange of species in solution.Useful insight into the hydrogen bond strength and geometry is also obtained by comparing experimental NMR parameters with theoretical predictions. 35,36lid-state NMR has recently been used to identify ribbonlike and quartet-like self-assembly in guanosine derivatives by means of 1 H chemical shis and proton-proton proximities, as identied in 1 H double-quantum/single-quantum correlation experiments (which used combined rotation and multiple-pulse spectroscopy (CRAMPS) to improve 1 H spectral resolution).The NH proton chemical shi was observed to be higher (13-15 ppm) for ribbon-like self-assembly compared to 10-11 ppm for a quartet-like arrangement, corresponding to a change from NH/N to NH/O intermolecular hydrogen bonding. 37ydrogen-bond networks in organosilicas based on adenine and thymine have also been studied by 1 H solid-state NMR; spatial connectivities between protons were established using 1 H-1 H double quantum MAS experiments, allowing the geometries of hydrogen-bonded base pairs to be determined. 38olid isocytosine provides an unusual opportunity to study two different tautomers of isocytosine, as they crystallize in a 1 : 1 ratio in a manner similar to that of the guanine and cytosine pairs in DNA.A combination of X-ray with solid-state NMR spectroscopic data and GIPAW calculations enabled precise structural parameters to be obtained, such as the geometries of intermolecular hydrogen bonds between isocytosine molecules, and by analogy Watson-Crick nucleic acid G-C base pairs. 1 H chemical shis of free NH and NH involved in the intermolecular hydrogen bond differ by 3 ppm (Fig. 3).In solution, the tautomers of isocytosine are in a fast equilibrium, and only averaged NMR parameters can be obtained. 15eak hydrogen bonding C-H/O in solid uracil has been investigated in a study that related experimentally determined 1 H, 13 C, and 15 N chemical shis with rst-principles calculations.The effects of intermolecular interactions were quantied by comparing shis calculated for isolated molecules, molecular planes, and a full crystal.Isolated molecule to plane changes in the 1 H chemical shis of 2 ppm were determined for the CH protons involved in the weak hydrogen bonding; this compares to changes of ca. 5 ppm for the NH protons involved in conventional NH/O hydrogen bonding. 39Similarly, the effects of conventional and weak hydrogen bonds on the principal components of 1 H, 13 C, and 15 N chemical shi tensors and 14 N and 17 O electric eld gradients of uracil atoms have been Fig. 2 31 P- 19 F REDOR dephasing curves for a selectively fluoroand phosphorothioate-substituted DNA and its 1 : 1 and 2 : 1 distamycin complexes.Solid lines represent expected decay curves based on simulations.Diamonds mark data for the unbound DNA, triangles for the 1 : 1 distamycin : DNA complex and the square for the 2 : 1 distamycin : DNA complex. 28Copyright Oxford University Press.Reproduced with permission.determined experimentally (for 13 C and 15 N) and computationally. 40he very low natural abundance of 17 O limits oxygen NMR studies of nucleic acids components to 17 O-enriched samples.2][43] The 17 O NMR tensors were found to be highly sensitive to the nature of the intermolecular interactions in the solid state.The solidstate NMR determination of NMR interaction tensors of the carbonyl oxygen (O6) of guanine in two 17 O-enriched guanosine derivatives has been reported.The 17 O chemical-shi tensor and quadrupolar-coupling tensor were found to be very sensitive to the presence of hydrogen bonding and ion-carbonyl interactions, with the effect from ion-carbonyl interactions being several times stronger than that from hydrogen-bonding interactions. 44lthough J couplings are not normally resolved in solid-state NMR spectra (because the observed linewidth is usually larger than the magnitude of the coupling), spin-echo based experiments oen allow J couplings as small as 3.8 Hz to be measured. 45,46A powerful application of the spin-echo MAS technique is the quantication of hydrogen-bond mediated 2h J NN couplings, since it allows the identication of hydrogenbonded partners, as well as the quantication of hydrogenbond strengths and geometries. 47The detection of hydrogen bonds in the solid state via correlation peaks due to hydrogenbond-mediated J coupling in a 15 N refocused INADEQUATE spectrum has been reported for two guanosine derivatives.It was demonstrated that different N-H/N intermolecular hydrogen-bonding arrangements (quartet and ribbon) can be unambiguously identied in the spectra of the supramolecular guanosine structures. 48The intermolecular coupling constants in these structures have later been quantied by a 15 N MAS spinecho experiment. 46J-coupling-based experiments, such as INADEQUATE, provide direct information on bonding pathways (including through hydrogen bonds).However, experiments which use dipolar (i.e. through space) couplings, either directly between two 15 N nuclei 49 or indirectly via proton-driven spin diffusion, 50 can also be used to identify inter-residue N-H/N hydrogen bonding e.g. in RNA.
