Theoretical study of the geometrical arrangement of GT and GA wobble pairs in two short duplexes, Proton NMR chemical shifts and interaction energy calculations

NMR shielding constants are calculated for the base protons of duplexes formed by the dodecamer d(CGTGAATTCGCG) and the decamer d(CCAAGATTGG). A good agreement with experimental data is obtained for B-DNA helices in which the wobble GT and GA pairs are in the plane of the corresponding GC pairs of the parent duplexes formed by d(CGCGAATTCGCG) and d(CCAAGCTTGG), if the glycosyl bonds of T and G or A and G are symmetrical with respect to the dyad axis of the Watson-Crick GC pair. Interaction energy calculations show that this type of geometrical arrangement, which implies a distortion of the ribonphosphate backbone of both strands of the duplexes are more stable than those in which only one strand has its conformation modified by the presence of the wobble pair. For the duplex containing the GA pair, NMR chemical shifts as well as interaction energy computations favour the Watson-Crick hydrogen bonding scheme. The variation of the different contributions (intrastrand, interstrand, pair-pair) to the interaction energy between the bases of the duplexes, with the geometrical arrangement of the wobble pairs, is reported.     

Hidden in plain sight: subtle effects of the 8-oxoguanine lesion on the structure, dynamics, and thermodynamics of a 15-base pair oligodeoxynucleotide duplex

The base lesion 8-oxoguanine is formed readily by oxidation of DNA, potentially leading to G → T transversion mutations. Despite the apparent similarity of 8-oxoguanine-cytosine base pairs to normal guanine-cytosine base pairs, cellular base excision repair systems effectively recognize the lesion base. Here we apply several techniques to examine a single 8-oxoguanine lesion at the center of a nonpalindromic 15-mer duplex oligonucleotide in an effort to determine what, if anything, distinguishes an 8-oxoguanine-cytosine (8oxoG-C) base pair from a normal base pair. The lesion duplex is globally almost indistinguishable from the unmodified parent duplex using circular dichroism spectroscopy and ultraviolet melting thermodynamics. The DNA mismatch-detecting photocleavage agent Rh(bpy)(2)chrysi(3+) cleaves only weakly and nonspecifically, revealing that the 8oxoG-C pair is locally stable at the level of the individual base pairs. Nuclear magnetic resonance spectra are also consistent with a well-conserved B-form duplex structure. In the two-dimensional nuclear Overhauser effect spectra, base-sugar and imino-imino cross-peaks are strikingly similar between parent and lesion duplexes. Changes in chemical shift due to the 8oxoG lesion are localized to its complementary cytosine and to the 2-3 bp immediately flanking the lesion on the lesion strand. Residues further removed from the lesion are shown to be unperturbed by its presence. Notably, imino exchange experiments indicate that the 8-oxoguanine-cytosine pair is strong and stable, with an apparent equilibrium constant for opening equal to that of other internal guanine-cytosine base pairs, on the order of 10(-6). This collection of experiments shows that the 8-oxoguanine-cytosine base pair is incredibly stable and similar to the native pair.     

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.     

Thermodynamics of RNA duplexes with tandem mismatches containing a uracil-uracil pair flanked by C.G/G.C or G.C/A.U closing base pairs

