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

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

The flexibility of A-form DNA.

We have determined the rise per base pair and persistence length of A-form DNA in trifluoroethanol solutions for fragments 350-900 base pairs in length that best describe rotational diffusion coefficients determined by transient electric birefringence. The 2.6 A spacing between base pairs found in crystal and fiber A-form structures is preserved in solution. The persistence length is about 1500 A, or about three times longer than for B-form DNA. There is no apparent electrostatic contribution to the persistence length in the salt concentration range 0.2-2.0 mM Na cacodylate. This suggests an even closer association between DNA and its neutralizing counterions than predicted by condensation theory, perhaps due to a sheath of trifluoroethanol excluded water surrounding the A-form helix.     

Alternation of DNA and solvent layers in the A form of d(GGCGCC) obtained by ethanol crystallization

We have determined by X-ray crystallography the structure of the hexamer duplex d(GGCGCC)2 in the A-form using ethanol as a precipitant. The same sequence had previously been crystallized in the B-form, but with 2-methyl-2,4-pentanediol as a precipitant. It appears that ethanol precipitation is a useful method to induce the formation of A-form crystals of DNA. Packing of the molecules in the crystal has unique features: the known interaction of A-DNA duplexes between terminal base-pairs and the minor groove of neighbor molecules is combined with a superstructure consisting in an alternation of DNA layers and solvent layers (water/ions). This organization in layers has been observed before, also with hexamers in the A conformation which crystallize in the same space group (C2221). The solvent layer has a precise thickness, although very few ordered water molecules can be detected. Another feature of this crystal is its large unit cell, which gives rise to an asymmetric unit with three hexamer duplexes. One of the three duplexes is quite different from the other two in several aspects: the number of base pairs per turn, the twist pattern, the mean value of the twist angle and the fact that one terminal base-pair is not stacked as part of the duplex and appears to be disordered. So the variability in conformation of this sequence is remarkable.     

Alternation of DNA and Solvent Layers in the A Form of d(GGCGCC) Obtainhted by Ethanol Crystallization

Abstract

We have determined by X-ray crystallography the structure of the hexamer duplex d(GGCGCC)₂ in the A-form using ethanol as a precipitant. The same sequence had previously been crystallized in the B-form, but with 2-methyl-2, 4-pentanediol as a precipitant. It appears that ethanol precipitation is a useful method to induce the formation of A-form crystals of DNA. Packing of the molecules in the crystal has unique features: the known interaction of A-DNA duplexes between terminal base-pairs and the minor groove of neighbor molecules is combined with a superstructure consisting in an alternation of DNA layers and solvent layers (water/ions). This organization in layers has been observed before, also with hexamers in the A conformation which crystallize in the same space group (C222 ₁). The solvent layer has a precise thickness, although very few ordered water molecules can be detected. Another feature of this crystal is its large unit cell, which gives rise to an asymmetric unit with three hexamer duplexes. One of the three duplexes is quite different from the other two in several aspects: the number of base pairs per turn, the twist pattern, the mean value of the twist angle and the fact that one terminal base-pair is not stacked as part of the duplex and appears to be disordered. So the variability in conformation of this sequence is remarkable.     

SYNTHESIS AND HYBRIDIZATION PROPERTIES OF RNA CONTAINING 8-CHLOROADENOSINE

ABSTRACT

8-Chloroadenosine (8-Cl-Ado) has shown potential as a chemotherapeutic agent for the treatment of multiple myeloma and certain leukemias. 8-Cl-Ado treatment leads to a decrease in global RNA levels and incorporation of the analog into cellular RNA in malignant cells. To investigate the effects of 8-Cl-Ado modifications on RNA structure and function, an 8-Cl-Ado phosphoramidite and controlled-pore glass support were synthesized and used to introduce 8-Cl-Ado at internal and 3′- terminal positions, respectively. RNA oligonucleotides containing 8-chloroadenine (8-Cl-A) residues were synthesized and hybridized with complementary RNA strands. Circular dichroism spectroscopy of the resulting RNA duplexes revealed that the modified nucleobase does not perturb the overall A-form helix geometry. The thermal stabilities of 8-Cl-Ado modified duplexes were determined by UV thermal denaturation analysis and were compared with analogous natural duplexes containing standard and mismatched base pairs. The 8-Cl-Ado modification destabilizes RNA duplexes by ～5 kcal/mole, approximately as much as a U:U mismatched base pair. The duplex destabilization of 8-Cl-A may result from perturbation of Watson-Crick base pairing induced by conformational preferences of 8-halogenated nucleosides.     