Solid-state NMR can distinguish between polymorphs and is particularly suited for characterising subtle differences in crystal packing.For example, ve polymorphs and one hydrate of 2-thiobarbituric acid have been characterised by 1D and 2D ( 1 H, 13 C, and 15 N) solid-state NMR spectroscopy.The polymorphs differ in the tautomeric form of the compound; an enol form, a keto form, or a 1 : 1 mixture of both are present in the crystals.The tautomeric form is easily recognised by 13 C CP-MAS spectroscopy, because the carbon chemical shi of C5 differs by ca.40 ppm (see Fig. 4). 51,52Complete assignments of 1 H and 13 C resonances were obtained by combining 1D and 2D (homo-and heteronuclear data). 1 H MAS NMR experiments provided information on hydrogen-bonded protons and their interaction strengths; the high 1 H chemical shi values (close to 15 ppm) in two polymorphs suggested the presence of strong interactions, which is consistent with short hydrogen bonds observed by X-ray crystallography. 51-crystals between a pharmaceutically active compound and a solid co-former are being widely investigated as an alternative to the use of drug salts for improving solid form properties (typically solubility). 13C NMR can straightforwardly verify the formation of a co-crystal, which will have an NMR spectrum which is distinct from that of the sum of the individual components.For example, 13 C CP-MAS has been used to conrm co-crystal formation between acyclovir (an acyclic nucleoside antiviral drug) with both glutaric and fumaric acids.The carbon chemical shis of acyclovir changed only slightly, which was rationalised in terms of the acyclovir molecule being involved in strong hydrogen bonding both in its pure and cocrystal forms.On the other hand, both glutaric and fumaric acid experience very different environments in the two cases, leading to changes in number of peaks due to changes of symmetry, and chemical shi changes of up to 5 ppm. 53

Dynamics
Nucleic acids are highly exible molecules that undergo functionally important structural transitions in response to external stimuli. 54Sequence-specic DNA exibility plays essential roles in a variety of cellular processes that are crucial for gene packaging, expression and regulation. 55,56For example, intrinsic sequence-specic DNA exibility is believed to play an important function in directing adaptive changes in DNA conformation that occur following protein and ligand recognition. 57,58It has also been proposed that a dynamic component or exibility of a lesion nucleotide plays a signicant role in the biomolecular recognition process of DNA lesions by repair enzymes. 59,60Similarly, many RNA functions are related to a multitude of functional dynamics. 61he dynamics of nucleic acids span a broad range of time scales from picoseconds, where fast vibrational and librational motions occur, up to seconds, where catalytic function and global refolding take place.X-ray crystallography and solution NMR have contributed high-resolution structures of nucleic acids, but neither technique is suitable for an investigation of dynamics over such a broad range.On the other hand, solidstate NMR can probe motions with correlation times ranging over several orders of magnitudes. 6,62A particular advantage of working in the solid state is that it is unnecessary to deconvolute the effects of overall molecular motions.Pre-1991 solidstate NMR studies of DNA structure and dynamics have been reviewed by Alam and Drobny. 9This review also describes dynamic and motional processes in DNA and basic principles of NMR determination of DNA dynamics.Solid-state NMR may also help the interpretation of solution-state relaxation times by providing experimental chemical shi anisotropies.For example, the 31 P chemical shi anisotropy of a 20mer RNA oligonucleotide under various salt and hydration conditions has been measured in order to interpret 31 P relaxation data in solution. 