Crystal structure of an RNA octamer duplex r(CCCIUGGG)2 incorporating tandem I.U wobbles.

The crystal structure of the RNA octamer duplex r(CCCIUGGG)2has been elucidated at 2.5 A resolution. The crystals belong to the space group P21and have unit cell constants a = 33.44 A, b = 43.41 A, c = 49.39 A and beta = 104.7 degrees with three independent duplexes (duplexes 1-3) in the asymmetric unit. The structure was solved by the molecular replacement method and refined to an Rwork/Rfree of 0.185/0.243 using 3765 reflections between 8.0 and 2.5 A. This is the first report of an RNA crystal structure incorporating I.U wobbles and three molecules in the asymmetric unit. Duplex 1 displays a kink of 24 degrees between the mismatch sites, while duplexes 2 and 3 have two kinks each of 19 degrees and 27 degrees, and 24 degrees and 29 degrees, respectively, on either side of the tandem mismatches. At the I.U/U.I mismatch steps, duplex 1 has a twist angle of 33.9 degrees, close to the average for all base pair steps, but duplexes 2 and 3 are underwound, with twist angles of 24.4 degrees and 26.5 degrees, respectively. The tandem I.U wobbles show intrastrand purine-pyrimidine stacking but exhibit interstrand purine-purine stacking with the flanking C.G pairs. The three independent duplexes are stacked non-coaxially in a head-to-tail fashion to form infinite pseudo-continuous helical columns which form intercolumn hydrogen bonding interactions through the 2'-hydroxyl groups where the minor grooves come together.     

Crystal structure of an RNA duplex r(G GCGC CC)2 with non-adjacent G*U base pairs

The crystal structure of a self-complementary RNA duplex r(GGGCGCUCC)2with non-adjacent G*U and U*G wobble pairs separated by four Watson-Crick base pairs has been determined to 2.5 A resolution. Crystals belong to the space group R3; a = 33.09 A,alpha = 87.30 degrees with a pseudodyad related duplex in the asymmetric unit. The structure was refined to a final Rworkof 17.5% and Rfreeof 24.0%. The duplexes stack head-to-tail forming infinite columns with virtually no twist at the junction steps. The 3'-terminal cytosine nucleosides are disordered and there are no electron densities, but the 3' penultimate phosphates are observed. As expected, the wobble pairs are displaced with guanine towards the minor groove and uracil towards the major groove. The largest twist angles (37.70 and 40.57 degrees ) are at steps G1*C17/G2*U16 and U7*G11/C8*G10, while the smallest twist angles (28.24 and 27.27 degrees ) are at G2*U16/G3*C15 and C6*G12/U7*G11 and conform to the pseudo-dyad symmetry of the duplex. The molecule has two unequal kinks (17 and 11 degrees ) at the wobble sites and a third kink at the central G5 site which may be attributed to trans alpha (O5'-P), trans gamma (C4'-C5') backbone conformations. The 2'-hydroxyl groups in the minor groove form inter-column hydrogen bonding, either directly or through water molecules.     

Crystal structure of 2'-O-Me(CGCGCG)2, an RNA duplex at 1.30 A resolution. Hydration pattern of 2'-O-methylated RNA.

The molecular and crystal structure of 2'-O-Me (CGCGCG)2 has been determined using synchrotron radiation at near-atomic resolution (1.30 A), the highest resolution to date in the RNA field. The crystal structure is a half-turn A-type helix with some helical parameters deviating from canonical A-RNA, such as low base pair rise, elevated helical twist and inclination angles. In CG steps, inter-strand guanines are parallel while cytosines are not parallel. In steps GC this motif is reversed. This type of regularity is not seen in other RNA crystal structures. The structure includes 44 water molecules and two hydrated Mg2+ions one of which lies exactly on the crystallographic 2-fold axis. There are distinct patterns of hydration in the major and the minor grooves. The major groove is stabilised by water clusters consisting of fused five- and six-membered rings. Minor groove contains only a single row of water molecules; each water bridges either two self-parallel cytosines or two self-parallel guanines by a pair of hydrogen bonds. The structure provides the first view of the hydration scheme of 2'-O-methylated RNA duplex.     

