Solution structure of the apical stem-loop of the human hepatitis B virus encapsidation signal

Hepatitis B virus (HBV) replication is initiated by HBV RT binding to the highly conserved encapsidation signal, epsilon, at the 5′ end of the RNA pregenome. Epsilon contains an apical stem–loop, whose residues are either totally conserved or show rare non-disruptive mutations. Here we present the structure of the apical stem–loop based on NOE, RDC and ¹H chemical shift NMR data. The ¹H chemical shifts proved to be crucial to define the loop conformation. The loop sequence 5′-CUGUGC-3′ folds into a UGU triloop with a CG closing base pair and a bulged out C and hence forms a pseudo-triloop, a proposed protein recognition motif. In the UGU loop conformations most consistent with experimental data, the guanine nucleobase is located on the minor groove face and the two uracil bases on the major groove face. The underlying helix is disrupted by a conserved non-paired U bulge. This U bulge adopts multiple conformations, with the nucleobase being located either in the major groove or partially intercalated in the helix from the minor groove side, and bends the helical stem. The pseudo-triloop motif, together with the U bulge, may represent important anchor points for the initial recognition of epsilon by the viral RT.     

Role of helical constraints of the EBS1–IBS1 duplex of a group II intron on demarcation of the 5′ splice site

Recognition of the 5′ splice site by group II introns involves pairing between an exon binding sequence (EBS) 1 within the ID3 stem–loop of domain 1 and a complementary sequence at the 3′ end of exon 1 (IBS1). To identify the molecular basis for splice site definition of a group IIB ai5γ intron, we probed the solution structure of the ID3 stem–loop alone and upon binding of its IBS1 target by solution NMR. The ID3 stem was structured. The base of the ID3 loop was stacked but displayed a highly flexible EBS1 region. The flexibility of EBS1 appears to be a general feature of the ai5γ and the smaller Oceanobacillus iheyensis (O.i.) intron and may help in effective search of conformational space and prevent errors in splicing as a result of fortuitous base-pairing. Binding of IBS1 results in formation of a structured seven base pair duplex that terminates at the 5′ splice site in spite of the potential for additional A-U and G•U pairs. Comparison of these data with conformational features of EBS1–IBS1 duplexes extracted from published structures suggests that termination of the duplex and definition of the splice site are governed by constraints of the helical geometry within the ID3 loop. This feature and flexibility of the uncomplexed ID3 loop appear to be common for both the ai5γ and O.i. introns and may help to fine-tune elements of recognition in group II introns.     

Unusual DNA duplex and hairpin motifs

Single-stranded DNA or double-stranded DNA has the potential to adopt a wide variety of unusual duplex and hairpin motifs in the presence (trans) or absence (cis) of ligands. Several principles for the formation of those unusual structures have been established through the observation of a number of recurring structural motifs associated with different sequences. These include: (i) internal loops of consecutive mismatches can occur in a B-DNA duplex when sheared base pairs are adjacent to each other to confer extensive cross- and intra-strand base stacking; (ii) interdigitated (zipper-like) duplex structures form instead when sheared G·A base pairs are separated by one or two pairs of purine·purine mismatches; (iii) stacking is not restricted to base, deoxyribose also exhibits the potential to do so; (iv) canonical G·C or A·T base pairs are flexible enough to exhibit considerable changes from the regular H-bonded conformation. The paired bases become stacked when bracketed by sheared G·A base pairs, or become extruded out and perpendicular to their neighboring bases in the presence of interacting drugs; (v) the purine-rich and pyrimidine-rich loop structures are notably different in nature. The purine-rich loops form compact triloop structures closed by a sheared G·A, A·A, A·C or sheared-like G_anti·C_syn base pair that is stacked by a single residue. On the other hand, the pyrimidine-rich loops with a thymidine in the first position exhibit no base pairing but are characterized by the folding of the thymidine residue into the minor groove to form a compact loop structure. Identification of such diverse duplex or hairpin motifs greatly enlarges the repertoire for unusual DNA structural formation.     

