Structure of an RNA hairpin from HRV-14.

The 5' noncoding region of the picornaviral genome begins with a cloverleaf which is required for viral replication, due at least in part to an interaction with the viral RNA polymerase as part of a fusion with the predominant viral protease. The necessary region of the cloverleaf has previously been narrowed to a highly conserved stem-loop. The solution structure of a 14-nucleotide RNA hairpin, which is part of the conserved stem-loop from human rhinovirus isotype 14, is presented here. The secondary structure of the hairpin is identical to predictions: a five base pair stem is bounded by a triloop with sequence UAU. However, the fold of the triloop is novel, with stacking of the second loop base onto the closing base pair of the stem, and deviations from A form geometry are introduced into the stem regions bordering the triloop, particularly on the 3' side. These deviations and the associated triloop structure could help to explain the distinct sequence conservation and mutational analysis data observed for the stem region of the hairpin, as compared to a second sequentially similar stem in the intact stem-loop.     

Structure of a small RNA hairpin.

The hairpin stem-loop form of the RNA oligonucleotide rCGC(UUU)GCG has been studied by NMR spectroscopy. In 10 mM phosphate buffer this RNA molecule forms a unimolecular hairpin with a stem of three base pairs and a loop of three uridines, as judged by both NMR and UV absorbance melting behavior. Distance and torsion angle restraints were determined using homonuclear proton-proton and heteronuclear proton-phosphorus 2-D NMR. These values were used in restrained molecular dynamics to determine the structure of the hairpin. The stem has characteristics of A-form geometry, although distortion from A-form occurs in the 3'-side of the stem, presumably to aid in accommodating the small loop. The loop nucleotides adopt C2'-endo conformations. NOE's strongly suggest stacking of the uracils with the stem, especially the first uracil on the 5'-side of the loop. The reversal of the chain direction in the loop seems to occur between U5 and U6. Loop structures produced by molecular dynamics simulations had a wide range of conformations and did not show stacking of the uracils. A flexible loop with significant dynamics is consistent with all the data.     

Effects of base substitutions in an RNA hairpin from molecular dynamics and free energy simulations

Contributions of individual interactions in the GGCGCAAGCC hairpin containing a GCAA tetraloop were studied by computer simulations using base substitutions. The G in the first tetraloop position was replaced by inosine (I) or adenosine (A), and the G in the C-G basepair closing the tetraloop was replaced by I. These substitutions eliminate particular hydrogen bonds proposed in the nuclear magnetic resonance model of the GCAA tetraloop. Molecular dynamics simulations of the GCAA tetraloop in aqueous solvent displayed a well-defined hydrogen pattern between the first and last loop nucleotides (G and A) stabilized by a bridging water molecule. Substitution of G-->I in the basepair closing the tetraloop did not significantly influence the loop structure and dynamics. The ICAA loop maintained the overall structure, but displayed variation in the hydrogen-bond network within the tetraloop itself. Molecular dynamics simulations of the ACAA loop led to conformational heterogeneity of the resulting structures. Changes of hairpin formation free energy associated with substitutions of individual bases were calculated by the free energy perturbation method. The calculated decrease of the hairpin stability upon G-->I substitution in the C-G basepair closing the tetraloop was in good agreement with experimental thermodynamic data. Our theoretical estimates for G-->I and G-->A mutations located in the tetraloop suggest larger loop destabilization than corresponding experimental results. The extent of conformational sampling of the structures resulting from base substitutions and its impact on the calculated free energy was discussed.     

Interactions of hairpin oligo-2'-O-methylribonucleotides containing methylphosphonate linkages with HIV TAR RNA

Methylphosphonate-modified oligo-2'-O-methylribonucleotides 15-20 nucleotides (nt) in length were prepared whose sequences are complementary to the 5' and 3' sides of the upper hairpin of HIV trans-acting response element (TAR) RNA. These anti-TAR oligonucleotides (ODNs) form stable hairpins whose melting temperatures (Tm) range from 55 degrees C to 80 degrees C. Despite their rather high thermal stabilities, the hairpin oligo-2'-O-methylribonucleotides formed very stable complexes with TAR RNA, with dissociation constants in the nanomolar concentration range at 37 degrees C. The affinities of the hairpin oligomers for TAR RNA were influenced by the positions of the methylphosphonate linkages. The binding affinity was reduced approximately 17-fold by the presence of two methylphosphonate linkages in the TAR loop complementary region (TLCR) of the oligomer, whereas methylphosphonate linkages outside this region increased binding affinity approximately 3-fold. The configurations of the methylphosphonate linkages in the TLCR also affected binding affinity, with the RpRp isomer showing significantly higher binding than the SpSp isomer. In addition to serving as probes of the interactions between the oligomer and TAR RNA, the presence of the methylphosphonate linkages in combination with the hairpin structure increases the resistance of these oligomers to degradation by exonucleases found in mammalian serum. The combination of high binding affinity and nuclease resistance of the hairpin ODNs containing methylphosphonate linkages suggests their potential utility as antisense compounds.     

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.     