The Flexibility of A-Form DNA

Abstract

We have determined the rise per base pair and persistence length of A-form DNA in trifluoroethanol solutions for fragments 350–900 base pairs in length that best describe rotational diffusion coefficients determined by transient electric birefringence. The 2.6 A spacing between base pairs found in crystal and fiber A-form structures is preserved in solution. The persistence length is about 1500 A, or about three times longer than for B-form DNA. There is no apparent electrostatic contribution to the persistence length in the salt concentration range 0.2–2.0 mM Na cacodylate. This suggests an even closer association between DNA and its neutralizing counterions than predicted by condensation theory, perhaps due to a sheath of trifluoroethanol excluded water surrounding the A-form helix.     

Synthesis and hybridization properties of RNA containing 8-chloroadenosine

8-Chloroadenosine (8-Cl-Ado) has shown potential as a chemotherapeutic agent for the treatment of multiple myeloma and certain leukemias. 8-Cl-Ado treatment leads to a decrease in global RNA levels and incorporation of the analog into cellular RNA in malignant cells. To investigate the effects of 8-Cl-Ado modifications on RNA structure and function, an 8-Cl-Ado phosphoramidite and controlled-pore glass support were synthesized and used to introduce 8-Cl-Ado at internal and 3'- terminal positions, respectively. RNA oligonucleotides containing 8-chloroadenine (8-Cl-A) residues were synthesized and hybridized with complementary RNA strands. Circular dichroism spectroscopy of the resulting RNA duplexes revealed that the modified nucleobase does not perturb the overall A-form helix geometry. The thermal stabilities of 8-Cl-Ado modified duplexes were determined by UV thermal denaturation analysis and were compared with analogous natural duplexes containing standard and mismatched base pairs. The 8-Cl-Ado modification destabilizes RNA duplexes by approximately 5 kcal/mole, approximately as much as a U:U mismatched base pair. The duplex destabilization of 8-Cl-A may result from perturbation of Watson-Crick base pairing induced by conformational preferences of 8-halogenated nucleosides.     

Structure of d(T)n.d(A)n.d(T)n: the DNA triple helix has B-form geometry with C2'-endo sugar pucker.

The polynucleotide helix d(T)n.d(A)n.d(T)n is the only deoxypolynucleotide triple helix for which a structure has been published, and it is generally assumed as the structural basis for studies of DNA triplexes. The helix has been assigned to an A-form conformation with C3'-endo sugar pucker by Arnott and Selsing [1974; cf. Arnott et al. (1976)]. We show here by infrared spectroscopy in D2O solution that the helix is instead B-form and that the sugar pucker is in the C2'-endo region. Distamycin A, which binds only to B-form and not to A-form helices, binds to the triple helix without displacement of the third strand, as demonstrated by CD spectroscopy and gel electrophoresis. Molecular modeling shows that a stereochemically satisfactory structure can be build using C2'-endo sugars and a displacement of the Watson-Crick base-pair center from the helix axis of 2.5 A. Helical constraints of rise per residue (h = 3.26 A) and residues per turn (n = 12) were taken from fiber diffraction experiments of Arnott and Selsing (1974). The conformational torsion angles are in the standard B-form range, and there are no short contacts. In contrast, we were unable to construct a stereochemically allowed model with A-form geometry and C3'-endo sugars. Arnott et al. (1976) observed that their model had short contacts (e.g., 2.3 A between the phosphate-dependent oxygen on the A strand and O2 in the Hoogsteen-paired thymine strand) which are generally known to be outside the allowed range.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nearest neighbor parameters for Watson-Crick complementary heteroduplexes formed between 2'-O-methyl RNA and RNA oligonucleotides

Results from optical melting studies of Watson–Crick complementary heteroduplexes formed between 2′-O-methyl RNA and RNA oligonucleotides are used to determine nearest neighbor thermodynamic parameters for predicting the stabilities of such duplexes. The results are consistent with the physical model assumed by the individual nearest neighbor-hydrogen bonding model, which contains terms for helix initiation, base pair stacking and base pair composition. The sequence dependence is similar to that for Watson–Crick complementary RNA/RNA duplexes, which suggests that the sequence dependence may also be similar to that for other backbones that favor A-form RNA conformations.     

Crystal structure analysis of an A-DNA fragment at 1.8 A resolution: d(GCCCGGGC).

