Crystal structure of an adenine bulge in the RNA chain of a DNA.RNA hybrid, d(CTCCTCTTC).r(gaagagagag)

Crystal structure of a DNA.RNA hybrid, d(CTCCTCTTC).r(gaagagagag), with an adenine bulge in the polypurine RNA strand was determined at 2.3 A resolution. The structure was solved by the molecular replacement method and refined to a final R-factor of 19.9% (Rfree 22.2%). The hybrid duplex crystallized in the space group I222 with unit cell dimensions, a = 46.66 A, b = 47.61 A and c = 54.05 A, and adopts the A-form conformation. All RNA and DNA sugars are in the C3'-endo conformation, the glycosyl angles in anti conformation and the majority of the C4'-C5' torsion angles in g+ except two trans angles, in conformity with the C3'-endo rigid nucleotide hypothesis. The adenine bulge is looped out and it is also in the anti C3'-endo conformation. The bulge is involved in a base-triple (C.g)*a interaction with the end base-pair (C9.g10) in the minor groove of a symmetry-related molecule. The 2' hydroxyl group of g15 is hydrogen bonded to O2P and O5' of g17, skipping the bulged adenine a16 and stabilizing the sugar-phosphate backbone of the hybrid. The hydrogen bonding and the backbone conformation at the bulged adenine site is very similar to that found in the crystal structure of a protein-RNA complex.     

Bulged adenosine influence on the RNA duplex conformation in solution

The RNA single bulge motif is an unpaired residue within a strand of several complementary base pairs. To gain insight into structural changes induced by the presence of the adenosine bulge on RNA duplex, the solution structures of RNA duplex containing a single adenine bulge (5'-GCAGAAGAGCG-3'/5'-CGCUCUCUGC-3') and a reference duplex with all Watson-Crick base pairs (5'-GCAGAGAGCG-3'/5'-CGCUCUCUGC-3') have been determined by NMR spectroscopy. The reference duplex structure is a regular right-handed helix with all of the attributes of an A-type helix. In the bulged duplex, single adenine bulge stacks into the helix, and the bulge region forms a well-defined structure. Both structures were analyzed by the use of calculated helical parameters. Distortions induced by the accommodation of unpaired residue into the helical structure propagate over the entire structure and are manifested as the reduced base pairs inclination and x-displacement. Intrahelical position of bulged adenine A5 is stabilized by efficient stacking with 5'-neighboring residues G4.     

Hydration of [d(CGC)r(aaa)d(TTTGCG)](2)

We have studied the hydration and dynamics of RNA C2'-OH in a DNA. RNA hybrid chimeric duplex [d(CGC)r(aaa)d(TTTGCG)](2). Long-lived water molecules with correlation time tau(c) larger than 0.3 ns were found close to the RNA adenine H2 and H1' protons in the hybrid segment. A possible long-lived water molecule was also detected close to the methyl group of 7T in the RNA-DNA junction but not to the other two thymine bases (8T and 9T). This result correlates with the structural studies that only DNA residue 7T in the RNA-DNA junction adopts an O4'-endo sugar conformation (intermediate between B-form and A-form), while the other DNA residues including 3C in the DNA-RNA junction, adopt C1'-exo or C2'-endo conformations (in the B-form domain). Based on the NOE cross-peak patterns, we have found that RNA C2'-OH tends to orient toward the O3' direction, forming a possible hydrogen bond with the 3'-phosphate group. The exchange rates for RNA C2'-OH were found to be around 5-20 s(-1), compared to 26.7(+/-13.8) s(-1) reported previously for the other DNA.RNA hybrid duplex. This slow exchange rate may be due to the narrow minor groove width of [d(CGC)r(aaa)d(TTTGCG)](2), which may trap the water molecules and restrict the dynamic motion of hydroxyl protons. The distinct hydration patterns of the RNA adenine H2 and H1' protons and the DNA 7T methyl group in the hybrid segment, as well as the orientation and dynamics of the RNA C2'-OH protons, may provide a molecular basis for further understanding the structure and recognition of DNA.RNA hybrid and chimeric duplexes.     

Solution structure of a trinucleotide A-T-A bulge loop within a DNA duplex.