The thermodynamics governing the denaturation of RNA duplexes containing 8 bp and a central tandem mismatch or 10 bp were evaluated using UV absorbance melting curves. Each of the eight tandem mismatches that were examined had one U-U pair adjacent to another noncanonical base pair. They were examined in two different RNA duplex environments, one with the tandem mismatch closed by G.C base pairs and the other with G.C and A.U closing base pairs. The free energy increments (Delta Gdegrees(loop)) of the 2 x 2 loops were positive, and showed relatively small differences between the two closing base pair environments. Assuming temperature-independent enthalpy changes for the transitions, (Delta Gdegrees(loop)) for the 2 x 2 loops varied from 0.9 to 1.9 kcal/mol in 1 M Na(+) at 37 degrees C. Most values were within 0.8 kcal/mol of previously estimated values; however, a few sequences differed by 1.2-2.0 kcal/mol. Single strands employed to form the RNA duplexes exhibited small noncooperative absorbance increases with temperature or transitions indicative of partial self-complementary duplexes. One strand formed a partial self-complementary duplex that was more stable than the tandem mismatch duplexes it formed. Transitions of the RNA duplexes were analyzed using equations that included the coupled equilibrium of self-complementary duplex and non-self-complementary duplex denaturation. The average heat capacity change (DeltaC(p)) associated with the transitions of two RNA duplexes was estimated by plotting DeltaH degrees and DeltaS degrees evaluated at different strand concentrations as a function of T(m) and ln T(m), respectively. The average DeltaC(p) was 70 +/- 5 cal K(-)(1) (mol of base pairs)(-)(1). Consideration of this heat capacity change reduced the free energy of formation at 37 degrees C of the 10 bp control RNA duplexes by 0.3-0.6 kcal/mol, which may increase Delta Gdegrees(loop) values by similar amounts.     

Thermodynamic analysis of 5' and 3' single- and 3' double-nucleotide overhangs neighboring wobble terminal base pairs

Thermodynamic parameters are reported for duplex formation of 40 self-complementary RNA duplexes containing wobble terminal base pairs with all possible 3′ single and double-nucleotide overhangs, mimicking the structures of short interfering RNAs (siRNA) and microRNAs (miRNA). Based on nearest neighbor analysis, the addition of a single 3′ dangling nucleotide increases the stability of duplex formation up to 1 kcal/mol in a sequence-dependent manner. The addition of a second dangling nucleotide increases the stability of duplexes closed with wobble base pairs in an idiosyncratic manner. The results allow for the development of a nearest neighbor model, which improves the predication of free energy and melting temperature for duplexes closed by wobble base pairs with 3′ single or double-nucleotide overhangs. Phylogenetic analysis of naturally occurring miRNAs was performed. Selection of the effector miR strand of the mature miRNA duplex appears to be dependent on the orientation of the GU closing base pair rather than the identity of the 3′ double-nucleotide overhang. Thermodynamic parameters for the 5′ single terminal overhangs adjacent to wobble closing base pairs are also presented.     

A nested double pseudoknot is required for self-cleavage activity of both the genomic and antigenomic hepatitis delta virus ribozymes.

The crystal structure of a genomic hepatitis delta virus (HDV) ribozyme 3' cleavage product predicts the existence of a 2 bp duplex, P1.1, that had not been previously identified in the HDV ribozymes. P1.1 consists of two canonical C-G base pairs stacked beneath the G.U wobble pair at the cleavage site and would appear to pull together critical structural elements of the ribozyme. P1.1 is the second stem of a second pseudoknot in the ribozyme, making the overall fold of the ribozyme a nested double pseudoknot. Sequence comparison suggests the potential for P1.1 and a similar fold in the antigenomic ribozyme. In this study, the base pairing requirements of P1.1 for cleavage activity were tested in both the genomic and antigenomic HDV ribozymes by mutagenesis. In both sequences, cleavage activity was severely reduced when mismatches were introduced into P1.1, but restored when alternative base pairing combinations were incorporated. Thus, P1.1 is an essential structural element required for cleavage of both the genomic and antigenomic HDV ribozymes and the model for the antigenomic ribozyme secondary structure should also be modified to include P1.1.     