Single crystals of the self-complementary octadeoxyribonucleotide d(GCCCGGGC) have been analysed by X-ray diffraction methods at a resolution of 1.8 A. The tetragonal unit cell of space group P4(3)2(1)2 has dimensions of a = 43.25 A and c = 24.61 A and contains eight strands of the oligonucleotide. The structure was refined by standard crystallographic techniques to an R factor of 17.1% using 1359 3 sigma structure factor observations. Two strands of the oligonucleotide are related by the crystallographic dyad axis to form a DNA helix in the A conformation. The d(GCCCGGGC) helix is characterized by a wide open major groove, a near perpendicular orientation of base pairs to the helix axis and an unusually small average helix twist angle of 31.3 degrees indicating a slightly underwound helix with 11.5 base pairs per turn. Extensive cross-strand stacking between guanine bases at the central cytosine-guanine step is made possible by a number of local conformational adjustments including a fully extended sugar-phosphate backbone of the central guanosine nucleotide.     

Kinetics for exchange of imino protons in the d(C-G-C-G-A-A-T-T-C-G-C-G) double helix and in two similar helices that contain a G . T base pair, d(C-G-T-G-A-A-T-T-C-G-C-G), and an extra adenine, d(C-G-C-A-G-A-A-T-T-C-G-C-G)

The relaxation lifetimes of imino protons from individual base pairs were measured in (I) a perfect helix, d(C-G-C-G-A-A-T-T-C-G-C-G), (II) this helix with a G . C base pair replaced with a G . T base pair, d(C-G-T-G-A-A-T-T-C-G-C-G), and (III) the perfect helix with an extra adenine base in a mismatch, d(C-G-C-A-G-A-A-T-T-C-G-C-G). The lifetimes were measured by saturation recovery proton nuclear magnetic resonance experiments performed on the imino protons of these duplexes. The measured lifetimes of the imino protons were shown to correspond to chemical exchange lifetimes at higher temperatures and spin-lattice relaxation times at lower temperatures. Comparison of the lifetimes in these duplexes showed that the destabilizing effect of the G . T base pair in II affected the opening rate of only the nearest-neighbor base pairs. For helix III, the extra adenine affected the opening rates of all the base pairs in the helix and thus was a larger perturbation for opening of the base pairs than the G . T base pair. The temperature dependence of the exchange rates of the imino proton in the perfect helix gives values of 14-15 kcal/mol for activation energies of A . T imino protons. These relaxation rates were shown to correspond to exchange involving individual base pair opening in this helix, which means that one base-paired imino proton can exchange independent of the others. For the other two helices that contain perturbations, much larger activation energies for exchange of the imino protons were found, indicating that a cooperative transition involving exchange of at least several base pairs was the exchange mechanism of the imino protons. The effects of a perturbation in a helix on the exchange rates and the mechanisms for exchange of imino protons from oligonucleotide helices are discussed.     

Crystal structure of the B-DNA hexamer d(CTCGAG): model for an A-to-B transition.

The crystal structure of the B-DNA hexamer d(CTCGAG) has been solved at 1.9 A resolution by iterative single isomorphous replacement, using the brominated derivative d(CG5BrCGAG), and refined to an R-factor of 18.6% for 120 nonhydrogen nucleic acid atoms and 32 water molecules. Although the central four base pairs form a typical B-form helix, several parameters suggest a transition to an A-like conformation at the termini. Based on this observation, a B-to-A transition was modeled, maintaining efficient base stacking across the junction. The wide minor groove (approximately 6.9 A) is reminiscent of that in the side-by-side double drug-DNA complexes and hosts a double spine of hydration. The global helix axes of the pseudo-continuous helices are at an acute angle of 60 degrees. The pseudocontinuous stacking is reinforced by the minor groove water structure extending between the two duplexes. The crossover point of two pairs of stacked duplexes is at the stacking junction, unlike that observed in the B-DNA decamers and dodecamers. This arrangement may have implications for the structure of a four-way DNA junction. The duplexes are arranged around a large (approximately 20 A diameter) channel centered on a 6(2) screw axis.     