We have synthesized an oligodeoxynucleotide duplex, d(G-C-A-T-C-G-A-T-A-G-C-T-A-C-G).d(C-G-T-A-G-C-C-G-A-T-C-G), with a three-base bulge loop (A-T-A) at a central site in the first strand. Nuclear Overhauser experiments (NOESY) in H2O indicate that the GC base pairs flanking the bulge loop are intact between 0 and 25 degrees C. Nuclear Overhauser effects in both H2O and D2O indicate that all bases within the bulge loop are stacked into the helix. These unpaired bases retain an anti conformation about their glycosidic bonds as they stack within the duplex. The absence of normal sequential connectivities between the two cytosine residues flanking the bulge site on the opposite strand indicates a disruption in the geometry of this base step upon insertion of the bulged bases into the helix. This conformational perturbation is more akin to a shearing apart of the bases, which laterally separates the two halves of the molecule, rather than the "wedge" model often invoked for single-base bulges. Using molecular dynamics calculations, with both NOE-derived proton-proton distances and relaxation matrix-calculated NOESY cross peak volumes as restraints, we have determined the solution structure of an A-T-A bulge loop within a DNA duplex. The bulged bases are stacked among themselves and with the guanine bases on either side of the loop. All three of the bulged bases are displaced by 2-3 A into the major groove, increasing the solvent accessibility of these residues. The ATA-bulge duplex is significantly kinked at the site of the lesion, in agreement with previously reported electron microscopy and gel retardation studies on bulge-containing duplexes [Hsieh, C.-H., & Griffith, J. D. (1989) Proc. Natl. Acad. Sci. U.S.A 86, 4833-4837; Bhattacharyya, A., & Lilley, D. M. J. (1989) Nucleic Acids Res. 17, 6821-6840]. Bending occurs in a direction away from the bulge-containing strand, and we find a significant twist difference of 84 degrees between the two base pairs flanking the bulge loop site. This value represents 58% of the twist difference for base pairs four steps apart in B-DNA. These results suggest a structural mechanism for the bending of DNA induced by unpaired bases, as well as accounting for the effect bulge loops may have on the secondary and tertiary structures of nucleic acids.     

Crystal structure of a model branchpoint-U2 snRNA duplex containing bulged adenosines

Bulged nucleotides play a variety of important roles in RNA structure and function, frequently forming tertiary interactions and sometimes even participating in RNA catalysis. In pre-mRNA splicing, the U2 snRNA base pairs with the intron branchpoint sequence (BPS) to form a short RNA duplex that contains a bulged adenosine that ultimately serves as the nucleophile that attacks the 5' splice site. We have determined a 2.18-A resolution crystal structure of a self-complementary RNA designed to mimic the highly conserved yeast (Saccharomyces cerevisiae) branchpoint sequence (5'-UACUAACGUAGUA with the BPS italicized and the branchsite adenosine underlined) base paired with its complementary sequence from U2 snRNA. The structure shows a nearly ideal A-form helix from which two unpaired adenosines flip out. Although the adenosine adjacent to the branchsite adenosine is the one bulged out in the structure described here, either of these adenosines can serve as the nucleophile in mammalian but not in yeast pre-mRNA splicing. In addition, the packing of the bulged RNA helices within the crystal reveals a novel RNA tertiary interaction in which three RNA helices interact through bulged adenosines in the absence of any divalent metal ions.     

Unusual nucleotide conformations in GNRA and UNCG type tetraloop hairpins: evidence from Raman markers assignments.

High resolution NMR data on UNCG and GNRA tetraloops (where N is any of the four nucleotides and R is a purine) have shown that they contain ribonucleosides with unusual 2'-endo/anti and 3'-endo/syn conformations, in addition to the 3'-endo/anti ones which are regularly encountered in RNA chains. In the current study, Raman spectroscopy has been used to probe these nucleoside conformations and follow the order (hairpin) to disorder (random chain) structural transitions in aqueous phase in the 5-80 degreesC temperature range. Spectral evolution of GCAA and GAAA tetraloops, as formed in very short hairpins with only three G.C base pairs in their stems (T m >60 degreesC), are reported and compared with those previously published on UUCG and UACG tetraloops, for which the syn orientation of the terminal guanine as well as the 2'-endo/anti conformation of the third rC residue have been confirmed by means of vibrational marker bands. Raman data obtained as a function of temperature show that the first uracil in the UUCG tetraloop is stacked and the two middle residues (rU and rC) are in the 2'-endo/anti conformation, in agreement with the previously published NMR results. As far as the new data concerning the GNRA type tetraloops are concerned, they lead us to conclude that: (i) in both cases (GCAA and GAAA tetraloops) the adenine bases are stacked; (ii) the second rC residue in the GCAA tetraloop has a 3'-endo/anti conformation; (iii) the sugar pucker associated with the third rA residue in both tetraloops possibly undergoes a 3'-endo/2'-endo interconversion as predicted by NMR results; (iv) the stem adopts a regular A-form structure; (v) all other nucleosides of these two GNRA tetraloops possess the usual 3'-endo/anti conformation.     