Probing the role of a secondary structure element at the 5'- and 3'-splice sites in group I intron self-splicing: the tetrahymena L-16 ScaI ribozyme reveals a new role of the G.U pair in self-splicing

Several ribozyme constructs have been used to dissect aspects of the group I self-splicing reaction. The Tetrahymena L-21 ScaI ribozyme, the best studied of these intron analogues, catalyzes a reaction analogous to the first step of self-splicing, in which a 5'-splice site analogue (S) and guanosine (G) are converted into a 5'-exon analogue (P) and GA. This ribozyme preserves the active site but lacks a short 5'-terminal segment (called the IGS extension herein) that forms dynamic helices, called the P1 extension and P10 helix. The P1 extension forms at the 5'-splice site in the first step of self-splicing, and P10 forms at the 3'-splice site in the second step of self-splicing. To dissect the contributions from the IGS extension and the helices it forms, we have investigated the effects of each of these elements at each reaction step. These experiments were performed with the L-16 ScaI ribozyme, which retains the IGS extension, and with 5'- and 3'-splice site analogues that differ in their ability to form the helices. The presence of the IGS extension strengthens binding of P by 40-fold, even when no new base pairs are formed. This large effect was especially surprising, as binding of S is essentially unaffected for S analogues that do not form additional base pairs with the IGS extension. Analysis of a U.U pair immediately 3' to the cleavage site suggests that a previously identified deleterious effect from a dangling U residue on the L-21 ScaI ribozyme arises from a fortuitous active site interaction and has implications for RNA tertiary structure specificity. Comparisons of the affinities of 5'-splice site analogues that form only a subset of base pairs reveal that inclusion of the conserved G.U base pair at the cleavage site of group I introns destabilizes the P1 extension >100-fold relative to the stability of a helix with all Watson-Crick base pairs. Previous structural data with model duplexes and the recent intron structures suggest that this effect can be attributed to partial unstacking of the P1 extension at the G.U step. These results suggest a previously unrecognized role of the G.U wobble pair in self-splicing: breaking cooperativity in base pair formation between P1 and the P1 extensions. This effect may facilitate replacement of the P1 extension with P10 after the first chemical step of self-splicing and release of the ligated exons after the second step of self-splicing.     

Synthesis and properties of 2'-O-methyl-2-thiouridine and oligoribonucleotides containing 2'-O-methyl-2-thiouridine

A new method for the synthesis of 2'-O-methyl-2-thiouridine (s2Um) found in thermophilic bacterial tRNA was developed. Structural properties of s2Um and s2Um(p)U were studied by using 1H NMR spectroscopy. A modified nonaribonucleotide (RNA*: 5'-CGUUs2UmUUGC-3') was synthesized to study the base-recognition ability of s2Um in formation of RNA-RNA and RNA DNA duplexes. The UV melting experiments revealed that RNA*-RNA and RNA*-DNA duplexes having an s2U-A base pair are more stable than those having a U-A base pair. On the contrary, the thermal stability of RNA*-RNA and RNA*-DNA duplexes having an s2U-G wobble base pair was much lower than that of the unmodified duplexes having a natural U-G base pair. It is concluded that s2Um has higher selectivity toward A over G than unmodified U.     

Crystal structure of the highly distorted chimeric decamer r(C)d(CGGCGCCG)r(G).spermine complex--spermine binding to phosphate only and minor groove tertiary base-pairing.

The crystal structure of the self-complementary chimeric decamer duplex r(C)d(CGGCGCCG)r(G), with RNA base pairs at both termini, has been solved at 1.9 A resolution by the molecular replacement method and refined to an R value of 0.145 for 2,314 reflections. The C3'-endo sugar puckers of the terminal riboses apparently drive the entire chimeric duplex into an A-DNA conformation, in contrast to the B-DNA conformation adopted by the all-deoxy decamer of the same sequence. Five symmetry related duplexes encapsulate a spermine molecule which interacts with ten phosphate groups, both directly and through water molecules to form multiple ionic and hydrogen bonding interactions. The spermine interaction severely bends the duplexes by 31 degrees into the major groove at the fourth base pair G(4).C(17), jolts it and slides the 'base plate' into the minor groove. This base pair, together with the adjacent base pair in the top half and the corresponding pseudo two-fold related base pairs in the bottom half, form four minor groove base-paired multiples with the terminal base pairs of two neighboring duplexes.     

Kinetics and energetics of base-pair opening in 5'-d(CGCGAATTCGCG)-3' and a substituted dodecamer containing G.T mismatches.

