Stacking interactions in RNA and DNA: Roll‐slide energy hyperspace for ten unique dinucleotide steps

Understanding dinucleotide sequence directed structures of nuleic acids and their variability from experimental observation remained ineffective due to unavailability of statistically meaningful data. We have attempted to understand this from energy scan along twist, roll, and slide degrees of freedom which are mostly dependent on dinucleotide sequence using ab initio density functional theory. We have carried out stacking energy analysis in these dinucleotide parameter phase space for all ten unique dinucleotide steps in DNA and RNA using DFT‐D by ωB97X‐D/6‐31G(2d,2p), which appears to satisfactorily explain conformational preferences for AU/AU step in our recent study. We show that values of roll, slide, and twist of most of the dinucleotide sequences in crystal structures fall in the low energy region. The minimum energy regions with large twist values are associated with the roll and slide values of B‐DNA, whereas, smaller twist values correspond to higher stability to RNA and A‐DNA like conformations. Incorporation of solvent effect by CPCM method could explain the preference shown by some sequences to occur in B‐DNA or A‐DNA conformations. Conformational preference of BII sub‐state in B‐DNA is preferentially displayed mainly by pyrimidine–purine steps and partly by purine–purine steps. The purine–pyrimidine steps show largest effect of 5‐methyl group of thymine in stacking energy and the introduction of solvent reduces this effect significantly. These predicted structures and variabilities can explain the effect of sequence on DNA and RNA functionality. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 134–147, 2015.     

Stacking geometry for non‐canonical G:U wobble base pair containing dinucleotide sequences in RNA: dispersion‐corrected DFT‐D study

Emergence of thousands of crystal structures of noncoding RNA molecules indicates its structural and functional diversity. RNA function is based upon a large variety of structural elements which are specifically assembled in the folded molecules. Along with the canonical Watson‐Crick base pairs, different orientations of the bases to form hydrogen‐bonded non‐canonical base pairs have also been observed in the available RNA structures. Frequencies of occurrences of different non‐canonical base pairs in RNA indicate their important role to maintain overall structure and functions of RNA. There are several reports on geometry and energetic stabilities of these non‐canonical base pairs. However, their stacking geometry and stacking stability with the neighboring base pairs are not well studied. Among the different non‐canonical base pairs, the G:U wobble base pair (G:U W:WC) is most frequently observed in the RNA double helices. Using quantum chemical method and available experimental data set we have studied the stacking geometry of G:U W:WC base pair containing dinucleotide sequences in roll‐slide parameters hyperspace for different values of twist. This study indicates that the G:U W:WC base pair can stack well with the canonical base pairs giving rise to large interaction energy. The overall preferred stacking geometry in terms of roll, twist and slide for the eleven possible dinucleotide sequences is seen to be quite dependent on their sequences. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 328–338, 2015.     

Different intrastrand and interstrand contributions to stacking account for roll variations at the alternating purine-pyrimidine sequences in A-DNA and A-RNA.

An explanation is suggested for the roll alternation between low and high values in A-type nucleic acid duplexes containing alternating sequences of purine and pyrimidine residues. The explanation combines two points. (1) Roll inevitably occurs in A-type duplexes due to geometrical reasons. (2) Intrastrand base stacking is much more impaired by roll than interstrand base stacking in A-type duplexes. Therefore purine-pyrimidine steps, whose bases mainly exhibit an intrastrand stacking, resist roll and decrease it. By contrast, bases at pyrimidine-purine steps exhibit a significant interstrand stacking that is tolerant to roll in A-type nucleic acid duplexes. In consequence, it is favourable if the purine-pyrimidine and pyrimidine-purine steps adopt low and high rolls, respectively in A-conformations of DNA and RNA molecules containing alternating purine-pyrimidine sequences. This is actually observed in the relevant molecular crystal structures.     