Loss of G-A base pairs is insufficient for achieving a large opening of U4 snRNA K-turn motif

Upon binding to the 15.5K protein, two tandem-sheared G–A base pairs are formed in the internal loop of the kink-turn motif of U4 snRNA (Kt-U4). We have reported that the folding of Kt-U4 is assisted by protein binding. Unstable interactions that contribute to a large opening of the free RNA (‘k–e motion’) were identified using locally enhanced sampling molecular dynamics simulations, results that agree with experiments. A detailed analysis of the simulations reveals that the k–e motion in Kt-U4 is triggered both by loss of G–A base pairs in the internal loop and backbone flexibility in the stems. Essential dynamics show that the loss of G–A base pairs is correlated along the first mode but anti-correlated along the third mode with the k–e motion. Moreover, when enhanced sampling was confined to the internal loop, the RNA adopted an alternative conformation characterized by a sharper kink, opening of G–A base pairs and modified stacking interactions. Thus, loss of G–A base pairs is insufficient for achieving a large opening of the free RNA. These findings, supported by previously published RNA structure probing experiments, suggest that G–A base pair formation occurs upon protein binding, thereby stabilizing a selective orientation of the stems.     

Requirement for canonical base pairing in the short pseudoknot structure of genomic hepatitis delta virus ribozyme

The tertiary structure of the 3′-cleaved product of the genomic hepatitis delta virus (HDV) ribozyme was solved by X-ray crystallographic analysis. In this structure, three single-stranded regions (SSrA, -B and -C) interact intricately with one another via hydrogen bonds between nucleotide bases, phosphate oxygens and 2′-OHs to form a nested double pseudoknot structure. Among these interactions, two Watson–Crick (W–C) base pairs, 726G–710C and 727G–709C, that form between SSrA and SSrC (P1.1) seem to be especially important for compact folding. To characterize the importance of these base pairs, ribozymes were subjected to in vitro selection from a pool of RNA molecules randomly substituted at positions 709, 710, 726 and 727. The results establish the importance of the two W–C base pairs for activity, although some mutants are active with one G–C base pair. In addition, the kinetic parameters were analyzed in all 16 combinations with two canonical base pairs. Comparison of variant ribozymes with the wild-type ribozyme reveals that the difference in reaction rates for these variants (ΔΔG^‡) is not simply accounted for by the differences in the stability of P1.1 (ΔΔG⁰₃₇). The role played by Mg²⁺ ions in formation of the P1.1 structure is also discussed.     

Non-nearest-neighbor dependence of stability for group III RNA single nucleotide bulge loops

Thirty-five RNA duplexes containing single nucleotide bulge loops were optically melted and the thermodynamic parameters for each duplex determined. The bulge loops were of the group III variety, where the bulged nucleotide is either a AG/U or CU/G, leading to ambiguity to the exact position and identity of the bulge. All possible group III bulge loops with Watson–Crick nearest-neighbors were examined. The data were used to develop a model to predict the free energy of an RNA duplex containing a group III single nucleotide bulge loop. The destabilization of the duplex by the group III bulge could be modeled so that the bulge nucleotide leads to the formation of the Watson–Crick base pair rather than the wobble base pair. The destabilization of an RNA duplex caused by the insertion of a group III bulge is primarily dependent upon non-nearest-neighbor interactions and was shown to be dependent upon the stability of second least stable stem of the duplex. In-line structure probing of group III bulge loops embedded in a hairpin indicated that the bulged nucleotide is the one positioned further from the hairpin loop irrespective of whether the resulting stem formed a Watson–Crick or wobble base pair. Fourteen RNA hairpins containing group III bulge loops, either 3′ or 5′ of the hairpin loop, were optically melted and the thermodynamic parameters determined. The model developed to predict the influence of group III bulge loops on the stability of duplex formation was extended to predict the influence of bulge loops on hairpin stability.     

Solution structure of the DNA decamer duplex containing a 3'-T x T basepair of the cis-syn cyclobutane pyrimidine dimer: implication for the mutagenic property of the cis-syn dimer

The cis–syn dimer is the major DNA photoproduct produced by UV irradiation. In order to determine the origin of the mutagenic property of the cis–syn dimer, we used NMR restraints and molecular dynamics to determine the solution structure of a DNA decamer duplex containing a wobble pair between the 3′-T of the cis–syn dimer and the opposite T residue (CS/TA duplex). The solution structure of the CS/TA duplex revealed that the 3′-T·T base pair of the cis–syn dimer had base pair geometry that was significantly different from the canonical Watson–Crick base pair and caused destabilization and conformational distortion of its 3′-region. However, a 3′-T·A base pair at the cis–syn dimer within this related DNA decamer maintains the normal Watson–Crick base pair geometry and causes little distortion in the conformation of its 3′-side. Our results show that in spite of its stable hydrogen bonding, the insertion of a T residue opposite the 3′-T of the cis–syn dimer is inhibited by structural distortion caused by the 3′-T·T base pair. This may explain why the frequency of the 3′-T→A transversion, which is the major mutation produced by the cis–syn dimer, is only 4%.     

