On loop folding in nucleic acid hairpin-type structures

In a series of studies, combining NMR, optical melting and T-jump experiments, it was found that DNA hairpins display a maximum stability when the loop part of the molecule comprises four or five nucleotide residues. This is in contrast with the current notion based on RNA hairpin studies, from which it had been established that a maximum hairpin stability is obtained for six or seven residues in the loop. Here we present a structural model to rationalize these observations. This model is based on the notion that to a major extent base stacking interactions determine the stability of nucleic acid conformations. The model predicts that loop folding in RNA is characterized by an extension of the base stacking at the 5'-side of the double helix by five or six bases; the remaining gap can then easily be closed by two nucleotides. Conversely, loop folding in DNA is characterized by extending base stacking at the 3'-side of the double helical stem by two or three residues; again bridging of the remaining gap can then be achieved by one or two nucleotides. As an example of loop folding in RNA the anticodon loop of yeast tRNAPhe is discussed. For the DNA hairpin formed by d(ATCCTAT4TAGGAT) it is shown that the loop structure obtained from molecular mechanics calculations obeys the above worded loop folding principles.     

Solution conformation of an RNA hairpin loop.

The hairpin conformation adopted by the RNA sequence 5'GCGAUUUCUGACCGCC3' has been studied by one- and two-dimensional NMR spectroscopy. Exchangeable imino spectra in 60 mM Na+ indicate that the hairpin has a stem of six base pairs (indicated by boldface type) and a loop of three nucleotides. NOESY spectra of nonexchangeable protons confirm the formation of the stem region. The duplex has an A-conformation and contains an A.C apposition; a G.U base pair closes the loop region. The stem nucleotides have C3'-endo sugar conformations, as expected of an A-form duplex, whereas the three loop nucleotides adopt C2'-endo sugar puckers. Stacking within the loop, C8 upon the sugar of U7, stabilizes the structure. The pH dependence of both the exchangeable and nonexchangeable NMR spectra is consistent with the formation of an A+.C base pair, protonated at the N1 position of adenine. The stability of the hairpin was probed by using absorbance melting curves. The hairpin structure with the A+.C base pair is about +2 kcal/mol less stable in free energy at 37 degrees C than the hairpin formed with an A.U pair replacing the A+.C pair.     

Conformational and model-building studies of the hairpin form of the mismatched DNA octamer d(m5C-G-m5C-G-T-G-m5C-G)

The hairpin form of the mismatched octamer d(m5C-G-m5C-G-T-G-m5C-G) was studied by means of NMR spectroscopy. In a companion study it is shown that the hairpin form of this DNA fragment consists of a structure with a stem of three Watson-Crick-type base pairs and a loop consisting of only two nucleotides. The non-exchangeable proton resonances were assigned by means of two-dimensional correlation spectroscopy and two-dimensional nuclear Overhauser effect spectroscopy. Proton-proton coupling constants were used for the conformational analysis of the deoxyribose ring and for some of the backbone torsion angles. From the two-dimensional NMR spectra and the coupling-constant analysis it is concluded that: (i) the stem of the hairpin exhibits B-DNA characteristics; (ii) the sugar rings are not conformationally pure, but display a certain amount of conformational flexibility; (iii) the stacking interaction in the stem of the hairpin is elongated from the 3'-side in a more or less regular fashion with the two loop nucleotides; (iv) at the 5'-side of the stem a stacking discontinuity occurs between the stem and the loop; (v) at the 5'-side of the stem the loop is closed by means of a sharp backbone turn which involves unusual gamma' and beta+ torsion angles in residue dG(6). The NMR results led to the construction of a hairpin-loop model which was energy-minimized by means of a molecular-mechanics program. The results clearly show that a DNA hairpin-loop structure in which the loop consists of only two nucleotides bridging the minor groove in a straightforward fashion, (i) causes no undue steric strain, and (ii) involves well-known conformational principles throughout the course of the backbone. The hairpin form of the title compound is compared with the hairpin form of d(A-T-C-C-T-A-T4-T-A-G-G-A-T), in which the central -T4- part forms a loop of four nucleotides. Both models display similarities as far as stacking interactions are concerned.     