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.     

The chemical stability of abasic RNA compared to abasic DNA

We describe the synthesis of an abasic RNA phosphoramidite carrying a photocleavable 1-(2-nitrophenyl)ethyl (NPE) group at the anomeric center and a triisopropylsilyloxymethyl (TOM) group as 2′-O-protecting group together with the analogous DNA and the 2′-OMe RNA abasic building blocks. These units were incorporated into RNA-, 2′-OMe-RNA- and DNA for the purpose of studying their chemical stabilities towards backbone cleavage in a comparative way. Stability measurements were performed under basic conditions (0.1 M NaOH) and in the presence of aniline (pH 4.6) at 37°C. The kinetics and mechanisms of strand cleavage were followed by High pressure liquid chromotography and ESI-MS. Under basic conditions, strand cleavage at abasic RNA sites can occur via β,δ-elimination and 2′,3′-cyclophosphate formation. We found that β,δ-elimination was 154-fold slower compared to the same mechanism in abasic DNA. Overall strand cleavage of abasic RNA (including cyclophosphate formation) was still 16.8 times slower compared to abasic DNA. In the presence of aniline at pH 4.6, where only β,δ-elimination contributes to strand cleavage, a 15-fold reduced cleavage rate at the RNA abasic site was observed. Thus abasic RNA is significantly more stable than abasic DNA. The higher stability of abasic RNA is discussed in the context of its potential biological role.     

Structure and dynamics of an RNA tetraloop: a joint molecular dynamics and NMR study

Molecular dynamics simulations of the RNA tetraloop 5'-CGCUUUUGCG-3' with high melting temperature and significant conformational heterogeneity in explicit water solvent are presented and compared to NMR studies. The NMR data allow for a detailed test of the theoretical model, including the quality of the force field and the conformational sampling. Due to the conformational heterogeneity of the tetraloop, high temperature (350 K) and locally enhanced sampling simulations need to be invoked. The Amber98 force field leads to a good overall agreement with experimental data. Based on NMR data and a principal component analysis of the 350 K trajectory, the dynamic structure of the tetraloop is revealed. The principal component free energy surface exhibits four minima, which correspond to well-defined conformational structures that differ mainly by their base stacking in the loop region. No correlation between the motion of the sugar rings and the stacking dynamics of the loop bases is found.     

RNA simulations: probing hairpin unfolding and the dynamics of a GNRA tetraloop

Simulations of an RNA hairpin containing a GNRA tetraloop were conducted to allow the characterization of its secondary structure formation and dynamics. Ten 10 ns trajectories of the folded hairpin 5'-GGGC[GCAA]GCCU-3' were generated using stochastic dynamics and the GB/SA implicit solvent model at 300 K. Overall, we find the stem to be a very stable subunit of this molecule, whereas multiple loop conformations and transitions between them were observed. These trajectories strongly suggest that extension of the C6 base away from the loop occurs cooperatively with an N-type-->S-type sugar pucker conversion in that residue and that similar pucker transitions are necessary to stabilize other looped-out bases. In addition, a short-lived conformer with an extended fourth loop residue (A8) lacking this stabilizing 2'-endo pucker mode was observed. Results of thermal perturbation at 400 K support this model of loop dynamics. Unfolding trajectories were produced using this same methodology at temperatures of 500 to 700 K. The observed unfolding events display three-state behavior kinetically (including native, globular, and unfolded populations) and, based on these observations, we propose a folding mechanism that consists of three distinct events: (i) collapse of the random unfolded structure and sampling of the globular state; (ii) passage into the folded region of configurational space as stem base-pairs form and gain helicity; and (iii) attainment of proper loop geometry and organization of loop pairing and stacking interactions. These results are considered in the context of current experimental knowledge of this and similar nucleic acid hairpins.     

Photochemically induced RNA and DNA abasic sites

Two phosphoramidite building blocks were synthesized that can easily be deprotected by UV light to reveal natural abasic sites in oligoribonucleotides as well as in oligodeoxyribonucleotides. Another building block which releases a 2'-O-methylated abasic site upon UV radiation is also described.     

Biased hypermutagenesis associated with mutations in an untranslated hairpin of an RNA virus

The mutation frequency of Turnip crinkle virus can increase 12-fold without inducing error catastrophe. Lesions in a hairpin repressor frequently reverted and led to second-site alterations biased for specific mutations. These results suggest that the hairpin may also function as an RNA chaperone to properly fold the RNA-dependent RNA polymerase.     