Proton nuclear magnetic resonance (NMR) spectroscopy is used to characterize the kinetics and energetics of base-pair opening in the dodecamers 5'-d(CGCGAATTCGCG)-3' and 5'-d(CGCGAATTTGCG)-3'. The latter dodecamer contains two symmetrical G.T mismatched base pairs. The exchange kinetics of imino protons is measured from resonance line widths and selective longitudinal relaxation times. For the G.T pair, the two imino protons (G-N1H and T-N3H) provide probes for the opening of each base in the mismatched pair. The lifetimes of individual base pairs in the closed state and the equilibrium constants for formation of the open state are obtained from the dependence of the exchange rates on the concentration of ammonia catalyst. The activation energies and standard enthalpy changes for base-pair opening are obtained from the temperature dependence of the lifetimes and equilibrium constants, respectively. The results indicate that the G.T mismatched pairs are kinetically and energetically destabilized relative to normal, Watson-Crick base pairs. The lifetimes of the G.T pairs are of the order of 1 ms or less, over the temperature range from 0 to 20 degrees C. The equilibrium constants for base-pair opening, at 20 degrees C, are increased up to 4000-fold, relative to those of normal base pairs. The energetic destabilization of the G.T base pairs is, at least in part, enthalpic in origin. The presence of the G.T mismatched base pairs destabilizes also neighboring base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural characterisation of the bromouracil.guanine base pair mismatch in a Z-DNA fragment.

The deoxyoligonucleotide d(BrU-G-C-G-C-G) was crystallised at pH 8.2 and its structure analysed by X-ray diffraction. The unit cell, of dimensions a = 17.94, b = 30.85, c = 49.94A contains four DNA duplexes in space group P2(1)2(1)2(1). The duplexes are in the Z conformation, with four Watson-Crick G.C base pairs and two BrU.G base pairs. The structure was refined to an R factor of 0.16 at a resolution of 2.2A with 64 solvent molecules located. The BrU.G base pair mismatch is of the wobble type, with both bases in the major tautomer form and hydrogen bonds linking 0-2 of BrU with N-1 of G and N3 of BrU with 0-6 of G. There is no indication of the presence of ionised base pairs, in spite of the high pH of crystallisation. The results are discussed in terms of the mutagenic properties of 5- bromouracil.     

DNA interstrand crosslink formation by mechlorethamine at a cytosine-cytosine mismatch pair: kinetics and sequence dependence

Expansion of the triplet repeat DNA sequence d[CGG]n.d[CCG]n is a characteristic of Fragile X syndrome, a human neurodegenerative disease. Stable intrastrand conformations formed by both d[CGG]n and d[CCG]n, and involving G-G and C-C mismatch pairs, respectively, are believed to be of importance in the development of the disease. We have shown previously that C-C mismatch pairs can be crosslinked covalently by mechlorethamine, a nitrogen mustard alkylating agent, and hence this reaction may be of value as a probe for conformers of d[CCG]n. To characterize the mechlorethamine C-C crosslink reaction further, here we report the kinetics and sequence dependence of formation of the crosslink species, using a series of model duplexes. The rate of reaction depends on the base sequence proximal to the C-C mismatch pair. Hence, in 19mer duplexes containing a central d[M4M3M2M1Cn1n2n3n4].d[N4N3N2N1Cm1m2m3m4] sequence, where M-m and N-n are complementary base pairs, the amount of crosslink increased with increasing G-C content of the eight base pairs neighboring the C-C mismatch and with the proximity of the G-C pairs to the C-C mismatch. Molecular dynamics simulations of the solvated duplexes provided an explanation of these data. Hence, for a C-C pair flanked by G-C base pairs the mismatched cytosine bases remain stacked within the duplex, but for a C-C pair flanked by A-T base pairs, the simulations suggested local opening of the duplex around the C-C pair, making it a less effective target for mechlorethamine.     