Synthesis of Modified RNA-Oligonucleotides for Structural Investigations

Abstract

RNA exhibits a higher structural diversity than DNA and is an important molecule in biology of life. It shows a number of secondary structures such as duplexes, hairpin loops, bulges, internal loops etc. However, in natural RNA, bases are limited to the four predominant structures U, C, A, and G and so the number of compounds that can be used for investigation of parameters of base stacking, base pairing and hydrogen bond, is limited. We synthesized different fluoromodifications of RNA building blocks: 1′-deoxy-1′-(2,4,6-trifluorophenyl)-ß-D-ribofuranose (F), 1′-deoxy-1′-(2,4,5-trifluorophenyl)-ß-D-ribofuranose (M) and 1′-deoxy-1′-(5-trifluoromethyl-1H-benzimidazol-1-yl)-ß-D-ribofuranose (D). Those amidites were incorporated and tested in a defined A, U- rich RNA sequence (12-mer, 5′-CUU UUC XUU CUU-3′ paired with 3′-GAA AAG YAA GAA-5’) (Schweitzer, B.A.; Kool, E.T. Aromatic nonpolar nucleosides as hydrophobic isosters of pyrimidine and purine nucleosides. J. Org. Chem. 1994, 59, 7238 pp.). Only one position was modified, marked as X and Y respectively. UV melting profiles of those oligonucleotides were measured.     

Stability of RNA hairpin loops closed by AU base pairs

Thermodynamic parameters are reported for hairpin formation in 1 M NaCl by RNA sequence of the type GCAXUAAUYUGC, where XY is the set of 10 possible mismatch base pairs. A nearest-neighbor analysis of the data indicates that the free energy of loop formation at 37 degrees C varies from 3.2 to 5.0 kcal/mol. These results combined with the model previously developed [Dale et al. (2000) RNA 6, 608] allow improvements in the model to predict the stability of RNA hairpin loops: DeltaG degrees (37L(n) = DeltaG degrees (37i(n)) + DeltaG degrees (37MM) - 0.8 (if first mismatch is GA or UU) - 0.8 (if first mismatch is GG and loop is closed on 5' side by a purine). Here, DeltaG degrees (37i(n) is the free energy for initiating a loop of n nucleotides, and DeltaG degrees (37MM) is the free energy for the interaction of the first mismatch with the closing base pair. Hairpins with GG first mismatches were found to vary in stability depending upon the orientation of the closing base pair (5' or 3' purine relative to the loop). The model gives good agreement when tested against four naturally occurring hairpin sequences.     

DNA polymorphism and local variation in base-pair orientation: a theoretical rationale.

Basepair stacking calculations have been carried out to understand the conformational polymorphism of DNA and its sequence dependence. The recently developed self-consistent parameter set, which is specially suitable for describing irregular DNA structures, has been used to describe the geometry of a basepair doublet. While for basepairs without any propeller, the favourable stacking patterns do not appear to have very strong features, much more noticeable sequence dependent stacking patterns emerge once a propeller is applied to the basepairs. The absolute minima for most sequences occurs for a doublet geometry close to the B-DNA fibre models. Hence in the B-DNA region, no strong sequence dependent features are found, but the range of doublet geometries observed in the crystal structures generally lie within the low energy contours, obtained from stacking energy calculations. The doublet geometry corresponding to the A-DNA fibre model is not energetically favourable for the purine-pyrimidine sequences, which prefer small roll angle values when the slide has a large negative value as in A-DNA. However positive roll with large negative slide is allowed for GG, GA, AG and the pyrimidine-purine steps. This is consistent with the observed geometries of various steps in A-DNA crystals. Thus the general features of the basepair doublets predicted from these theoretical studies agree very well with the results from crystal structure analysis. However, since most sequences show an overall preference for B-type doublet geometry, the B----A transition for random sequence DNA cannot be explained on the basis of basepair stacking interactions.     

The dynamic structural basis of differential enhancement of conformational stability by 5'- and 3'-dangling ends in RNA

Unpaired bases at the end of an RNA duplex (dangling ends) can stabilize the core duplex in a sequence-dependent manner and are important determinants of RNA folding, recognition, and functions. Using 2-aminopurine as a dangling end purine base, we have employed femtosecond time-resolved fluorescence spectroscopy, combined with UV optical melting, to quantitatively investigate the physical and structural nature of the stacking interactions between the dangling end bases and the terminal base pairs. A 3'-dangling purine base has a large subpopulation that stacks on the guanine base of the terminal GC or UG pair, either intrastrand or cross-strand depending on the orientation of the pair, thus providing stabilization of different magnitudes. On the contrary, a 5'-dangling purine base only has a marginal subpopulation that stacks on the purine of the same strand (intrastrand) but has little cross-strand stacking. Thus a 5'-dangling purine does not provide significant stabilization. These stacking structures are not static, and a dangling end base samples a range of stacked and unstacked conformations with respect to the terminal base pair. Femtosecond time-resolved anisotropy decay reveals certain hindered base conformational dynamics that occur on the picosecond to nanosecond time scales, which allow the dangling base to sample these substates. When the dangling purine is opposite to a U and is able to form a potential base pair at the end of the duplex, there is an interplay of base stacking and hydrogen-bonding interactions that depends on the orientation of the base pair relative to the adjacent GC pair. By resolving these populations that are dynamically exchanging on fast time scales, we elucidated the correlation between dynamic conformational distributions and thermodynamic stability.     