Sequence and conformational effects on imino proton exchange in A.T- and A.U-containing DNA and RNA duplexes

¹H-nmr relaxation has been used to study the effect of sequence and conformation on imino proton exchange in adenine–thymine (A · T) and adenine–uracil (A · U) containing DNA and RNA duplexes. At low temperature, relaxation is caused by dipolar interactions between the imino and the adenine amino and AH2 protons, and at higher temperature, by exchange with the solvent protons. Although room temperature exchange rates vary between 3 and 12s^?1, the exchange activation energies (E_α) are insensitive to changes in the duplex sequence (alternating vs homopolymer duplexes), the conformation (B-form DNA vs A-form RNA), and the identity of the pyrimidine base (thymine vs uracil). The average value of the activation energy for the five duplexes studied, poly[d(A-T)], poly[d(A) · d(T)], poly[d(A-U)], Poly[d(A) · d(U)], and poly[r(A) · r(U)], was 16.8 ± 1.3 kcal/mol. In addition, we find that the average E_α for the A.T base pairs in a 43-base-pair restriction fragment is 16.4 ± 1.0 kcal/mol. This result is to be contrasted with the observation that the E_α of cytosine-containing duplexes depends on the sequence, conformation, and substituent groups on the purine and pyrimidine bases. Taken together, the data indicate that there is a common low-energy pathway for the escape of the thymine (uracil) imino protons from the double helix. The absolute values of the exchange rates in the simple sequence polymers are typically 3–10 times faster than in DNAs containing both A · T and G · C base pairs.     

Double helix conformation, groove dimensions and ligand binding potential of a G/C stretch in B-DNA.

The self-complementary DNA fragment CCGGCGCCGG crystallizes in the rhombohedral space group R3 with unit cell parameters a = 54.07 A and c = 44.59 A. The structure has been determined by X-ray diffraction methods at 2.2 A resolution and refined to an R value of 16.7%. In the crystal, the decamer forms B-DNA double helices with characteristic groove dimensions: compared with B-DNA of random sequence, the minor groove is wide and deep and the major groove is rather shallow. Local base pair geometries and stacking patterns are within the range commonly observed in B-DNA crystal structures. The duplex bears no resemblance to A-form DNA as might have been expected for a sequence with only GC base pairs. The shallow major groove permits an unusual crystal packing pattern with several direct intermolecular hydrogen bonds between phosphate oxygens and cytosine amino groups. In addition, decameric duplexes form quasi-infinite double helices in the crystal by end-to-end stacking. The groove geometries and accessibilities of this molecule as observed in the crystal may be important for the mode of binding of both proteins and drug molecules to G/C stretches in DNA.     

The crystal structure of an RNA oligomer incorporating tandem adenosine-inosine mismatches.

The X-ray crystallographic structure of the RNA duplex [r(CGCAIGCG)]2 has been refined to 2.5 A. It shows a symmetric internal loop of two non-Watson-Crick base pairs which form in the middle of the duplex. The tandem A-I/I-A pairs are related by a crystallographic two-fold axis. Both A(anti)-I(anti) mismatches are in a head-to-head conformation forming hydrogen bonds using the Watson-Crick positions. The octamer duplexes stack above one another in the cell forming a pseudo-infinite helix throughout the crystal. A hydrated calcium ion bridges between the 3'-terminal of one molecule and the backbone of another. The tandem A-I mismatches are incorporated with only minor distortion to the backbone. This is in contrast to the large helical perturbations often produced by sheared G-A pairs in RNA oligonucleotides.     

Polarized electronic spectra of Z-DNA single crystals

Polarized electronic absorption spectra of the (100) face of single crystals of the Z-form double helical duplex of d(m5CGUAm5CG) have been obtained from Kramers-Kronig analysis of reflection data. The c crystallographic axis is parallel to the helix axis and shows but weak absorption. The b axis is perpendicular to the helix axis and shows a structureless absorption band centered at 270 nm with an oscillator strength of 0.26. Calculations of the crystal spectra utilizing available transition moment data for the individual chromophores are carried through using the oriented gas model (no interbase interactions) and, again, employing all base-base interactions (point dipole) in the duplex. The calculated hypochromism of the 270 nm band is much less than the experimental value obtained from the crystal data. The crystal spectra appear to be representative of Z-form double helices of essentially infinite length and not of a collection of twelve base duplexes. No evidence for n pi* transitions polarized parallel to the helix axis is found.     