High-resolution NMR studies of chimeric DNA-RNA-DNA duplexes, heteronomous base pairing, and continuous base stacking at junctions.

Two symmetrical DNA-RNA-DNA duplex chimeras, d(CGCG)r(AAUU)d(CGCG) (designated rAAUU) and d(CGCG)r(UAUA)d(CGCG) (designated rUAUA), and a nonsymmetrical chimeric duplex, d(CGTT)r(AUAA)d(TGCG)/d(CGCA)r(UUAU)d(A ACG) (designated rAUAA), as well as their pure DNA analogues, containing dU instead of T, have been synthesized by solid-phase phosphoramidite methods and studied by high-resolution NMR techniques. The 1D imino proton NOE spectra of these d-r-d chimeras indicate normal Watson-Crick hydrogen bonding and base stacking at the junction region. Preliminary qualitative NOESY, COSY, and chemical shift data suggest that the internal RNA segment contains C3'-endo (A-type) sugar conformations except for the first RNA residues (position 5 and 17) following the 3' end of the DNA block, which, unlike the other six ribonucleotides, exhibit detectable H1'-H2' J coupling. The nucleosides of the two flanking DNA segments appear to adopt a fairly normal C2'-endo B-DNA conformation except at the junction with the RNA blocks (residues 4 and 16), where the last DNA residue appears to adopt an intermediate sugar conformation. The DNA-RNA junction residues exhibit quite different COSY, chemical shift, and NOE behavior, but these effects do not appear to propagate into the DNA or RNA segments. The circular dichroism spectra of these d-r-d chimeras also display a mixture of characteristic A-type and B-type absorption bands. The data indicate that A-type and B-type conformations can coexist in a single short continuous nucleic acid duplex, but our results differ somewhat from previous theoretical model studies.     

Comparative NMR study of A(n)-bulge loops in DNA duplexes: intrahelical stacking of A, A-A, and A-A-A bulge loops.

We have prepared a series of deoxyoligonucleotide duplexes of the sequence d(G-C-A-T-C-G-X-G-C-T-A-C-G).d(C-G-T-A-G-C-C-G-A-T-G-C), in which X represents either one (A), two (A-A), or three (A-A-A) unpaired adenine basis. Using two-dimensional proton and phosphorus NMR spectroscopy, we have characterized conformational features of these bulge-loop duplexes in solution. We find that Watson-Crick hydrogen bonding is intact for all 12 base pairs, including the GC bases that flank the bulge loop. Observation of NOE connectivities in both H2O and D2O allows us to unambiguously localize all of the bulged adenine residues to intrahelical positions within the duplex. This is in contrast to an earlier model for multiple-base bulge loops in DNA [Bhattacharyya, A., & Lilley, D. M. J. (1989) Nucleic Acids Res. 17, 6821-6840], in which all but the most 5' bulged base are looped out into solution. We find that insertion of two or three bases into the duplex results in the disruption of specific sequential NOEs for the base step across from the bulge loop site on the opposite strand. This disruption is characterized by a partial shearing apart of these bases, such that certain sequential NOEs for this base step are preserved. We observe a downfield-shifted phosphorus resonance, which we assign in the A-A-A bulge duplex to the 3' side of the last bulged adenine residue. Proton and phosphorus chemical shift trends within the An-bulge duplex series indicate that there is an additive effect on the structural perturbations caused by additional unpaired bases within the bulge loop. This finding parallels previous observations [Bhattacharyya, A., & Lilley, D. M. J. (1989) Nucleic Acids Res. 17, 6821-6840; Hsieh, C.-H., & Griffith, J. D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4833-4837] on the magnitude of the induced bending of DNA duplexes by multiple-base bulge loops.     

Z-RNA: the solution NMR structure of r(CGCGCG).