3-D models of the antigenomic ribozyme of the hepatitis delta agent with eight new contacts suggested by sequence analysis of 188 cDNA clones.

We mapped 359 mutations at 25 positions in synthetic variants of the antigenomic ribozyme of the hepatitis delta agent by analyzing the sequences of 188 cDNA clones. These data were used to identify three features of the ribozyme: highly conserved nucleotides, positions with restricted nucleotide substitutions and three-dimensional relationships between nucleotides. The distribution of mutations at the 25 positions was as follows: G-11 (the eleventh nucleotide from the cleavage site) was mutated in 56 clones; G-12 in 36; U-15 in 33; C-13 in 26; G-28 in 23; C-27 in 21; C-29 in 19; U-26 in 17; C-18 in 14; A-14 in 13; C-16 in 13; C-19 in 12; U-17 in 11; A-20 in 10; G-42 in 9; G-40 in 7; G-41 in 7; C-24 in 6; U-32 in 6; U-23 in 5; C-25 in 4; C-21 in 3; G-30 in 3; G-31 in 3; C-22 in 1. All clones containing a mutation at C-25 had an A at this position, suggesting that the extra cyclic amino group present in adenine and cytosine may function during the cleavage event. Mutations at certain positions were common in simple clones (containing only one or two mutations), while mutations at other positions were over-represented in more complex clones. Both compensatory base changes and co-mutational frequencies were used to identify eight pairs of nucleotides which may interact with each other: G-11 and C-18, G-12 and C-27, C-13 and G-28, C-21 and U-23/C-24, C-21 and G-30, U-23 and G-31/U-32, C24 and G-30, C-27 and G-42. These pairs, which involve some of the most conserved positions in the molecule, suggest interactions among nucleotides previously depicted in open-loop structures. The newly proposed points of contact between pairs of nucleotides are compatible with both the axehead and pseudoknot secondary structural models and were combined with previously proposed Watson-Crick base paired helices to produce two three dimensional models. In both of these, C-25 and C-76 are placed near the cleavage site.     

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.     

Effects of internal nonbonded bases and a G.U base pair on the stability of a short ribonucleic acid helix.

Proton nuclear magnetic resonance has been used to examine the effect of both noncomplementary and G.U oppositions in the duplexes formed by the synthetic pentaribonucleotides CpApApUpG, CpApUpUpG, CpApGpUpG, and CpApCpUpG. The lack of any sigmoidal behavior in the chemical shift vs. temperature plots of the base protons in the individual pentaribonucleotides indicates that duplexes with noncomplementary base oppositions of the type: formula: (see text), (where X = A, U, G, or C) do not form. Variable temperature spectra of the mixture of CpApGpUpG and CpApUpUpG were recorded over the range of 70--10 degrees C. The chemical shift vs. temperature plot of the purine aromatic protons displayed sigmoidal curves. This demonstrated both duplex formation and the presence of a G.U. base pair. The average Tm of the duplex was found to be 23.4 +/- 2.0 degrees C. This is similar to that of the duplex formed by CpApUpG (24.0 +/- 1.0 degrees C) but less than the Tm of the following duplexes: CpApApUpG:CpApUpUpG (Tm = 28.5 +/- 2.1 degrees C), CpApGpUpG:CpApCpUpG (Tm = 38.4 +/- 0.6 degrees C) and CpApUpApUpG (Tm = 41.5 +/- 1.1 degrees C). The G.U base pair has a Tm (20.0 degrees C) significantly lower than the rest of the duplex (24 +/- 1 degree C) and is a region of local instability within the double helix. This 1H NMR study is the first to investigate both the formation and relative stability of an internal G.U. base pair neighboring regular Watson--Crick base pairs.     