Conformational feasibility of a hairpin with two purines in the loop. 5'-d-GGTACIAGTACC-3'

Structural feasibility and conformational requirements for the sequence 5'-d-GGTACIAGTACC-3' to adopt a hairpin loop with I6 and A7 in the loop are studied. It is shown that a hairpin loop containing only two nucleotides can readily be formed without any unusual torsional angles. Stacking is continued on the 5'-side of the loop, with the I6 stacked upon C5. The base A7, on the 3'-side of the loop, can either be partially stacked with I6 or stick outside without stacking. Loop closure can be achieved for both syn and anti conformations of the glycosidic torsions for G8 while maintaining the normal Watson-Crick base pairing with the opposite C5. All torsional angles in the stem fall within the standard B-family of DNA helical structures. The phosphodiesters of the loop have trans,trans conformations. Loop formation might require the torsion about the C4'-C5' bond of G8 to be trans as opposed to the gauche+ observed in B-DNA. These results are discussed in relation to melting temperature studies [Howard et al. (1991) Biochemistry (preceding paper in this issue)] that suggest the formation of very stable hairpin structures for this sequence.     

NMR structure and dynamics of the RNA-binding site for the histone mRNA stem-loop binding protein

The 3' end of replication-dependent histone mRNAs terminate in a conserved sequence containing a stem-loop. This 26-nt sequence is the binding site for a protein, stem-loop binding protein (SLBP), that is involved in multiple aspects of histone mRNA metabolism and regulation. We have determined the structure of the 26-nt sequence by multidimensional NMR spectroscopy. There is a 16-nt stem-loop motif, with a conserved 6-bp stem and a 4-nt loop. The loop is closed by a conserved U.A base pair that terminates the canonical A-form stem. The pyrimidine-rich 4-nt loop, UUUC, is well organized with the three uridines stacking on the helix, and the fourth base extending across the major groove into the solvent. The flanking nucleotides at the base of the hairpin stem do not assume a unique conformation, despite the fact that the 5' flanking nucleotides are a critical component of the SLBP binding site.     

Structural characterization of three RNA hexanucleotide loops from the internal ribosome entry site of polioviruses.

Structural characteristics of three RNA hairpins from the internal ribosome entry site of poliovirus mRNAs have been determined in solution by NMR. Complete proton, phosphorus and carbon resonance assignments were made for the three 16 nt hairpins. The loop sequences, 5'-AAUCCA , AAACCA and GAACCA, have been shown to be essential for viral mRNA translation. NOESY spectra for the three oligomers were very similar indicating a common three dimensional structure. Stems were A-type duplexes with C3'-endo sugar pucker. In the loops, sequential base stacking interactions were detected for all bases except between U8/A8 and C9, indicating a turn in the phosphodiester backbone at this point. Only one nucleotide, U8/A8, had a sugar pucker which deviated appreciably from C3'-endo. The final base in the loop, A11, exhibited an unusual gauche (-) gamma angle. An ensemble of 10 structures calculated for one hairpin using restrained molecular dynamics shows that the first three bases of the loop are turned so as to be exposed to the exterior of the molecule, while the remaining three bases are in an orientation approximating a continuation of the stem helix. Structure calculations and NMR relaxation measurements indicate that the loop apex is subject to considerable local dynamics.     

Solution structure of a purine rich hexaloop hairpin belonging to PGY/MDR1 mRNA and targeted by antisense oligonucleotides

A preferential target of antisense oligonucleotides directed against human PGY/MDR1 mRNA is a hairpin containing a stem with a G*U wobble pair, capped by the purine-rich 5'r(GGGAUG)3' hexaloop. This hairpin is studied by multidimensional NMR and restrained molecular dynamics, with special emphasis on the conformation of south sugars and non-standard phosphate linkages evidenced in both the stem and the loop. The hairpin is found to be highly structured. The G*U wobble pair, a strong counterion binding site, displays structural particularities that are characteristic of this type of mismatch. The upper part of the stem undergoes distortions that optimize its interactions with the beginning of the loop. The loop adopts a new fold in which the single-stranded GGGA purine tract is structured in A-like conformation stacked in continuity of the stem and displays an extensive hydrogen bonding surface for recognition. The remarkable hairpin stability results from classical inter- and intra-strand interactions reinforced by numerous hydrogen bonds involving unusual backbone conformations and ribose 2'-hydroxyl groups. Overall, this work emphasizes numerous features that account for the well-ordered structure of the whole hairpin and highlights the loop properties that facilitate interaction with antisense oligonucleotides.     

Structure and dynamics of the DNA hairpins formed by tandemly repeated CTG triplets associated with myotonic dystrophy.