Efficient aminoacylation of the tRNA(Ala) acceptor stem: dependence on the 2:71 base pair

Specific aminoacylation by aminoacyl-tRNA synthetases requires accurate recognition of cognate tRNA substrates. In the case of alanyl-tRNA synthetase (AlaRS), RNA duplexes that mimic the acceptor stem of the tRNA are efficient substrates for aminoacylation in vitro. It was previously shown that recognition by AlaRS is severely affected by a simple base pair transversion of the G2:C71 pair at the second position in the RNA helix. In this study, we determined the aminoacylation efficiencies of 50 variants of the tRNA(Ala) acceptor stem containing substitutions at the 2:71 position. We find that there is not a single functional group of the wild-type G2:C71 base pair that is critical for positive recognition. Rather, we observed that base-pair orientation plays an important role in recognition. In particular, pyrimidine2:purine71 combinations generally resulted in decreased aminoacylation efficiency compared to the corresponding purine:pyrimidine pair. Moreover, the activity of a pyrimidine:purine variant could be partially restored by the presence of a major groove amino group at position 71. In an attempt to understand this result further, dielectric continuum electrostatic calculations were carried out, in some cases with additional inclusion of van der Waals interaction energies, to determine interaction potentials of the wild-type duplexAla and seven 2:71 variants. This analysis revealed a positive correlation between major groove negative electrostatic potential in the vicinity of the 3:70 base pair and measured aminoacylation efficiency.     

Local conformational variations observed in B-DNA crystals do not improve base stacking: computational analysis of base stacking in a d(CATGGGCCCATG)(2) B<-->A intermediate crystal structure

The crystal structure of d(CATGGGCCCATG)₂ shows unique stacking patterns of a stable B↔A-DNA intermediate. We evaluated intrinsic base stacking energies in this crystal structure using an ab initio quantum mechanical method. We found that all crystal base pair steps have stacking energies close to their values in the standard and crystal B-DNA geometries. Thus, naturally occurring stacking geometries were essentially isoenergetic while individual base pair steps differed substantially in the balance of intra-strand and inter-strand stacking terms. Also, relative dispersion, electrostatic and polarization contributions to the stability of different base pair steps were very sensitive to base composition and sequence context. A large stacking flexibility is most apparent for the CpA step, while the GpG step is characterized by weak intra-strand stacking. Hydration effects were estimated using the Langevin dipoles solvation model. These calculations showed that an aqueous environment efficiently compensates for electrostatic stacking contributions. Finally, we have carried out explicit solvent molecular dynamics simulation of the d(CATGGGCCCATG)₂ duplex in water. Here the DNA conformation did not retain the initial crystal geometry, but moved from the B↔A intermediate towards the B-DNA structure. The base stacking energy improved in the course of this simulation. Our findings indicate that intrinsic base stacking interactions are not sufficient to stabilize the local conformational variations in crystals.     