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

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

Base sequence effects in double-helical DNA. II. Configurational statistics of rodlike chains

Matrix generator techniques have been adapted to account for precise structural features of the nucleotide repeating unit and to translate the primary sequence of DNA base pairs into three-dimensional structures. Chains have been constructed to reflect the local sequence-dependent differences of bending and twisting of adjacent residues and various overall chain properties, including the average unperturbed moments of the end-to-end vector r and the mean angular orientation (〈γ〉 between base pair normals, 〈?₁〉 between long axes, and 〈?₂〉 between short axes) of terminal chain residues, have been computed. The chain backbone is treated implicitly in terms of the spatial fluctuations of successive base pairs. Motions are limited to low-energy perturbations of the standard B-DNA helix. Approximate potential energy schemes are used to represent the rules governing the patterns of local base–base morphology and flexibility. Theoretical predictions are compared with experimental observations at both the local and the macro-molecular level. Initial applications are limited to the rodlike poly(dA) · poly(dT) and poly(dG) · poly(dC) helices. The former duplex is found to be more compressed and the latter more extended than random-sequence DNA of the same chain length. The flexibility of the duplexes as a whole is described in terms of the average higher moments of the displacement vector ρ = r - 〈r〉 and the likelihood of chain cyclization is estimated from the three-dimensional Hermite series expansions of the displacement tensors. Emphasis is placed on theoretical methodology and the practical relevance of the calculated chain moments to observed physical properties.     

Crystal structures of A-DNA duplexes

All crystal structures of A-DNA duplexes exhibit a typical crystal packing, with the termini of one molecule abutting the shallow grooves of symmetry related neighbors, while all other forms (B, Z, and RNA) tend to form infinitely stacked helices. The A-DNA arrangement leads to the formation of shallow groove base multiples that have implications for the structure of DNA in compacted states. The characteristic packing leaves big solvent channels, which can be sometimes occupied by B-DNA duplexes. Comparisons of the structures of the same oligomer crystallizing in two different space groups and of different sequences crystallizing in the same space group show that the lattice forces dominate the A-DNA conformation in the crystals, complicating the effort to elucidate the influence of the base sequence on the structures. Nevertheless, in both alternating and nonalternating fragments some sequence effects can still be uncovered. Furthermore, several studies have started to define the minimal sequence changes or chemical modifications that can interconvert the oligomers between different double-helical conformers (A-, B-, and Z-form). Overall, it is seen that the rigid nucleotide principle applies to the oligomeric fragments. Besides the structures of the naked DNAs, their interactions with water, polyamines, and metal ions have attracted considerable attention. There are conserved patterns in the hydration, involving both the grooves and the backbone, which are different from those of B-DNA or Z-DNA. Overall, A-DNA seems to be more economically hydrated than B-DNA, particularly around the sugar-phosphate backbone. Spermine was found to be able to bind exclusively to either of the grooves or to the phosphate groups of the backbone, or exhibit a mixed binding mode. The located metal cations prefer binding to guanine bases and phosphate groups. The only mispairs investigated in A-DNA are the wobble pairs, yielding structural insight into their effects on helix stabilities and hydration. G · T wobble pairs have been determined in various sequence contexts, where they differentially affect the conformations and stableness of the duplexes. The structure of a G · ^m5C base pair, which surprisingly also adopted the wobble conformation, suggests that a similar geometry may transiently exist for G · C pairs. These results from the crystalline state will be compared to the solution state and discussed in relation to their relevance in biology. © 1997 John Wiley & Sons, Inc. Biopoly 44: 45–63, 1997     

Influence of a fluorobenzene nucleobase analogue on the conformational flexibility of RNA studied by molecular dynamics simulations

Chemically modified bases are frequently used to stabilize nucleic acids, to study the driving forces for nucleic acid structure formation and to tune DNA and RNA hybridization conditions. In particular, fluorobenzene and fluorobenzimidazole base analogues can act as universal bases able to pair with any natural base and to stabilize RNA duplex formation. Although these base analogues are compatible with an A-form RNA geometry, little is known about the influence on the fine structure and conformational dynamics of RNA. In the present study, nano-second molecular dynamics (MD) simulations have been performed to characterize the dynamics of RNA duplexes containing a central 1'-deoxy-1'-(2,4-difluorophenyl)-beta-D-ribofuranose base pair or opposite to an adenine base. For comparison, RNA with a central uridine:adenine pair and a 1'-deoxy-1'-(phenyl)-beta-D-ribofuranose opposite to an adenine was also investigated. The MD simulations indicate a stable overall A-form geometry for the RNAs with base analogues. However, the presence of the base analogues caused a locally enhanced mobility of the central bases inducing mainly base pair shear and opening motions. No stable 'base-paired' geometry was found for the base analogue pair or the base analogue:adenine pairs, which explains in part the universal base character of these analogues. Instead, the conformational fluctuations of the base analogues lead to an enhanced accessibility of the bases in the major and minor grooves of the helix compared with a regular base pair.