Anomalous expansion of the DNA triplet (CTG)n causes myotonic dystrophy. Structural studies have been carried out on (CTG)n repeats in an attempt to better understand the molecular mechanism of repeat expansion. NMR and gel electrophoretic studies demonstrate the presence of hairpin structures for (CTG)5 and (CTG)6 in solution. The monomeric hairpin structure remains invariant over a wide range of salt concentrations (10-200 mM NaCl), DNA concentrations (micromolar to millimolar in DNA strand) and pH (6.0-7.5). The (CTG)n hairpin contains three bases in the loop when n is odd and four bases when n is even. For both odd and even n the stacking and pairing in the stem remain the same, i.e, two hydrogen bond T.T pairs stack with the neighboring G.C pairs. All the nucleotides in (CTG)5 and (CTG)6 adopt C2'-endo, anti conformations. Full-relaxation matrix analysis has been performed to derive the NOE distance constraints from NOESY experiments at seven different mixing times (25, 50, 75, 100, 125, 200 and 500 ms). NOESY-derived distance constraints were subsequently used in restrained molecular dynamics simulations to obtain a family of structures consistent with the NMR data. The theoretical order parameters are computed for H5-H6(cytosines) and H2'-H2" dipolar correlations for both (CTG)5 and (CTG)6 by employing the Lipari-Szabo formalism. Experimental data show that the cytosine in the loop of the (CTG)5 hairpin is slightly more flexible than those in the stem. The cytosine in the loop of the (CTG)6 hairpin is extremely flexible, implying that the dynamics of the four base loop is intrinsically different from that of the three base loop.     

NMR structure of 2′-O-(2-methoxyethyl) modified and C5-methylated RNA dodecamer duplex

The solution-state structure of 2′-O-(2-methoxyethly) substituted dodecamer r(*CG*CGAA*U*U*CG*C)d(G), 2′-MOE RNA, with all cytosines and uracils methylated at the C5-position has been determined by NMR spectroscopy. The chemical modifications were used to improve the oligonucleotide's drug-like properties. The 2′-MOE group drives pseudorotational equilibrium of the ribofuranose moiety to the N-type conformation and supposedly results in structural preorganization leading to high affinity of a modified oligonucleotide towards its complementary biological target, improved pharmacokinetic and toxicological properties. The high melting temperature of the antiparallel duplex structure adopted by 2′-MOE RNA was explained through the formation of a stable A-form RNA consistent with effective base-pairing and stacking interactions. The comparison of the solution-state structure with the crystal structure of a non-methylated analogue shows an increase in the stacking at the base pair steps for the C5-methylated 2′-MOE RNA duplex. The MOE substituents adopt a well-defined structure in the minor groove with the predominant gauche conformations around the ethylene bond.     

Loop and stem dynamics during RNA hairpin folding and unfolding

2-Aminopurine (2AP) is a fluorescent adenine analog that probes mainly base stacking in nucleic acids. We labeled the loop or the stem of the RNA hairpin gacUACGguc with 2AP to study folding thermodynamics and kinetics at both loci. Thermal melts and fast laser temperature jumps detected by 2AP fluorescence monitored the stability and folding/unfolding kinetics. The observed thermodynamic and kinetic traces of the stem and loop mutants, though strikingly different at a first glance, can be fitted to the same free-energy landscape. The differences between the two probe locations arise because base stacking decreases upon unfolding in the stem, whereas it increases in the loop. We conclude that 2AP is a conservative adenine substitution for mapping out the contributions of different RNA structural elements to the overall folding process. Molecular dynamics (MD) totaling 0.6 μsec were performed to look at the conformations populated by the RNA at different temperatures. The combined experimental data, and MD simulations lead us to propose a minimal four-state free-energy landscape for the RNA hairpin. Analysis of this landscape shows that a sequential folding model is a good approximation for the full folding dynamics. The frayed state formed initially from the native state is a heterogeneous ensemble of structures whose stem is frayed either from the end or from the loop.     

Multinuclear NMR studies of DNA hairpins. 2. Sequence-dependent structural variations

The solution conformation of three related DNA hairpins, each with five bases in the loop, is investigated by proton and phosphorus 2D NMR methods. The sequences of the three oligomers are d(CGCGTTGTTCGCG), d(CGCGTTTGTCGCG), and d(CTGCTCTTGTTGAGCAG). One pair of hairpins shares the same stem sequence but differs in the loop, and the appearance of an unusual phosphate torsion in the stem is found to depend on the sequence in the loop of the hairpin. The second pair of hairpins shares the same loop region but differs in the stem sequence in that the base pair which closes the loop is a C-G or G-C pair. The pattern of NOEs reveals that the stacking arrangement in the loop region depends on the base pair that closes the stem. These results suggest that hairpin loop conformation and dynamics are sensitive to small changes in the loop and adjacent stem sequences. These findings are discussed in relation to sequence-dependent thermodynamic changes that have been observed in RNA hairpins.     