Left-handed double-helical Z-RNA has been studied using the ribohexanucleotide pentaphosphate r(CpGpCpGpCpG). One-dimensional and two-dimensional proton nmr experiments were used to probe the structural details of the left-handed helix in concentrated sodium perchlorate solution. In 1M NaClO4 the RNA adopts the normal A-form double helix, and in 6M NaClO4 it is nearly all in the Z form. In 4M NaClO4 it exists as nearly equal parts of A form and Z form. Resonances corresponding to both A and Z form appear in the nmr spectrum, indicating that the duplex exchanges slowly between forms. Spin-spin coupling constants between protons in the ribose rings were used to determine the sugar-pucker conformations of the individual nucleotides. Quantitative nuclear Overhauser experiments were used to determine proton-proton distances within the nucleoside, and from these distances values for the glycosidic torsion angle were determined. The results show that the cytidines adopt C2'-endo sugar puckers (S type) with pseudo-rotation phase values (P) of approximately 165 degrees. The bases are in the anti conformation, with chi values of approximately -140 degrees. The internal guanosines adopt C3'-endo sugar puckers (N type) with P approximately 18 degrees, while the 3'-terminal guanosine ribose exists in an equilibrium between S- and N-type conformations. All three guanosine bases adopt the syn conformation, with chi approximately 70 degrees. The results indicate that the solution structure of Z-RNA is very similar to that of Z-DNA.     

X-ray structures of U2 snRNA-branchpoint duplexes containing conserved pseudouridines

A pseudouridine-modified region of the U2 small nuclear (sn)RNA anneals with the intronic branchpoint sequence and positions a bulged adenosine to serve as the nucleophile in the first chemical step of pre-mRNA splicing. We have determined three X-ray structures of RNA oligonucleotides containing the pseudouridylated U2 snRNA and the branchpoint consensus sequences. The expected adenosine branchpoint is extrahelical in a 1.65 A resolution structure containing the mammalian consensus sequence variant and in a 2.10 A resolution structure containing a shortened Saccharomyces cerevisiae consensus sequence. The adenosine adjacent to the expected branchpoint is extrahelical in a third structure, which contains the intact yeast consensus sequence at 1.57 A resolution. The hydration and base stacking interactions mediated by the U2 snRNA pseudouridines correlate with the identity of the unpaired adenosine. The expected adenosine bulge is associated with a well-stacked pseudouridine, which is linked via an ordered water molecule to a neighboring nucleotide. In contrast, the bulge of the adjacent adenosine shifts the base stacking and disrupts the water-mediated interactions of the pseudouridine. These structural differences may contribute to the ability of the pseudouridine modification to promote the bulged conformation of the branch site adenosine and to enhance catalysis by snRNAs. Furthermore, iodide binding sites are identified adjacent to the unconventional bulged adenosine, and the structure of the mammalian consensus sequence variant provides a high-resolution view of a hydrated magnesium ion bound in a similar manner to a divalent cation binding site of the group II intron.     

HU protein employs similar mechanisms of minor-groove recognition in binding to different B-DNA sites: demonstration by Raman spectroscopy

The sequence isomers d(CGCAAATTTGCG) and d(TCAAGGCCTTGA) form self-complementary duplexes that present distinct targets for binding of the homodimeric architectural protein HU of Bacillus stearothermophilus (HUBst). Raman spectroscopy shows that although each duplex structure is of the B-DNA type, there are subtle conformational dissimilarities between them, involving torsion angles of the phosphodiester backbone and the arrangements of stacked bases. Each DNA duplex forms a stable stoichiometric (1:1) complex with HUBst, in which the structure of the HUBst dimer is largely conserved. However, the Raman signature of each DNA duplex is perturbed significantly and similarly with HUBst binding, as reflected in marker bands assigned to localized vibrations of the phosphodiester moieties and base residues. The spectral perturbations identify a reorganization of the DNA backbone and partial unstacking of bases with HUBst binding, which is consistent with non-sequence-specific minor-groove recognition. Prominent among the HUBst-induced perturbations of B-DNA are a conversion of approximately one-third of the alpha/beta/gamma torsions from the canonical g(-)/t/g(+) conformation to an alternative conformation, an equivalent conversion of deoxyadenosyl moieties from the C2'-endo/anti to the C3'-endo/anti conformation, and appreciable unstacking of purines. The results imply that each solution complex is characterized by structural perturbations extending throughout the 12-bp sequence. Comparison with previously studied protein/DNA complexes suggests that binding of HUBst bends DNA by approximately 70 degrees.     

X-ray crystal structure of a dimethylene sulfone-bridged ribonucleotide dimer in a single-stranded state.