63The principal components of 13 C and 15 N chemical-shi tensors in solid 3-, 7-, and 9-benzyladenine isomers have been determined and the inuence of the substitution on the magnitude and orientation of the tensors has been discussed. 64everal isotopes can be utilised as probes for measuring dynamics in the solid state.One of the most useful isotopes is deuterium, because the solid-state NMR line shape and relaxation of deuterium spins are essentially dominated by a single mechanismthe interaction of the nuclear quadrupole moment with local electric eld gradients.Using systematic isotopic labelling schemes, the local dynamics of the base, sugar, and backbone moieties of individual nucleotides within a sequence can be investigated with deuterium solid-state NMR experiments.A combination of deuterium line shape and relaxation data probes a wide range of motions from nanosecond time-scale dynamics (probed by relaxation measurements) to micro/millisecond time scales (from line shape measurements). 65or example, the internal motions of the nucleoside 2 0 -deoxythymidine in the solid state have been investigated by deuterium SS-NMR.The base position was found to be essentially rigid, even at elevated temperatures.On the other hand, T 1 measurements on 2 0 ,2 00 -dideuterothymidine indicated the presence of two kinds of motion: (1) small-amplitude librations on the nanosecond time scale and (2) large amplitude jumping motions on the millisecond to microsecond time scale, which were hypothesised to be 2 0 -endo-3 0 -endo interconversion. 66imilarly two kinds of motion have been observed in a hydrated, non-crystalline sample of D-ribose selectively 2 H-labelled at the 2 0 position. 67ethylation of nucleotide bases is important for many biological processes.The HhaI system is a restriction-modication system consisting of a methyltransferase and endonuclease, which together act as a defense mechanism in prokaryotic systems, protecting the cell from invasive DNA.Deuterium SS-NMR has been used to understand and quantify the extent to which dynamics may assist proteins to recognise methylation sites distributed within DNA double helix, and to quantify the degree to which methylation perturbs the local dynamics of DNA (see Fig. 5 for an example of deuterium line shape analysis). 65,68The spectra obtained from DNAs selectively deuterated on the furanose ring within the GCGC moiety, recognised by the HhaI methyltransferase, indicated that all of these positions were structurally exible.The furanose ring within the deoxycytidine that is the methylation target displayed the largest amplitude motion and ca. 10 times higher jump rates obtained by tting the deuterium line shapes, whereas the furanose rings of nucleotides more remote from the methylation site had less mobile furanose rings.Furthermore, deuterium solid-state NMR revealed that methylation of the cytidine base reduces the amplitudes of motions of the phosphatesugar backbone 68,69 and changes the direction of the motions, 70 even though the crystal structures displayed only small perturbations from unmethylated DNA.The deuterium solid-state NMR data were later compared with 13 C solution relaxation measurements 71 and with variable temperature solution 31 P NMR 72 and it was concluded that the local internal motions of the studied DNA oligomer in solution and in the hydrated solid state were virtually the same.On the basis of these results, it was hypothesized that local DNA dynamics promotes methylation by lowering energetic barriers for the conformational changes required for HhaI binding.