Structural effects of m6A modification of the Xist A-repeat AUCG tetraloop and its recognition by YTHDC1

The A-repeat region of the lncRNA Xist is critical for X inactivation and harbors several N⁶-methyladenosine (m⁶A) modifications. How the m⁶A modification affects the conformation of the conserved AUCG tetraloop hairpin of the A-repeats and how it can be recognized by the YTHDC1 reader protein is unknown. Here, we report the NMR solution structure of the (m⁶A)UCG hairpin, which reveals that the m⁶A base extends 5′ stacking of the A-form helical stem, resembling the unmethylated AUCG tetraloop. A crystal structure of YTHDC1 bound to the (m⁶A)UCG tetraloop shows that the (m⁶A)UC nucleotides are recognized by the YTH domain of YTHDC1 in a single-stranded conformation. The m⁶A base inserts into the aromatic cage and the U and C bases interact with a flanking charged surface region, resembling the recognition of single-stranded m⁶A RNA ligands. Notably, NMR and fluorescence quenching experiments show that the binding requires local unfolding of the upper stem region of the (m⁶A)UCG hairpin. Our data show that m⁶A can be readily accommodated in hairpin loop regions, but recognition by YTH readers requires local unfolding of flanking stem regions. This suggests how m⁶A modifications may regulate lncRNA function by modulating RNA structure.     

Solution structure of the HIV-1 exon splicing silencer 3

Alternative splicing of the human immunodeficiency virus type 1 (HIV-1) genomic RNA is necessary to produce the complete viral protein complement, and aberrations in the splicing pattern impair HIV-1 replication. Genome splicing in HIV-1 is tightly regulated by the dynamic assembly/disassembly of trans host factors with cis RNA control elements. The host protein, heterogeneous nuclear ribonucleoprotein (hnRNP) A1, regulates splicing at several highly conserved HIV-1 3′ splice sites by binding 5′-UAG-3′ elements embedded within regions containing RNA structure. The physical determinants of hnRNP A1 splice site recognition remain poorly defined in HIV-1, thus precluding a detailed understanding of the molecular basis of the splicing pattern. Here, the three-dimensional structure of the exon splicing silencer 3 (ESS3) from HIV-1 has been determined using NMR spectroscopy. ESS3 adopts a 27-nucleotide hairpin with a 10-bp A-form stem that contains a pH-sensitive A⁺C wobble pair. The seven-nucleotide hairpin loop contains the high-affinity hnRNP-A1-responsive 5′-UAGU-3′ element and a proximal 5′-GAU-3′ motif. The NMR structure shows that the heptaloop adopts a well-organized conformation stabilized primarily by base stacking interactions reminiscent of a U-turn. The apex of the loop is quasi-symmetric with UA dinucleotide steps from the 5′-GAU-3′ and 5′-UAGU-3′ motifs stacking on opposite sides of the hairpin. As a step towards understanding the binding mechanism, we performed calorimetric and NMR titrations of several hnRNP A1 subdomains into ESS3. The data show that the UP1 domain forms a high-affinity (K_d = 37.8 ± 1.1 nM) complex with ESS3 via site-specific interactions with the loop.     

Folding of a DNA hairpin loop structure in explicit solvent using replica-exchange molecular dynamics simulations

Hairpin loop structures are common motifs in folded nucleic acids. The 5'-GCGCAGC sequence in DNA forms a characteristic and stable trinucleotide hairpin loop flanked by a two basepair stem helix. To better understand the structure formation of this hairpin loop motif in atomic detail, we employed replica-exchange molecular dynamics (RexMD) simulations starting from a single-stranded DNA conformation. In two independent 36 ns RexMD simulations, conformations in very close agreement with the experimental hairpin structure were sampled as dominant conformations (lowest free energy state) during the final phase of the RexMDs ( approximately 35% at the lowest temperature replica). Simultaneous compaction and accumulation of folded structures were observed. Comparison of the GCA trinucleotides from early stages of the simulations with the folded topology indicated a variety of central loop conformations, but arrangements close to experiment that are sampled before the fully folded structure also appeared. Most of these intermediates included a stacking of the C(2) and G(3) bases, which was further stabilized by hydrogen bonding to the A(5) base and a strongly bound water molecule bridging the C(2) and A(5) in the DNA minor groove. The simulations suggest a folding mechanism where these intermediates can rapidly proceed toward the fully folded hairpin and emphasize the importance of loop and stem nucleotide interactions for hairpin folding. In one simulation, a loop motif with G(3) in syn conformation (dihedral flip at N-glycosidic bond) accumulated, resulting in a misfolded hairpin. Such conformations may correspond to long-lived trapped states that have been postulated to account for the folding kinetics of nucleic acid hairpins that are slower than expected for a semiflexible polymer of the same size.     