Thermodynamic insights into 2-thiouridine-enhanced RNA hybridization

Nucleobase modifications dramatically alter nucleic acid structure and thermodynamics. 2-thiouridine (s²U) is a modified nucleobase found in tRNAs and known to stabilize U:A base pairs and destabilize U:G wobble pairs. The recently reported crystal structures of s²U-containing RNA duplexes do not entirely explain the mechanisms responsible for the stabilizing effect of s²U or whether this effect is entropic or enthalpic in origin. We present here thermodynamic evaluations of duplex formation using ITC and UV thermal denaturation with RNA duplexes containing internal s²U:A and s²U:U pairs and their native counterparts. These results indicate that s²U stabilizes both duplexes. The stabilizing effect is entropic in origin and likely results from the s²U-induced preorganization of the single-stranded RNA prior to hybridization. The same preorganizing effect is likely responsible for structurally resolving the s²U:U pair-containing duplex into a single conformation with a well-defined H-bond geometry. We also evaluate the effect of s²U on single strand conformation using UV- and CD-monitored thermal denaturation and on nucleoside conformation using ¹H NMR spectroscopy, MD and umbrella sampling. These results provide insights into the effects that nucleobase modification has on RNA structure and thermodynamics and inform efforts toward improving both ribozyme-catalyzed and nonenzymatic RNA copying.     

Base pair mismatches and carcinogen-modified bases in DNA: an NMR study of G.T and G.O4meT pairing in dodecanucleotide duplexes

High-resolution two-dimensional NMR studies have been completed on the self-complementary d(C-G-C-G-A-G-C-T-T-G-C-G) duplex (designated G.T 12-mer) and the self-complementary d(C-G-C-G-A-G-C-T-O4meT-G-C-G) duplex (designated G.O4meT 12-mer) containing G.T and G.O4meT pairs at identical positions four base pairs in from either end of the duplex. The exchangeable and nonexchangeable proton resonances have been assigned from an analysis of two-dimensional nuclear Overhauser enhancement (NOESY) spectra for the G.T 12-mer and G.O4meT 12-mer duplexes in H2O and D2O solution. The guanosine and thymidine imino protons in the G.T mismatch resonate at 10.57 and 11.98 ppm, respectively, and exhibit a strong NOE between themselves and to imino protons of flanking base pairs in the G.T 12-mer duplex. These results are consistent with wobble pairing at the G.T mismatch site involving two imino proton-carbonyl hydrogen bonds as reported previously [Hare, D. R., Shapiro, L., & Patel, D. J. (1986) Biochemistry 25, 7445-7456]. In contrast, the guanosine imino proton in the G.O4meT pair resonates at 8.67 ppm. The large upfield chemical shift of this proton relative to that of the imino proton resonance of G in the G.T mismatch or in G.C base pairs indicates that hydrogen bonding to O4meT is either very weak or absent. This guanosine imino proton has an NOE to the OCH3 group of O4meT across the pair and NOEs to the imino protons of flanking base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of sheared G:A base pairs in an RNA duplex modelled after ribozymes, as revealed by NMR.

The thermal stability and structure of an RNA duplex, r(GGACGAGUCC)2, the base sequence of which was modelled after both a hammerhead ribozyme and a lead ribozyme, were studied by CD and NMR. We previously demonstrated that the corresponding DNA duplex, d(GGACGAGTCC)2, formed unique 'sheared' G:A base pairs, where an amino proton, instead of an imino proton, of G is involved in the hydrogen bonding, and G and A bases are arranged 'side by side' instead of 'head to head' (Nucleic Acids Res. (1993) 21, 5418-5424). CD melting profiles showed that the RNA duplex is thermally more stable than the corresponding DNA duplex. NMR studies revealed that sheared G:A base pairs are formed in the RNA duplex, too, although the overall structure of the RNA is the A form, which differs from the B form taken on by the corresponding DNA. A model building study confirmed that sheared G:A base pairs can be accommodated in the double helical structure of the A form. A difference between the RNA and DNA duplexes in the stacking interaction involving G:A mismatch bases is also suggested. The demonstration that sheared G:A base pairs can be formed not only in DNA but also in RNA suggests that this base pairing plays an important role regarding the RNA structure.