A crystal structure has been solved for an analog of the r(ApU) ribodinucleotide, r(Aso2U), where a bridging non-ionic dimethylene sulfone linker replaces the phosphodiester linking group found in natural RNA. Crystals of the single-stranded state of r(Aso2U) were obtained from water at 50 degrees C. In these crystals, one hydrogen bond is formed between bases from different strands and base stacking occurs in intermolecular 'homo-A' and 'homo-U' stacks. Similar to typical oligoribonucleotides, the ribose rings adopt N-type conformations and dihedral angles chi are in the anti range. The all-trans rotamer of the CH2-SO2-CH2-CH2 bridge was found, which leads to a large adenine-uracil distance. Qualitative analysis of a NOESY spectrum of the Aso2U part in r(Uso2Cso2Aso2U) dissolved in a dimethylsulfoxide-D2O mixture indicates that the conformation observed in the crystal is also populated in solution. Comparison with the structure of r(Gso2C), which has been crystallized in the Watson-Crick paired state, shows that a rotation around zeta by +112 degrees leads from the observed, single-stranded state to a conformation that is compatible with formation of a duplex. A concerted trans/gauche flip of alpha and gamma then yields the standard conformer of A-type RNA helices. From the observed structure of r(Gso2C) and other oligonucleotides it is anticipated that this flip will also revert the ribose pucker from C2'-exo to C3'-endo.     

Neomycin, spermine and hexaamminecobalt (III) share common structural motifs in converting B- to A-DNA.

The (dG)n.(dC)n-containing 34mer DNA duplex [d(A2G15C15T2)]2 can be effectively converted from the B-DNA to the A-DNA conformation by neomycin, spermine and Co(NH3)6(3+). Conversion is demonstrated by a characteristic red shift in the circular dichroism spectra and dramatic NMR spectral changes in chemical shifts. Additional support comes from the substantially stronger CH6/GH8-H3'NOE intensities of the ligand-DNA complexes than those from the native DNA duplex. Such changes are consistent with a deoxyribose pucker transition from the predominate C2'-endo (S-type) to the C3'-endo (N-type). The changes for all three ligand-DNA complexes are identical, suggesting that those three complex cations share common structural motifs for the B- to A-DNA conversion. The A-DNA structure of the 4:1 complex of Co(NH3)6(3+)/d(ACCCGCGGGT) has been analyzed by NOE-restrained refinement. The structural basis of the transition may be related to the closeness of the two negatively charged sugar-phosphate backbones along the major groove in A-DNA, which can be effectively neutralized by the multivalent positively charged amine functions of these ligands. In addition, ligands like spermine or Co(NH3)6(3+) can adhere to guanine bases in the deep major groove of the double helix, as is evident from the significant direct NOE cross-peaks from the protons of Co(NH3)6(3+) to GH8, GH1 (imino) and CH4 (amino) protons. Our results point to future directions in preparing more potent derivatives of Co(NH3)6(3+) for RNA binding or the induction of A-DNA.     

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.     

An oligodeoxyribonucleotide N3'--> P5' phosphoramidate duplex forms an A-type helix in solution.

The solution conformations of the dinucleotide d(TT) and the modified duplex d(CGCGAATTCGCG)2 with N3'--> P5' phosphoramidate internucleoside linkages have been studied using circular dichroism (CD) and NMR spectroscopy. The CD spectra indicate that the duplex conformation is similar to that of isosequential phosphodiester RNA, a A-type helix, and is different from that of DNA, a B-type helix, NMR studies of model dimers d(TpT) and N3'--> P5' phosphoramidate d(TnpT) show that the sugar ring conformation changes from predominantly C2'-endo to C3'-endo when the 3'-phosphoester is replaced by a phosphoramidate group. Two-dimensional NMR (NOESY, DQF-COSY and TOCSY spectra) studies of the duplex provide additional details about the A-type duplex conformation of the oligonucleotide phosphoramidate and confirm that all furanose rings of 3'-aminonucleotides adopt predominantly N-type sugar puckering.     