In a similar study, solid-state 2 H line shape and inversion recovery data were collected for six DNA samples containing deuterons at the H2 00 positions of various residues of a DNA dodecamer.The DNA was hydrated to 11-13 waters per nucleotide by vapour diffusion in a humidity chamber containing saturated salts in 2 H-depleted water, to reach conditions where motions in the solid are very close to those observed in solution 73 and to establish that the line shapes do not differ simply as a result of differential hydration.Remarkable variations in line shape and longitudinal relaxation times (T 1Z ) were observed between residues framing the methylation site and their neighbours.The residues close to the methylation site had shorter T 1Z values of 20-30 ms and a noticeable modulation of the line shape, suggesting considerable motional averaging.Nearby residues were not nearly as exible, as demonstrated by the line shapes and signicantly larger T 1Z (59-82 ms).It was established that all H2 00 nuclei experienced small amplitude librations (10 ) of the C-D bond at frequencies faster than the quadrupolar interaction (174 kHz), and, in addition, H2 00 nuclei on the nucleotides close to the methylation site experienced large amplitude motions (36 ) at similar frequencies.These effects are specic for the methylation target DNA sequence as other DNAs revealed no signicant variation in T 1Z or line shapes between individual residues. 74n the other hand, no signicant differences in the local dynamics of the furanose ring within a uracil : adenine (U : A) base pair and a normal thymine : adenine (T : A) base pair have been revealed by deuterium solid-state NMR.The relaxation times were identical within the experimental error and the solid lineshapes were essentially indistinguishable.Therefore, U : A base pair furanose rings appeared to have identical dynamic properties as a normal T : A base pair, and the local dynamics of the furanose ring are unlikely to be the sole arbiter for uracil recognition and specicity in U : A base pairs. 75 solid-state deuterium NMR study of localised mobility at the C9pG10 step, the EcoRI restriction endonuclease target, in the DNA Dickerson dodecamer has been described both in crystalline and amorphous state.76,77 The furanose ring and helix backbone of dC9 display large amplitudes of motion on the 0.1 ms time scale, which contrasts with much smaller local dynamics in other nucleotides (dA5, dA6, dT7, and dT8) of the same dodecamer derived by earlier 2 H NMR studies.78,79 The large amplitude motions occur only close to the site where the EcoRI restriction endonuclease binds and cleaves.
NMR interactions can be signicantly inuenced by fast molecular motions, such as vibrations.A theoretical study that combined DFT molecular dynamics simulations of a set of amino acids and nucleic acid bases with calculations of NMR parameters revealed the impact of vibrational motions on isotropic chemical shis, chemical shi anisotropies (CSAs) and quadrupolar interactions.Re-orientation of the NMR tensors by molecular motion reduces the magnitudes of the NMR anisotropies, and inclusion of molecular dynamics signicantly improved the agreement between calculated quadrupolar couplings and experimental values. 80MR experiments together with molecular dynamics simulations and NMR calculations have been used to investigate mobility of water molecules and sodium ions in solid hydrates of two nucleotides.The structure of guanosine monophosphate system was found to be relatively rigid, with a well-ordered solvation shell of the nucleotide, while the water molecules in the uridine monophosphate system were shown to be remarkably mobile even at À80 C. The disorder of water molecules was observed in the 13 C, 31 P, and 23 Na solid-state NMR experiments as multiple signals for equivalent sites of the nucleotide corresponding to different local arrangements of the solvation shell.Deuterium NMR spectra of the samples recrystallized from D 2 O and molecular dynamics simulations also conrmed differences in water mobility between the two systems.The disordered solvation shell in UMP was proposed to be a good model for solvated nucleotides in general, with fast reorientation of water molecules and uctuations in the hydrogen-bond network.81

Interactions with metal ions
Because (oligo)nucleotides are polyanions, their structure and biological function depends strongly on their association with metal ions.Metal ions are involved in almost every aspect of nucleic acid chemistry, ranging from neutralization of the anionic nucleic acids 82 through specic stabilization of threedimensional structures of nucleic acid molecules, up to their effect as cofactors in RNA-mediated catalysis. 83However, the dynamic non-covalent nature of these interactions has hampered the development of accurate and quantitative descriptions. 84irect detection of light alkali metal ions by diffraction techniques is challenging, especially for sodium cations, because their X-ray scattering contributions are virtually identical to those of water, and Na + /O distances are only slightly shorter than strong hydrogen bonds between well-ordered water molecules. 85his oen renders it impossible to identify Na + ions, even with state-of-the-art diffraction techniques.