Crystal structure of an RNA 16-mer duplex R(GCAGAGUUAAAUCUGC)2 with nonadjacent G(syn).A+(anti) mispairs

G.A mispairs are one of the most common noncanonical structural motifs of RNA. The 1.9 A resolution crystal structure of the RNA 16-mer r(GCAGAGUUAAAUCUGC)2 has been determined with two isolated or nonadjacent G.A mispairs. The molecule crystallizes with one duplex in the asymmetric unit in space group R3 and unit cell dimensions a = b = c = 49.24 A and alpha = beta = gamma = 51.2 degrees. It is the longest known oligonucleotide duplex at this resolution and isomorphous to the 16-mer duplex with the C.A+ mispairs [Pan, et al., (1998) J. Mol. Biol. 283, 977-984]. The C.A+ mispair behaves like a wobble pair while the G.A+ does not. The G.A mispairs are protonated at N1 of the adenines as in the C.A+ mispairs, and two hydrogen bonds in the G(syn).A+(anti) conformation are formed. The syn guanine is stabilized by an intranucleotide hydrogen bond between the 2-amino and the 5'-phosphate groups. The G(syn).A+(anti) conformation can provide a different surface for recognition in the grooves compared to other G.A hydrogen bonding schemes. The major groove is widened between the two mispairs allowing access to ligands. One of the 3-fold axes is occupied by a sodium ion and a water molecule, while a second is occupied by another water molecule.     

Hydration of the RNA duplex r(CGCAAAUUUGCG)2 determined by NMR.

The so-called spine of hydration in the minor groove of AnTn tracts in DNA is thought to stabilise the structure, and kinetically bound water detected in the minor groove of such DNA species by NMR has been attributed to a narrow minor groove [Liepinsh, E., Leupin, W. and Otting, G. (1994) Nucleic Acids Res. 22, 2249-2254]. We report here an NMR study of hydration of an RNA dodecamer which has a wide, shallow minor groove. Complete assignments of exchangeable protons, and a large number of non-exchangeable protons in r(CGCAAAUUUGCG)2 have been obtained. In addition, ribose C2'-OH resonances have been detected, which are probably involved in hydrogen bonds. Hydration at different sites in the dodecamer has been measured using ROESY and NOESY experiments at 11.75 and 14.1 T. Base protons in both the major and minor grooves are in contact with water, with effective correlation times for the interaction of approximately 0.5 ns, indicating weak hydration, in contrast to the hydration of adenine C2H in the homologous DNA sequence. NOEs to H1' in the minor groove are consistent with hydration water present that is not observed in the analogous DNA sequence. Hydration kinetics in nucleic acids may be determined by chemical factors such as hydrogen-bonding more than by simple conformational factors such as groove width.     

Crystal structure of a left-handed RNA tetramer, r(C-br8G)2

The crystal structure of the modified RNA tetramer, r(C-br8G-C-br8G), was determined by x-ray methods. The crystals are trigonal and belong to the space group P3212. There are three independent tetramers in the unit cell and each forms a left-handed duplex similar to Z-DNA regarding the orientations of the base moiety and the sugar puckerings in guanosine and cytidine. The effect of the additional bromine atom and 2'-hydroxy group on the stabilization of the Z-form structure are also described.     

The crystal structure of an alternating RNA heptamer r(GUAUACA) forming a six base-paired duplex with 3'-end adenine overhangs

The crystal structure of an alternating RNA heptamer r(GUAUACA) has been determined to 2.0 Å resolution and refined to an R_work of 17.1% and R_free of 18.5% using 2797 reflections. The heptamer crystallized in the space group C222 with a unit cell of a = 25.74, b = 106.58, c = 30.26 Å and two independent strands in the asymmetric unit. Each heptamer forms a duplex with its symmetry-related strand and each duplex contains six Watson–Crick base pairs and 3′-end adenosine overhangs. Therefore, two kinds of duplex (duplex 1 and duplex 2) are formed. Duplexes 1 stack on each other forming a pseudo-continuous column, which is typical of the RNA packing mode, while duplex 2 is typical of A-DNA packing with its termini in abutting interactions. Overhang adenine residues stack within the duplexes with C3′-endo sugar pucker and C2′-endo sugar pucker in duplexes 1 and 2, respectively. A Na⁺ ion in the crystal lattice is water bridged to two N1 atoms of symmetry-related A7 bases.     