Modifications of the 3'-UTR stem-loop of infectious bursal disease virus are allowed without influencing replication or virulence

Many questions regarding the initiation of replication and translation of the segmented, double-stranded RNA genome of infectious bursal disease virus (IBDV) remain to be solved. Computer analysis shows that the non-polyadenylated extreme 3′-untranslated regions (UTRs) of the coding strand of both genomic segments are able to fold into a single stem–loop structure. To assess the determinants for a functional 3′-UTR, we mutagenized the 3′-UTR stem–loop structure of the B-segment. Rescue of infectious virus from mutagenized cDNA plasmids was impaired in all cases. However, after one passage, the replication kinetics of these viruses were restored. Sequence analysis revealed that additional mutations had been acquired in most of the stem–loop structures, which compensated the introduced ones. A rescued virus with a modified stem–loop structure containing four nucleotide substitutions, but preserving its overall secondary structure, was phenotypically indistinguishable from wild-type virus, both in vitro (cell culture) and in vivo (chickens, natural host). Sequence analysis showed that the modified stem–loop structure of this virus was fully preserved after four serial passages. Apparently, it is the stem–loop structure and not the primary sequence that is the functional determinant in the 3′-UTRs of IBDV.     

Structural effect of the anticancer agent 6-thioguanine on duplex DNA

The incorporation of 6-thioguanine (S6G) into DNA is an essential step in the cytotoxic activity of thiopurines. However, the structural effects of this substitution on duplex DNA have not been fully characterized. Here, we present the solution structures of DNA duplexes containing S6G opposite thymine (S6G·T) and opposite cytosine (S6G·C), solved by high-resolution NMR spectroscopy and restrained molecular dynamics. The data indicate that both duplexes adopt right-handed helical conformations with all Watson–Crick hydrogen bonding in place. The S6G·T structures exhibit a wobble-type base pairing at the lesion site, with thymine shifted toward the major groove and S6G displaced toward the minor groove. Aside from the lesion site, the helices, including the flanking base pairs, are not highly perturbed by the presence of the lesion. Surprisingly, thermal dependence experiments suggest greater stability in the S6G-T mismatch than the S6G-C base pair.     

Noncanonical G(syn)-G(anti) base pairs stabilized by sulphate anions in two X-ray structures of the (GUGGUCUGAUGAGGCC) RNA duplex

The structures of two crystal forms of the RNA 16-mer with the sequence GUGGUCUGAUGAGGCC, grown in the presence of a high concentration of sulphate ions, have been determined using synchrotron radiation at 1.4- and 2.0-Å resolution. RNA with this sequence is known as one of the two strands of the noncleavable form of the hammerhead ribozyme. In both crystal structures, two G(syn)–G(anti) noncanonical base pairs are observed in the middle of a 14 base-pair (bp) duplex having 5′-dangling GU residues. Both structures contain sulphate anions interacting with the G–G bp stabilizing G in its syn conformation and bridging the two RNA strands. In both cases the interactions take place in the major groove, although the anions are accommodated within different helix geometries, most pronounced in the changing width of the major groove. In one structure, where a single sulphate spans both G–G pairs, the major groove is closed around the anion, while in the other structure, where each of the two G–G pairs is associated with a separate sulphate, the groove is open. This work provides the first examples of a G–G pair in syn-anti conformation, which minimizes the purine–purine clash in the center of the duplex, while utilizing its residual hydrogen bonding potential in specific interactions with sulphate anions.     

Discriminating duplex and hairpin oligonucleotides using chemical shifts: application to the anticodon stem–loop of Escherichia coli tRNAPhe

A sensitive NMR spectroscopic method for detection of duplex forms of self-complementary nucleic acid sequences has been implemented. The G·U wobble base pair formed between a ¹⁵N-labeled strand and an unlabeled probe strand is used to identify the duplex. The guanine imino resonance, with its characteristic chemical shift, is detected using a 2D ¹⁵N–¹H heteronuclear multiple quantum coherence (HMQC) spectrum and provides a sensitive and unambiguous route to hairpin–duplex discrimination. The method has been used to identify the duplex and hairpin forms of an RNA oligonucleotide at concentrations of ~20 µM. This method has also been used to rule out possible duplex formation of an RNA oligonucleotide corresponding to the unmodified anticodon stem–loop of Escherichia coli tRNA^Phe and suggests that this hairpin has a 3 nt loop.     