Helix bending as a factor in protein/DNA recognition

Normal vectors perpendicular to individual base pairs are a powerful tool for studying the bending behavior of B-DNA, both in the form of normal vector plots and in matrices that list angles between vectors for all possible base pair combinations. A new analysis program, FREEHELIX, has been written for this purpose, and applied to 86 examples of sequence-specific protein/DNA complexes whose coordinates are on deposit in the Nucleic Acid Data Base. Bends in this sample of 86 structures almost invariably follow from roll angles between adjacent base pairs; tilt makes no net contribution. Roll in a direction compressing the broad major groove is much more common than that which compresses the minor groove. Three distinct types of B-DNA bending are observed, each with a different molecular origin: (1) Localized kinking is produced by large roll at single steps or at two steps separated by one turn of helix. (2) Smooth, planar curvature is produced by positive and negative roll angles spaced a half-turn apart, with random side-to-side zigzag roll at intermediate points, rather than a tilt contribution that might have been expected theoretically. (3) Three-dimensional writhe results from significant roll angles at a continuous series of steps. Writhe need not change the overall direction of helix axis, if it is continued indefinitely or for an integral number of helical turns. A-DNA itself can be formally considered as possessing uniform, continuous writhe that yields no net helix bending. Smooth curvature is the most intricate deformation of the three, and is least common. Writhe is the simplest deformation and is most common; indeed, a low level of continuous writhe is the normal condition of an otherwise unbent B-DNA helix of general sequence. With one exception, every example of major kinking in this sample of 86 structures involves a pyrimidine–purine step: C–A/T–G, T–A, or C–G. Purine–purine steps, especially A–A, show the least tendency toward roll deformations. © 1998 John Wiley & Sons, Inc. Biopoly 44: 361–403, 1997     

Effects of base sequence on the loop folding in DNA hairpins

High-resolution NMR and UV-melting experiments have been used to study the hairpin formation of partly self-complementary DNA fragments in an attempt to derive rules that describe the folding in these molecules. Earlier experiments on the hexadecanucleotide d(ATCCTA-TTTT-TAGGAT) had indicated that within the loop of four thymidines a wobble T-T pair is formed (Blommers et al., 1987). In the present paper it is shown that if the first and the last thymines of the intervening sequence are replaced by complementary bases, sometimes base pairs can be formed. Thus for the intervening sequences -CTTG- and -TTTA- with the pyrimidine in the 5'-position and the purine in the 3'-position, a base pair is formed leading to a loop consisting of two residues. For the intervening sequences -GTTC- and -ATTT- with the purine in the 5'-position and the pyrimidine in the 3'-position, this turns out not to be the case. It was found that it made no difference when the four-membered sequence was closed by a G-C base pair or an A-T base pair. Replacement of the two central thymidine residues by the more bulky adenine residues limits the hairpin to a four-membered loop scheme. Very surprisingly, it was found from 2D NOE experiments that the T-A base pair, formed in the loop consisting of the -TTTA- sequence, is a Hoogsteen pair. It is argued that the pairing of the bases in this scheme may facilitate the formation of a loop of two residues, since the distance of the C1' atoms in this base pair is 8.6 A instead of 10.4 A found in the canonical Watson-Crick base pair. Combination of the data obtained for the series of DNA fragments studied shows that the results can be explained by a simple, earlier proposed, loop folding principle which assumes that the folding of the four-membered loop is dictated by the stacking of the double-helical stem of the hairpin.     

Hybrid simulation approach incorporating microscopic interaction along with rigid body degrees of freedom for stacking between base pairs

Stacking interaction between the aromatic heterocyclic bases plays an important role in the double helical structures of nucleic acids. Considering the base as rigid body, there are total of 18 degrees of freedom of a dinucleotide step. Some of these parameters show sequence preferences, indicating that the detailed atomic interactions are important in the stacking. Large variants of non‐canonical base pairs have been seen in the crystallographic structures of RNA. However, their stacking preferences are not thoroughly deciphered yet from experimental results. The current theoretical approaches use either the rigid body degrees of freedom where the atomic information are lost or computationally expensive all atom simulations. We have used a hybrid simulation approach incorporating Monte‐Carlo Metropolis sampling in the hyperspace of 18 stacking parameters where the interaction energies using AMBER‐parm99bsc0 and CHARMM‐36 force‐fields were calculated from atomic positions. We have also performed stacking energy calculations for structures from Monte‐Carlo ensemble by Dispersion corrected density functional theory. The available experimental data with Watson–Crick base pairs are compared to establish the validity of the method. Stacking interaction involving A:U and G:C base pairs with non‐canonical G:U base pairs also were calculated and showed that these structures were also sequence dependent. This approach could be useful to generate multiscale modeling of nucleic acids in terms of coarse‐grained parameters where the atomic interactions are preserved. This method would also be useful to predict structure and dynamics of different base pair steps containing non Watson–Crick base pairs, as found often in the non‐coding RNA structures. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 212–226, 2016.     