The 1.19 A X-ray structure of 2'-O-Me(CGCGCG)(2) duplex shows dehydrated RNA with 2-methyl-2,4-pentanediol in the minor groove

The crystal and molecular structure of 2′-O-Me(CGCGCG)₂ has been determined at 1.19 Å resolution, at 100 K, using synchrotron radiation. The structure in space group P3₂12 is a half-turn right-handed helix that includes two 2-methyl-2,4-pentanediol (MPD) molecules bound in the minor groove. The structure deviates from A-form RNA. The duplex is overwound with an average value of 9.7 bp per turn, characterised as having a C3′-endo sugar pucker, very low base pair rise and high helical twist and inclination angles. The structure includes 65 ordered water molecules. Only a single row of water molecules is observed in the minor groove due to the presence of hydrophobic 2′-O-methyl groups. As many as five magnesium ions are located in the structure. Two are in the major groove and interact with O⁶ and N⁷ of guanosine and N⁴ of cytidine residues through their hydration spheres. This work provides the first example of molecular interactions of nucleic acids with MPD, which was used as a precipitant, cryo-solvent and resolution enhancing agent. The two MPD molecules intrude into the hydration network in the minor groove, each forming hydrogen bonds between their secondary hydroxyl group and exo-amino functions of guanosine residues. Comparison of the 2′-O-Me(CGCGCG)₂ structure in the P3₂12 and P6₁22 crystals delineates stability of the water network within the minor groove to dehydration by MPD and is of interest for evaluating factors governing small molecule binding to RNA. Intrusion of MPD into the minor groove of 2′-O-Me(CGCGCG)₂ is discussed with respect to RNA dehydration, a prerequisite of Z-RNA formation.     

Conformational properties and thermodynamics of the RNA duplex r(CGCAAAUUUGCG)2: comparison with the DNA analogue d(CGCAAATTTGCG)2.

The thermodynamic stability of nine dodecamers (four DNA and five RNA) of the same base composition has been compared by UV-melting. TheDeltaG of stabilisation were in the order: r(GACUGAUCAGUC)2>r(CGCAAATTTGCG)2 approximately r(CGCAUAUAUGCG)2>d(CGCAAATTTGCG)2 approximately r(CGCAAAUUUGCG)2>d(CGCATATATGCG)2 approximately d(GACTGATCAGTC)2>r(CGCUUUAAAGCG)2 approximately d(CGCTTTAAAGCG)2. Compared with the mixed sequences, both r(AAAUUU) and r(UUUAAA) are greatly destablising in RNA, whereas in DNA, d(TTTAAA) is destabilising but d(AAATTT) is stabilising, which has been attributed to the formation of a special B'structure involving large propeller twists of the A-T base pairs. The solution structure of the RNA dodecamer r(CGCAAAUUUGCG)2has been determined using NMR and restrained molecular dynamics calculations to assess the conformational reasons for its stability in comparison with d(CGCAAATTTGCG)2. The structures refined to a mean pairwise r.m.s.d. of 0.89+/-0.29 A. The nucleotide conformations are typical of the A family of structures. However, although the helix axis displacement is approximately 4.6 A into the major groove, the rise (3.0 A) and base inclination ( approximately 6 degrees ) are different from standard A form RNA. The extensive base-stacking found in the AAATTT tract of the DNA homologue that is largely responsible for the higher thermodynamic stability of the DNA duplex is reduced in the RNA structure, which may account for its low relative stability.     

Solution structure of the aminoacyl-capped oligodeoxyribonucleotide duplex (W-TGCGCAC)(2).