Solution structure of a let-7 miRNA:lin-41 mRNA complex from C. elegans

let-7 microRNA (miRNA) regulates heterochronic genes in developmental timing of the nematode Caenorhabditis elegans. Binding of miRNA to messenger RNA (mRNA) and structural features of the complex are crucial for gene silencing. We herein present the NMR solution structure of a model mimicking the interaction of let-7 miRNA with its complementary site (LCS 2) in the 3′ untranslated region (3′-UTR) of the lin-41 mRNA. A structural study was performed by NMR spectroscopy using NOE restraints, torsion angle restraints and residual dipolar couplings. The 33-nt RNA construct folds into a stem–loop structure that features two stem regions which are separated by an asymmetric internal loop. One of the stems comprises a GU wobble base pair, which does not alter its overall A-form RNA conformation. The asymmetric internal loop adopts a single, well-defined structure in which three uracils form a base triple, while two adenines form a base pair. The 3D structure of the construct gives insight into the structural aspects of interactions between let-7 miRNA and lin-41 mRNA.     

Solution structure of a modified 2',5'-linked RNA hairpin involved in an equilibrium with duplex

The isomerization of phosphodiester functionality of nucleic acids from 3′,5′- to a less common 2′,5′-linkage influences the complex interplay of stereoelectronic effects that drive pseudorotational equilibrium of sugar rings and thus affect the conformational propensities for compact or more extended structures. The present study highlights the subtle balance of non-covalent forces at play in structural equilibrium of 2′,5′-linked RNA analogue, 3′-O-(2-methoxyethyl) substituted dodecamer *CG*CGAA*U*U*CG*CG, 3′-MOE-2′,5′-RNA, where all cytosines and uracils are methylated at C5. The NMR and UV spectroscopic studies have shown that 3′-MOE-2′,5′-RNA adopts both hairpin and duplex secondary structures, which are involved in a dynamic exchange that is slow on the NMR timescale and exhibits strand and salt concentration as well as pH dependence. Unusual effect of pH over a narrow physiological range is observed for imino proton resonances with exchange broadening observed at lower pH and relatively sharp lines observed at higher pH. The solution structure of 3′-MOE-2′,5′-RNA hairpin displays a unique and well-defined loop, which is stabilized by Watson–Crick A5·*U8 base pair and by n → π* stacking interactions of O4′ lone-pair electrons of A6 and *U8 with aromatic rings of A5 and *U7, respectively. In contrast, the stem region of 3′-MOE-2′,5′-RNA hairpin is more flexible. Our data highlight the important feature of backbone modifications that can have pronounced effects on interstrand association of nucleic acids.     

Mismatching base-pair dependence of the kinetics of DNA-DNA hybridization studied by surface plasmon fluorescence spectroscopy

Two single-stranded DNAs consisting of complementary base pairs except for one mismatching base pair (MM1) can form double-stranded DNA by molecular recognition. This type of duplex is not as stable as that formed by MM0. In order to add to a better understanding of the physical mechanism of the hybridization and dissociation processes at sensor (chip) surfaces, we studied the kinetics of the MM1 hybridization by surface plasmon fluorescence spectroscopy. Target DNA strands labelled with a fluorescent molecule Cy5 at the 5′ end and hybridizing with the surface-attached probe DNA can be excited by the strong optical field of a surface plasmon resonance mode. The emitted fluorescence can be detected with high sensitivity. The affinity of a duplex was found to depend on the chemical nature, i.e. G–G, G–T etc., and on the position of the mismatching base pair along the 15mer duplex.     

Structure–function analysis of the ribozymes of chrysanthemum chlorotic mottle viroid: a loop–loop interaction motif conserved in most natural hammerheads

Loop–loop tertiary interactions play a key role in the folding and catalytic activity of natural hammerhead ribozymes. Using a combination of NMR spectroscopy, site-directed mutagenesis and kinetic and infectivity analyses, we have examined the structure and function of loops 1 and 2 of the (+) and (–) hammerheads of chrysanthemum chlorotic mottle viroid RNA. In both hammerheads, loop 1 is a heptanucleotide hairpin loop containing an exposed U at its 5′ side and an extrahelical U at its 3′-side critical for the catalytic activity of the ribozyme in vitro and for viroid infectivity in vivo, whereas loop 2 has a key opened A at its 3′-side. These structural features promote a specific loop–loop interaction motif across the major groove. The essential features of this tertiary structure element, base pairing between the 5′ U of loop 1 and the 3′ A of loop 2, and interaction of the extrahelical pyrimidine of loop 1 with loop 2, are likely shared by a significant fraction of natural hammerheads.     