Most of the metals that bind to nucleic acids are diamagnetic and possess signicantly abundant isotopes that are NMRactive, making them potential targets for NMR.Unfortunately, the majority of these biologically signicant isotopes also involve half-integer quadrupolar nuclei that provide limited information by solution-state NMR experiments due to the efficient quadrupolar relaxation that signicantly broadens the NMR spectral lines.Moreover exchange of metals between bound sites and bulk solution is fast on the NMR timescale.These problems can be circumvented by carrying out the NMR experiments in the solid state, where the chemical exchange is stopped (or signicantly reduced) and relaxation broadening quenched by the absence of rapid reorientations. 86olid-state NMR has been frequently used for the characterisation of metal-ion interactions with nucleic acid components.For example, natural abundance 15 N solid-state NMR spectra of complexes formed between Na + , Ba 2+ , and Cd 2+ and guanosine-5 0 -monophosphate and inosine-5 0 -monophosphate demonstrated the great sensitivity of 15 N shieldings to metal ion coordination.It was also shown that changes in the 15 N chemical shi upon ion binding could be correlated with the strength and directionality of metal to nitrogen coordination. 87 23 a NMR has been applied in several studies of the sodium salts of nucleotides.Usually, 1D 23 Na MAS spectra do not exhibit resolved features from which information on the number of sodium sites and the associated NMR parameters can be readily extracted.In contrast, 2D 23 Na multiple-quantum MAS (MQMAS) spectra usually display clearly distinct spectral regions corresponding to distinct sodium sites in the crystal lattice (see Fig. 6).From individual spectral cross-sections, it is possible to obtain three 23 Na NMR parameters: the isotropic chemical shi d iso , quadrupolar coupling C Q and quadrupolar asymmetry h Q .In some cases, the assignment of the NMR parameters to individual sites has been made on the basis of a simple correlation between C Q and the local ion-binding geometry. 88,89A partial assignment of the four non-equivalent sodium sites of Na 2 ATP was accomplished by incorporating 31 P- 23 Na rotational echo double resonance (REDOR), variable temperature and relaxation methodologies onto the basic MQMAS high-resolution experiment. 90In the same paper, 23 Na spin-lattice relaxation times were also determined and related to local mobility around the individual sodium sites.Highresolution 1D and 2D 23 Na NMR spectra of deoxyguanosine-5 0monophosphate have also been obtained with the doublerotation (DOR) technique. 91 solid-state 23 Na NMR study of monovalent cation (Li + , Na + , K + , Rb + , Cs + , and NH 4 + ) binding to double-stranded calf thymus DNA at low relative humidity has been reported.Results from 23 Na-31 P REDOR experiment established that monovalent cations are directly bound to the phosphate groups of DNA and are partially dehydrated under these conditions.Quantitative thermodynamic parameters concerning the cation-binding affinity for the phosphate group were obtained by 23 Na NMR titration experiments.These binding affinities were shown to be signicantly different from those observed for the DNA in solution.92 Although magnesium is essential for the proper physiological folding of polynucleotides, direct NMR studies on this ion are complicated by its unfavourable nuclear properties (low natural abundance of 25 Mg, low magnetogyric ratio, large quadrupolar moment).One possible magnesium analogue is [Co(NH 3 ) 6 ] 3+ , which binds to nucleic acids and is of similar size and shape as hexaaquamagnesium.The 59 Co nuclide is a 100% naturally abundant isotope with relatively high magnetogyric ratio and moderate quadrupole moment. It ha been demonstrated that 59 Co MAS experiments on relatively small amount of tRNA can distinguish resonances corresponding to different metal binding environments.These characterisations were assisted by studies on model compounds and by 31 P- 59 Co recoupling experiments.86 G-quadruplexes are DNA and RNA structural motifs composed of stacked G-quartets in which four guanine residues form a planar arrangement (Fig. 7).Because of their relevance to biological processes, such as DNA replication and transcription, these uncanonical structures are considered to be novel therapeutic targets and have also been identied as promising building blocks for DNA-based nanomaterials and nanodevices.