Reported here is the solution structure of the aminoacyl-DNA duplex (W-TGCGCAC)(2). This duplex forms a continuously pi-stacked helix consisting of both nucleobases and amino acid side chains. According to NMR and UV analyses, the duplex melts in a cooperative transition and with 1.3-1.8% greater hyperchromicity than the control duplex (TGCGCAC)(2). A van't Hoff analysis of UV melting points at different concentrations shows that the two tryptophan residues contribute 4.8 kcal/mol to the DeltaH degrees of complex formation at 10 mM salt concentration and less than 1 kcal/mol at 150 mM salt. The entropic cost for duplex association in the presence of the amino acid residues is 13 cal/molK greater than that for the control at 10 mM salt concentration, and 3 cal/molK lower than that of the control at 0.15 ionic strength. The conformation of W-TGCGCAC in duplex form, determined via restrained torsion angle molecular dynamics, shows an undisturbed B-form DNA duplex with dangling 3'-termini. The tryptophanyl residue at the 5'-terminus packs tightly against T2 and the proximal part of adenine, without engaging in hydrogen bonding. While not providing strong enthalpic net stabilization of the duplex, the tryptophan "cap" on the duplex does seem to reduce the fraying at the termini, indicating a subtle balance of entropic and enthalpic factors contributing to the molecular dynamics. The structure also shows that, at least in the present sequence context, stacking on the terminal base pair is more favorable than intercalation, probably because the enthalpic cost associated with breaking up the stacking between DNA base pairs cannot be paid for by favorable pi-stacking interactions with the indole ring of tryptophan. These results are of importance for understanding stacking interactions in protein-DNA complexes, particularly those in enzyme-substrate complexes involving exposed nucleobases.     

Crystal structure of an RNA duplex containing phenyl-ribonucleotides, hydrophobic isosteres of the natural pyrimidines

Chemically modified nucleotide analogs have gained widespread popularity for probing structure-function relationships. Among the modifications that were incorporated into RNAs for assessing the role of individual functional groups, the phenyl nucleotide has displayed surprising effects both in the contexts of the hammerhead ribozyme and pre-mRNA splicing. To examine the conformational properties of this hydrophobic base analog, we determined the crystal structure of an RNA double helix with incorporated phenyl ribonucleotides at 1.97 A resolution. In the structure, phenyl residues are engaged in self-pairing and their arrangements suggest energetically favorable stacking interactions with 3'-adjacent guanines. The presence of the phenyl rings in the center of the duplex results in only moderate changes of the helical geometry. This finding is in line with those of earlier experiments that showed the phenyl analog to be a remarkably good mimetic of natural base function. Because the stacking interactions displayed by phenyl residues appear to be similar to those for natural bases, reduced conformational restriction due to the lack of hydrogen bonds with phenyl as well as alterations in its solvent structure may be the main causes of the activity changes with phenyl-modified RNAs.     

Crystal structure of d(GCGAAAGCT) containing a parallel-stranded duplex with homo base pairs and an anti-parallel duplex with Watson-Crick base pairs

A DNA fragment d(GCGAAAGCT), known to adopt a stable mini-hairpin structure in solution, has been crystallized in the space group I4₁22 with the unit-cell dimensions a = b = 53.4 Å and c = 54.0 Å, and the crystal structure has been determined at 2.5 Å resolution. The four nucleotide residues CGAA of the first half of the oligomer form a parallel duplex with another half through the homo base pairs, C₂:C₂⁺ (singly-protonated between the Watson– Crick sites), G₃:G₃ (between the minor groove sites), A₄:A₄ (between the major groove sites) and A₅:A₅ (between the Watson–Crick sites). The two strands remaining in the half of the parallel duplex are split away in different directions, and they pair in an anti-parallel B-form duplex with the second half extending from a neighboring parallel duplex, so that an infinite column is formed in a head-to-tail fashion along the c-axis. It seems that a hexa-ammine cobalt cation supports such a branched and bent conformation of the oligomer. One end of the parallel duplex is stacked on the corresponding end of the adjacent parallel duplex; between them, the guanine base of the first residue is stacked on the fourth ribose of another duplex.     

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.     

Crystal structures of two forms of a 14-mer RNA/DNA chimer duplex with double UU bulges: a novel intramolecular U*(A x U) base triple.

The RNA/DNA 14-mer, (gguauuucgguaCc)2 with consecutive uridine bulges (underlined) on each strand has been determined in two crystal forms, spermine bound (Sp-form) and spermine free (Sp-free). The former was solved by the MAD method with three-wavelength data collected at Brookhaven National Laboratory (BNL); the later isomorphous structure was solved by the molecular replacement method using data collected on our Raxis IIc imaging plate system. The two crystal forms belong to the space group C2 with one molecule of double-stranded 14 mer in the asymmetric unit. The Sp-form has cell constants, a = 60.06, b = 29.10, c = 52.57 A, beta = 120.79 degrees and was refined to 1.7 A resolution with a final Rwork/Rfree of 19.8%/22.7% using 8,549 independent reflections. The Sp-free structure has cell constants, a = 60.06, b = 29.58, c = 52.50 A, beta = 120.85 degrees and was refined to 1.8 A with a final Rwork/ Rfree of 20.8%/23.2% using 6,285 unique reflections. The two structures are identical, except that the Sp-form has a spermine bound in the major groove, parallel to the RNA helical axis. One of the uridine bulges forms a novel intramolecular U*(A x U) base triple. The helices are in the C3'-endo conformation (A-form), but the bulges adopt the C2'-endo sugar pucker. Furthermore, the bulges induce a kink (30 degrees) in the helix axis and a very large twist (55 degrees) between the base pairs flanking the bulges. The Sp-form has one Mg2+ ion whereas the Sp-free form has two Mg2+ ions.     