A directional nucleation-zipping mechanism for triple helix formation

A detailed kinetic study of triple helix formation was performed by surface plasmon resonance. Three systems were investigated involving 15mer pyrimidine oligonucleotides as third strands. Rate constants and activation energies were validated by comparison with thermodynamic values calculated from UV-melting analysis. Replacement of a T·A base pair by a C·G pair at either the 5′ or the 3′ end of the target sequence allowed us to assess mismatch effects and to delineate the mechanism of triple helix formation. Our data show that the association rate constant is governed by the sequence of base triplets on the 5′ side of the triplex (referred to as the 5′ side of the target oligopurine strand) and provides evidence that the reaction pathway for triple helix formation in the pyrimidine motif proceeds from the 5′ end to the 3′ end of the triplex according to the nucleation-zipping model. It seems that this is a general feature for all triple helices formation, probably due to the right-handedness of the DNA double helix that provides a stronger base stacking at the 5′ than at the 3′ duplex–triplex junction. Understanding the mechanism of triple helix formation is not only of fundamental interest, but may also help in designing better triple helix-forming oligonucleotides for gene targeting and control of gene expression.     

Identification of cis-Acting Nucleotides and a Structural Feature in West Nile Virus 3′-Terminus RNA That Facilitate Viral Minus Strand RNA Synthesis

The 3′-terminal nucleotides (nt) of West Nile virus (WNV) genomic RNA form a penultimate 16-nt small stem-loop (SSL) and an 80-nt terminal stem-loop (SL). These RNA structures are conserved in divergent flavivirus genomes. A previous in vitro study using truncated WNV 3′ RNA structures predicted a putative tertiary interaction between the 5′ side of the 3′-terminal SL and the loop of the SSL. Although substitution or deletion of the 3′ G (nt 87) within the SSL loop, which forms the only G-C pair in the predicted tertiary interaction, in a WNV infectious clone was lethal, a finding consistent with the involvement in a functionally relevant pseudoknot interaction, extensive mutagenesis of nucleotides in the terminal SL did not identify a cis-acting pairing partner for this SSL 3′ G. However, both the sequence and the structural context of two adjacent base pairs flanked by symmetrical internal loops in the 3′-terminal SL were shown to be required for efficient viral RNA replication. Nuclear magnetic resonance analysis confirmed the predicted SSL and SL structures but not the tertiary interaction. The SSL was previously reported to contain one of three eEF1A binding sites, and G87 in the SSL loop was shown to be involved in eEF1A binding. The nucleotides at the bottom part of the 3′-terminal SL switch between 3′ RNA-RNA and 3′-5′ RNA-RNA interactions. The data suggest that interaction of the 3′ SL RNA with eEF1A at three sites and a unique metastable structural feature may participate in regulating structural changes in the 3′-terminal SL.     

Isolation and characterization of thermodynamically stable and unstable RNA hairpins from a triloop combinatorial library

Hairpins are the most common elements of RNA secondary structure, playing important roles in RNA tertiary architecture and forming protein binding sites.Triloops are common in a variety of naturally occurring RNA hairpins, but little is known about their thermodynamic stability. Reported here are the sequences and thermodynamic parameters for a variety of stable and unstable triloop hairpins. Temperature gradient gel electrophoresis (TGGE) can be used to separate a simple RNA combinatorial library based on thermal stability [Bevilacqua, J. M., and Bevilacqua, P. C. (1998) Biochemistry 45, 15877-15884]. Here we introduce the application of TGGE to separating and analyzing a complex RNA combinatorial library based on thermal stability, using an RNA triloop library. Several rounds of in vitro selection of an RNA triloop library were carried out using TGGE, and preferences for exceptionally stable and unstable closing base pairs and loop sequences were identified. For stable hairpins, the most common closing base pair is CG, and U-rich loop sequences are preferred. Closing base pairs of GC and UA result in moderately stable hairpins when combined with a stable loop sequence. For unstable hairpins, the most common closing base pairs are AU and UG, and U-rich loop sequences are no longer preferred. In general, the contributions of the closing base pair and loop sequence to overall hairpin stability appear to be additive. Thermodynamic parameters for individual hairpins determined by UV melting are generally consistent with outcomes from selection experiments, with hairpins containing a CG closing base pair having a DeltaDeltaG degrees (37) 2.1-2.5 kcal/mol more favorable than hairpins with other closing base pairs. Sequences and thermodynamic rules for triloop hairpins should aid in RNA structure prediction and determination of whether naturally occurring triloop hairpins are thermodynamically stable.