[93][94][95] Alkali metal ions such as Na + and K + are known to play important roles in the formation, stability, and structural polymorphism of G-quadruplexes.Solution NMR has been used for studying alkali metal ion binding to G-quadruplexes. Alhough it is generally difficult to obtain site-specic information, with exceptions where spin-1/2 nuclear probes of 205 Tl + and 15 NH 4 + were used (see, for example, ref. 96-98), it has been recently shown that insight into the binding of sodium and potassium ions can also be obtained by NMR in solution.99 Solid-state NMR has emerged as a method for directly detecting alkali metal ions in these and related systems.Recently, solidstate NMR techniques have been successfully developed for the determination of cation coordination within G-quartet.Wu's group has studied the solid-state 23 Na, 39 K, and 87 Rb NMR of guanosine complexes. Foexample, solid-state 23 Na NMR has been used to determine the mode of Na + binding to an Oxytricha nova telomeric DNA repeat.Using a 2D MQMAS 23 Na experiment, three sodium cations residing inside the quadruplex channel were observed.Each of these sodium cations was sandwiched between two G-quartets.100 The utility of 2D MQMAS 23 Na experiment in obtaining accurate site-specic information about ion binding in G-quadruplexes has also been conrmed in other studies.[101][102][103] The relative affinity of monovalent cations for a stacking G-quartet structure was studied by solid-state NMR.Two major types of cations were found to be bound to the structure: one at the surface and the other within the channel cavity between two G-quartets. Onthe basis of solid-state 23 Na results from a series of ion titration experiments, quantitative thermodynamic parameters concerning the relative cation binding affinity for each of the two major binding sites have been obtained.104 23 Na NMR and quantum chemical calculations have also been used to determine the coordination of the sodium ion in a calix [4]arene-guanosine conjugate dimer, which was shown to form a single G-quartet at the centre of the structure with pentacoordinated sodium ion.105 23 Na spin-echo experiments have been used to selectively suppress the phosphate-bound Na + ions in a solid G-quadruplex, because they have shorter decoherence times than the G-quartet-bound sodium atoms.99 The presence of K + ions in cells is believed to be crucial for the stability of telomeric G-quadruplex structures.The rather weak association between K + ions and biological structures together with the low gyromagnetic ratio of 39 K (spin 3/2) renders solution 39 K NMR spectroscopy of limited utility.However, solid-state NMR detection of K + ions bound to G-quadruplex structures has been shown to provide an unambiguous signature of potassium ions bound to G-quadruplex.106 It has been also proposed that 87 Rb can be used as a surrogate of potassium for detecting K + binding by solid-state NMR, because 87 Rb has a much higher NMR sensitivity than 39 K, but a similar radius.107

Conclusions
Despite their biological importance, many important issues related to the structure, dynamics and function of nucleic acids are not well understood.In this review, we have described recent applications of solid-state NMR and NMR crystallography to the study of nucleic acid components, focussing on applications where SS-NMR provides structural or dynamic information that is not accessible by other methods.Recent advances in experimental SS-NMR methods and DFT computations have opened new ways for studying nucleic acid systems.Limited motion in solids allows direct characterisation of individual conformations and intra-and intermolecular interactions.Furthermore, the local dynamics and interactions with the solvation shell and metal ions in solid hydrates are close to the hydration environment in solution, without the complication of overall molecular motion.Moreover, progress in the calculation of NMR parameters of solids enables NMR observables to be linked with structural models, greatly helping the interpretation of the experimental data.Solid-state NMR and NMR crystallography have thus become viable methods of determining the structure and local dynamics of nucleic acids and their components.

Fig. 3 1 H
Fig. 3 1 H NMR spectrum of solid isocytosine acquired at 65 kHz MAS.Two tautomers in 1 : 1 ratio are present in the solid form.The chemical shift of the hydrogen-bound NH is substantially different from that of the free NH.

Fig. 5
Fig. 5 Six experimental deuterium line shapes (black) for individual selectively labelled sites in a DNA dodecamer with the simulation (blue) of each overlaid.The differences between the line shapes are caused by different local dynamics of the individual sites.Reprinted with permission from (ref.65) Copyright 2008 American Chemical Society.

Fig. 7
Fig. 7 Diagram illustrating the cyclic hydrogen bonding in a G-quartet.The monovalent cation, which resides in or out of the guanine plane, is omitted.