Crystal structure of an RNA helix recognized by a zinc-finger protein: an 18-bp duplex at 1.6 A resolution

The crystal structure of the 19-mer RNA, 5'-GAAUGCCUGCGAGCAUCCC-3' has been determined from X-ray diffraction data to 1.6 A resolution by the multiwavelength anomalous diffraction method from crystals containing a brominated uridine. In the crystal, this RNA forms an 18-mer self-complementary double helix with the 19th nucleotide flipped out of the helix. This helix contains most of the target stem recognized by the bacteriophage Mu Com protein (control of mom), which activates translation of an unusual DNA modification enzyme, Mom. The 19-mer duplex, which contains one A.C mismatch and one A.C/G.U tandem wobble pair, was shown to bind to the Com protein by native gel electrophoresis shift assay. Comparison of the geometries and base stacking properties between Watson-Crick base pairs and the mismatches in the crystal structure suggest that both hydrogen bonding and base stacking are important for stabilizing these mismatched base pairs, and that the unusual geometry adopted by the A.C mismatch may reveal a unique structural motif required for the function of Com.     

Effects of N2,N2-dimethylguanosine on RNA structure and stability: crystal structure of an RNA duplex with tandem m2 2G:A pairs

Methylation of the exocyclic amino group of guanine is a relatively common modification in rRNA and tRNA. Single methylation (N²-methylguanosine, m²G) is the second most frequently encountered nucleoside analog in Escherichia coli rRNAs. The most prominent case of dual methylation (N²,N²-dimethylguanosine, m²₂G) is found in the majority of eukaryotic tRNAs at base pair m²₂G26:A44. The latter modification eliminates the ability of the N² function to donate in hydrogen bonds and alters its pairing behavior, notably vis-à-vis C. Perhaps a less obvious consequence of the N²,N²-dimethyl modification is its role in controlling the pairing modes between G and A. We have determined the crystal structure of a 13-mer RNA duplex with central tandem m²₂G:A pairs. In the structure both pairs adopt an imino-hydrogen bonded, pseudo-Watson–Crick conformation. Thus, the sheared conformation frequently seen in tandem G:A pairs is avoided due to a potential steric clash between an N²-methyl group and the major groove edge of A. Additionally, for a series of G:A containing self-complementary RNAs we investigated how methylation affects competitive hairpin versus duplex formation based on UV melting profile analysis.     

NMR structure of an antisense DNA.RNA hybrid duplex containing a 3'-CH(2)N(CH(3))-O-5' or an MMI backbone linker.

The solution structure of an antisense DNA.RNA hybrid duplex, d(CGCGTT-MMI-TTGCGC).r(GCGCAAAACGCG) (designated R4), containing an MMI backbone linker [3'-CH(2)N(CH(3))-O5'], is elucidated. The structural details of the MMI linker, its structural effects on the neighboring residues, and the molecular basis of the MMI effects are examined. The lipophilic N-methyl group of MMI is peripheral to the helix, assuming a conformation that is most stable with regard to the N-O torsion angle. The MMI linker promotes a 3'-endo conformation for the sugar moieties at both 3'- and 5'-adjacent positions and a backbone kink involving distant residues along the 3'-direction. Comparison of R4 with other analogous hybrid duplexes previously studied in this laboratory reveals a new family of low-energy helical conformations that can be accommodated in stable duplexes and a common feature of C3'-modified sugars for adopting a C3'-endo pucker. The results of these studies emphasize the interplay of several factors that govern the formation of stable hybrid duplexes and provide a basis for the understanding of the biological role of the MMI modifications, which are important building blocks for a family of promising chimeric antisense oligonucleotides.