Structural properties of DNA:RNA duplexes containing 2'-O-methyl and 2'-S-methyl substitutions: a molecular dynamics investigation

The physical properties of a DNA:RNA hybrid sequence d(CCAACGTTGG)*(CCAACGUUGG) with modifications at the C2'-positions of the DNA strand by 2'-O-methyl (OMe) and 2'-S-methyl (SMe) groups are studied using computational techniques. Molecular dynamics simu-lations of SMe_DNA:RNA, OMe_DNA:RNA and standard DNA:RNA hybrids in explicit water indicate that the nature of the C2'-substituent has a significant influence on the macromolecular conformation. While the RNA strand in all duplexes maintains a strong preference for C3'-endo sugar puckering, the DNA strand shows considerable variation in this parameter depending on the nature of the C2'-substituent. In general, the preference for C3'-endo puckering follows the following trend: OMe_DNA>DNA>SMe_DNA. These results are further corroborated using ab initio methods. Both gas phase and implicit solvation calculations show the C2'-OMe group stabilizes the C3'-endo conformation while the less electronegative SMe group stabilizes the C2'-endo conformation when compared to the standard nucleoside. The macromolecular conformation of these nucleic acids also follows an analogous trend with the degree of A-form character decreasing as OMe_DNA:RNA>DNA:RNA>SMe_DNA:RNA. A structural analysis of these complexes is performed and compared with experimental melting point temper-atures to explain the structural basis to improved binding affinity across this series. Finally, a possible correlation between RNase H activity and conformational changes within the minor groove of these complexes is hypothesized.     

High-resolution NMR study of a synthetic DNA-RNA hybrid dodecamer containing the consensus pribnow promoter sequence: d(CGTTATAATGCG).r(CGCAUUAUAACG)

The nonsymmetrical double-helical hybrid dodecamer d(CGTTATAATGCG).r(CGCAUUAUAACG) was synthesized with solid-phase phosphoramidite methods and studied by high-resolution 2D NMR. The imino protons were assigned by one-dimensional nuclear Overhauser methods. All the base protons and H1', H2', H2", H3', and H4' sugar protons of the DNA strand and the base protons, H1', H2', and most of the H3'-H4' protons of the RNA strand were assigned by 2D NMR techniques. The well-resolved spectra allowed a qualitative analysis of relative proton-proton distances in both strands of the dodecamer. The chemical shifts of the hybrid duplex were compared to those of the pure DNA double helix with the same sequence (Wemmer et al., 1984). The intrastrand and cross-strand NOEs from adenine H2 to H1' resonances of neighboring base pairs exhibited characteristic patterns that were very useful for checking the spectral assignments, and their highly nonsymmetric nature reveals that the conformations of the two strands are quite different. Detailed analysis of the NOESY and COSY spectra, as well as the chemical shift data, indicate that the RNA strand assumes a normal A-type conformation (C3'-endo) whereas the DNA strand is in the general S domain but not exactly in the normal C2'-endo conformation. The overall structure of this RNA-DNA duplex is different from that reported for hybrid duplexes in solution by other groups (Reid et al., 1983a; Gupta et al., 1985) and is closer to the C3'-endo-C2'-endo hybrid found in poly(dA).poly(dT) and poly(rU).poly(dA) in the fiber state (Arnott et al., 1983, 1986).     

Crystal structure of an RNA duplex r(gugucgcac)(2) with uridine bulges.

The crystal structure of a nonamer RNA duplex with a uridine bulge in each strand, r(gugucgcac)(2), was determined at 1.4 A resolution. The structure was solved by multiple anomalous diffraction phasing method using a three-wavelength data set collected at the Advanced Protein Source and refined to a final R(work)/R(free) of 21.2 %/23.4 % with 33,271 independent reflections (Friedel pairs unmerged). The RNA duplex crystallized in the tetragonal space group P4(1)22 with two independent molecules in the asymmetric unit. The unit cell dimensions are a=b=47.18 A and c=80.04 A. The helical region of the nonamer adopts the A-form conformation. The uridine bulges assume similar conformations, with uracils flipping out and protruding into the minor groove. The presence of the bulge induces very large twist angles (approximately +50 degrees) between the base-pairs flanking the bulges while causing profound kinks in the helix axis at the bulges. This severe twist and the large kink in turn produces a very narrow major groove at the middle of the molecule. The ribose sugars of the guanosines before the bulges adopt the C2'-endo conformation while the rest, including the bulges, are in the C3'-endo conformation. The intrastrand phosphate-phosphate (P-P) distance of the phosphate groups flanking the bulges (approximately 4.4 A) are significantly shorter than the average P-P distance in the duplex (6.0 A). This short distance between the two phosphate groups brings the non-bridging oxygen atoms close to each other where a calcium ion is bound to each strand. The calcium ions in molecule 1 are well defined while the calcium ions in molecule 2 are disordered.