An acid ribonuclease from rye germ cytosol

Acid ribonuclease from rye germ cytosol was purified 1200-fold. The enzyme is homogeneous on polyacrylamide gel. The optimum pH for ribonuclease activity is 5.8, its MW is 28 500. The enzyme is an endonuclease yielding in the first step of its activity oligonucleotides with a free —OH group in the 3′ position. The end products of RNA hydrolysis are cyclic purine and pyrimidine nucleoside phosphates and the corresponding nucleoside 3′-phosphates. This ribonuclease preferentially attacks sites close to the adenine base and shows a lag in the release of the cytosine base. Specificity tests on natural and synthetic substrates are in good agreement.     

Configuration of wobble base pairs having pyrimidines as anticodon wobble bases: significance for codon degeneracy

Degeneracy of the genetic code was attributed by Crick to imprecise hydrogen-bonded base-pairing at the wobble position during codon–anticodon pairing. The Crick wobble rules define but do not explain the RNA base pair combinations allowed at this position. We select six pyrimidine bases functioning as anticodon wobble bases (AWBs) to study their H-bonded pairing properties with the four major RNA bases using density functional theory at the B3LYP/6-31G(d,p) level. This is done to assess the extent to which the configuration of a solitary RNA wobble base pair may in itself determine specificity and degeneracy of the genetic code by allowing or disallowing the given base pair during codon–anticodon pairing. Calculated values of select configuration markers for the base pairs screen well between allowed and disallowed base pairs for most cases examined here, where the base pair width emerges as an important factor. A few allowed wobble pairs invoke the involvement of RNA nucleoside conformation, as well as involvement of the exocyclic substituent in H-bonding. This study, however, cannot explain the disallowed status of the Ura?Gua wobble pair on the basis of configuration alone. Explanation of the allowed status of the V?Ura pair requires further study on the mediatory role of water molecules. Apart from these two cases, these computational results are sufficient, on the basis of base pair configuration alone, to account for the specificity and degeneracy of the genetic code for all known cases of codon–anticodon pairing which involve the pyrimidine AWBs studied here.     

Molecular mechanical studies of base-pair opening in d(CGCGC):d(GCGCG), dG5·dC5, d(TATAT):d(ATATA), and dA5·dT5 in the B and Z forms of DNA

Molecular mechanical energy refinement of double-helical pentanucleotide tetra-phosphates, d(CGCGC):d(GCGCG), dG₅·dC₅, d(TATAT):d(ATATA), and dA₅ ·dT₅ geometries, are presented in order to examine the energy required to open the Nl(purine) …? N3(pyrimidine) distance (base-pair opening) of a Watson-Crick base pair from its normal value of 3 Å to a value of 6 Å. The structural consequences of forcing base-pair opening is sequence dependent. For both dA₅ ·dT₅ and d(TATAT):d(ATATA), forcing the Nl (AdeKN3 (Thy) distance of the central base pair to a value of 6 Å slides the bases perpendicular to the helix axis forming a low-energy non-Watson-Crick base pair having an adenine amine hydrogen …? thymine carbonyl oxygen hydrogen bond. The two GC sequences behave differently from both AT sequences and differently from each other. Forcing the Nl(Gua) …? N3(Cyt) distance to 6 Å leads to unconventional structures in which hydrogen bonds are formed between the separated bases and the bases above or below them. These structures appear to be trapped in true local minima 6–10 kcal/mol higher in energy than the Watson-Crick structures. Preliminary simulations on d(CGCGC):d(GCGCG) in the Z geometry suggest the reason the Z form may be more refractory to proton exchange than the B form, consistent with experimental observations.     

Base pair buckling can eliminate the interstrand purine clash at the CpG steps in B-DNA caused by the base pair propeller twisting

Results of calculations using various empirical potentials suggest that base pair buckling, which commonly occurs in DNA crystal structures, is sufficient to eliminate the steric clash at CpG steps in B-DNA, originating from the base pair propeller twisting. The buckling is formed by an inclination of cytosines while deviations of guanines from a plane perpendicular to the double helix axis are unfavorable. The buckling is accompanied by an increased vertical separation of the base pair centers but the buckled arrangement of base pairs is at least as stable as when the vertical separation is normal and buckle zero. In addition, room is created by the increased vertical separation for the bases to propeller twist as is observed in DNA crystal structures. Further stabilization of base stacking is introduced into the buckled base pair arrangement by roll opening the base pairs into the double helix minor groove. The roll may lead to the double helix bending and liberation of guanines from the strictly perpendicular orientation to the double helix axis. The liberated guanines further contribute to the base pair buckling and stacking improvement. This work also suggests a characteristic very stable DNA structure promoted by nucleotide sequences in which runs of purines follow runs of pyrimidine bases.     