Solution structure of the pseudo-5' splice site of a retroviral splicing suppressor

Control of Rous sarcoma virus RNA splicing depends in part on the interaction of U1 and U11 snRNPs with an intronic RNA element called the negative regulator of splicing (NRS). A 23mer RNA hairpin (NRS23) of the NRS directly binds U1 and U11 snRNPs. Mutations that disrupt base-pairing between the loop of NRS23 and U1 snRNA abolish its negative control of splicing. We have determined the solution structure of NRS23 using NOEs, torsion angles, and residual dipolar couplings that were extracted from multidimensional heteronuclear NMR spectra. Our structure showed that the 6-bp stem of NRS23 adopts a nearly A-form duplex conformation. The loop, which consists of 11 residues according to secondary structure probing, was in a closed conformation. U913, the first residue in the loop, was bulged out or dynamic, and loop residues G914-C923, G915-U922, and U916-A921 were base-paired. The remaining UUGU tetraloop sequence did not adopt a stable structure and appears flexible in solution. This tetraloop differs from the well-known classes of tetraloops (GNRA, CUYG, UNCG) in terms of its stability, structure, and function. Deletion of the bulged U913, which is not complementary to U1 snRNA, increased the melting temperature of the RNA hairpin. This hyperstable hairpin exhibited a significant decrease in binding to U1 snRNP. Thus, the structure of the NRS RNA, as well as its sequence, is important for interaction with U1 snRNP and for splicing suppression.     

Structural characterisation of a uracil containing hairpin DNA by NMR and molecular dynamics.

Three-dimensional (3D) structure of a hairpin DNA d-CTAGAGGATCCTTTUGGATCCT (22mer; abbreviated as U4-hairpin), which has a uracil nucleotide unit at the fourth position from the 5' end of the tetra-loop has been solved by NMR spectroscopy. The(1)H resonances of this hairpin have been assigned almost completely. NMR restrained molecular dynamics and energy minimisation procedures have been used to describe the 3D structure of the U4 hairpin. This study establishes that the stem of the hairpin adopts a right handed B-DNA conformation while the T(12)and U(15)nucleotide stack upon 3' and 5' ends of the stem, respectively. Further, T(14)stacks upon both T(12)and U(15)while T(13)partially stacks upon T(14). Very weak stacking interaction is observed between T(13)and T(12). All the individual nucleotide bases adopt ' anti ' conformation with respect to their sugar moiety. The turning phosphate in the loop is located between T(13)and T(14). The stereochemistry of U(15)mimics the situation where uracil would stack in a B-DNA conformation. This could be the reason as to why the U4-hairpin is found to be the best substrate for its interaction with uracil DNA glycosylase (UDG) compared to the other substrates in which the uracil is at the first, second and third positions of the tetra-loop from its 5' end, as reported previously.     

Molecular dynamics simulations of the complex between human U1A protein and hairpin II of U1 small nuclear RNA and of free RNA in solution.

RNA-protein interactions are essential to a wide range of biological processes. In this paper, a 0.6-ns molecular dynamics simulation of the sequence-specific interaction of human U1A protein with hairpin II of U1 snRNA in solution, together with a 1.2-ns simulation of the free RNA hairpin, is reported. Compared to the findings in the x-ray structure of the complex, most of the interactions remained stable. The nucleotide U8, one of the seven conserved nucleotides AUUGCAC in the loop region, was unusually flexible during the simulation, leading to a loss of direct contacts with the protein, in contrast to the situation in the x-ray structure. Instead the sugar-phosphate backbone of nucleotide C15 was found to form several interactions with the protein. Compared to the NMR structure of U1A protein complexed with the 3'-untranslated region of its own pre-mRNA, the protein core kept the same conformation, and in the two RNA molecules the conserved AUUGCAC of the loop and the closest CG base pair were located in very similar positions and orientations, and underwent very similar interactions with the protein. Therefore, a common sequence-specific interaction mechanism was suggested for the two RNA substrates to bind to the U1A protein. Conformational analysis of the RNA hairpin showed that the conformational changes of the RNA primarily occurred in the loop region, which is just involved in the sites of binding to the protein and in agreement with experimental observation. Both the loop and stem of the RNA became more ordered upon binding to the protein. It was also demonstrated that the molecular dynamics method could be successfully used to simulate the dynamical behavior of a large RNA-protein complex in aqueous solution, thus opening a path for the exploration of the complex biological processes involving RNA at a molecular level.     

Structure of 2',5'-linked tetraribonucleotide loops: a novel RNA motif

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.