The structure of a full turn of an A-DNA duplex d(CGCGGGTACCCGCG)2

We report the 2.6 Å resolution crystal structure of the tetra-decamer d(CGCGGGTACCCGCG) in the tetragonal space group P4₃. This sequence contains the KpnI restriction site GGTACC in the centre which is flanked by alternating ‘CG’ sequences, and has a ‘TA’ step at the centre. These are features could favour the left-handed Z type helix. Despite this, overall the molecule has the A form. This is the first tetra-decamer crystallized in the A-DNA conformation, i.e. more than one full turn of the A helix. The crystallographic asymmetric unit consists of one tetra-decamer duplex. The helical twist and slide, as well as the base pair–base pair stacking interactions show alternations at the alternating pyrimidine–purine and purine–pyrimidine base steps. This variation is reminiscent of the dinucleotide repeat in left-handed Z-DNA helices. The crystal packing is unlike other A-DNA crystal structures, with each helix having a large number of contacts of many different types with symmetry-related neighbours.     

Sequence dependence for the energetics of dangling ends and terminal base pairs in ribonucleic acid

Stability increments of terminal unpaired nucleotides (dangling ends) and terminal base pairs on the core helixes AUGCAU and UGCGCA are reported. Enthalpy, entropy, and free energy changes of helix formation were measured spectrophotometrically for 18 oligoribonucleotides containing the core sequences. The results indicate 3' dangling purines add more stability than 3' dangling pyrimidines. In most cases, the additional stability from a 3' dangling end on an AU base pair is less than that on a GC base pair [Freier, S.M., Burger, B.J., Alkema, D., Neilson, T., & Turner, D.H. (1985) Biochemistry 22, 6198-6206]. The sequence dependence provides a test for the importance of dangling ends for various RNA interactions. Correlations are suggested with codon context effects and with the three-dimensional structure of yeast phenylalanine transfer RNA. In the latter case, all terminal unpaired nucleotides having stability increments more favorable than -1 kcal/mol are stacked on the adjacent base pair. All terminal unpaired nucleotides having stability increments less favorable than -0.3 kcal/mol are not stacked on the adjacent base pair. In several cases, this lack of stacking is associated with a turn in the sugar-phosphate backbone. This suggests stability increments measured on oligoribonucleotides may be useful for predicting tertiary structure in large RNA molecules. Comparison of the stability increments for terminal dangling ends and base pairs, and of terminal GC and AU base pairs, indicates the free energy increment associated with forming a hydrogen bond can be about -1 kcal/mol of hydrogen bond.     

Stabilities of nearest-neighbor doublets in double-helical DNA determined by fitting calculated melting profiles to observed profiles

Melting profiles were calculated for restriction fragments of ?X174 and fd phage DNAs and compared with experimental profiles. The algorithm of Fixman and Freire was slightly modified so that a stability parameter was assigned not to a base pair but to each nearest-neighbor doublet. Stabilities of the 10 kinds of nearest-neighbor doublets were estimated by fitting the calculated profiles to the observed ones. Agreement of the calculated and observed profiles was much improved by this modification. The most interesting finding was that purine (3′-5′) pyrimidine stackings are much more stable than their respective reverses. The order of nearest-neighbor stabilities is in excellent agreement with that of negative stacking energies calculated by Rein and coworkers by a quantum-chemical method.     

Structure of 2′,5′-Linked Tetraribonucleotide Loops: A Novel RNA Motif

Abstract

We report on the three dimensional structure of an RNA hairpin containing a 2′,5′-linked tetraribonucleotide loop, namely, 5′-rGGAC(UUCG)GUCC-3′ (where UUCG = U_2′p5′U_2′p5′C_2′p5′G_2′p5′). We show that the 2′,5′-linked RNA loop adopts a conformation that is quite different from that previously observed for the native 3′,5′-linked RNA loop. The 2′,5′- RNA loop is stabilized by (a) U:G wobble base pairing, with both bases in the anti conformation, (b) extensive base stacking, and (c) sugar–base contacts, all of which contribute to the extra stability of this hairpin structure.