Molecular mechanics and dynamics of an abasic frameshift in DNA and comparison to NMR data

In a previous publication (Ph. Cuniasse, L.C. Sowers, R. Eritja, B. Kaplan, M.F. Goodman, J.A.H. Cognet, M. Le Bret, W. Guschlbauer and G.V. Fazakerley, Biochemistry 28, 2018 (1989), we determined by two dimensional NMR studies and molecular mechanics calculations the three-dimensional structure of a non-selfcomplementary oligonucleotide: [sequence; see text] where dr, at the center of the first strand, is a model abasic site. In order to explain all the results arising from NMR measurements, we found that an equilibrium between two conformations was necessary. These conformations differ mainly by the sugar pucker of G5 which is C2' endo or C3' endo. The latter is stabilized by addition of counterions between phosphate residues P3 and P4. In this paper, we have constructed systematically, all possible structures as a function of torsion angles delta of dr4 and of G5 by molecular mechanics in the presence or absence of counterions. Since these conformations were not forced with NMR distance measurements, this method allows detailed comparisons between all possible conformations and NMR data. Maps of contour lines of the potential energy, of fits to NMR distance measurements, and of helical twist as a function of torsion angles delta of dr4 and of G5 unravel the difficulties associated with the study of the G5 sugar pucker conformation equilibrium. Sugar puckers and proton distances are very sensitive criteria to monitor molecular dynamics. Relying on these experimental criteria, we have tested many molecular dynamics preparation phases and we propose a new warm-up and equilibration procedure for molecular dynamics. Thus we show with a 290 ps molecular dynamic run that G5 is in conformational equilibrium and that all NMR data are well reproduced.     

NMR (13)C-relaxation study of base and sugar dynamics in GCAA RNA hairpin tetraloop

Intramolecular dynamics of a 14-mer RNA hairpin including GCAA tetraloop was investigated by (13)C NMR relaxation. R(1) and R(1rho) relaxation rates were measured for all protonated base carbons as well as for C1' carbons of ribose sugars at several magnetic field strengths. The data has been interpreted in the framework of modelfree analysis [G. Lipari and A. Szabo. J Am Chem Soc 104, 4546-4559 (1982); G. Lipari and A. Szabo. J Am Chem Soc 104, 4559-4570 (1982)] characterizing the internal dynamics of the molecule by order parameters and correlation times for fast motions on picosecond to nanosecond time scale and by contributions of the chemical exchange. The fast dynamics reveals a rather rigid stem and a significantly more flexible loop. The cytosine and the last adenine bases in the loop as well as all the loop sugars exhibit a significant contribution of conformational equilibrium on microsecond to millisecond time scale. The high R(1rho) values detected on both base and sugar moieties of the loop indicate coordinated motions in this region. A semiquantitative analysis of the conformational equilibrium suggests the exchange rates on the order of 10(4) s(-1). The results are in general agreement with dynamics studies of GAAA loops by NMR relaxation and fluorescent spectroscopy and support the data on the GCAA loop dynamics obtained by MD simulations.     

Probing the structural hierarchy and energy landscape of an RNA T-loop hairpin

The T-loop motif is an important recurrent RNA structural building block consisting of a U-turn sub-motif and a UA trans Watson–Crick/Hoogsteen base pair. In the presence of a hairpin stem, the UA non-canonical base pair becomes part of the UA-handle motif. To probe the hierarchical organization and energy landscape of the T-loop, we performed replica exchange molecular dynamics (REMD) simulations of the T-loop in isolation and as part of a hairpin. Our simulations reveal that the isolated T-loop adopts coil conformers stabilized by base stacking. The T-loop hairpin shows a highly rugged energy landscape featuring multiple local minima with a transition state for folding consisting of partially zipped states. The U-turn displays a high conformational flexibility both when the T-loop is in isolation and as part of a hairpin. On the other hand, the stability of the UA non-canonical base pair is enhanced in the presence of the UA-handle. This motif is apparently a key component for stabilizing the T-loop, while the U-turn is mostly involved in long-range interaction. Our results suggest that the stability and folding of small RNA motifs are highly dependent on local context.     

Understanding the thermodynamic stability of an RNA hairpin and its mutant.

The thermodynamic stability of RNA hairpin loops has been a subject of considerable interest in the recent past (Wimberly et al., 1991). There have been experimental reports indicating that the hairpins with a C(UUCG)G loop sequence are thermodynamically very stable (Wimberly et al., 1991). We used the solution structure of GGAC(UUCG)GUCC (Cheong et al., 1990; Varani et al., 1991) as the starting conformation in our attempt to understand its thermodynamic stability. We carried out molecular dynamics/free energy simulations to understand the basis for the destabilization of the C(UUCG)G loop by mutating cytosine (C7)-->uracil. Because of the limited length of simulation and the presence of kinetic barriers (solvent intervention) to the uracil-->cytosine mutation, all of our computed free energy differences are based on multiple forward simulations. Based on these calculations we find that the cytosine-->uracil mutation in the loop destabilizes it by approximately 1.5kcal/mol relative to that of the reference state, an A-form RNA but with cytosine (C7) looped out. This is the same sign and magnitude as that observed in the thermodynamic studies carried out by Varani et al.(1991). We have carried out free energy component analysis to understand the effect of mutating the cytosine residue to uracil on the thermodynamic stability of the C(UUCG)G hairpin loops. Our calculations show that the most significant contribution to the stability is from the phosphate group linking U5 and U6, which favors the cytosine residue over uracil by about 6.0 kcal/mol. The residues U5, U6, and G8 in the loop region also contribute significantly to the stability. The contributions from the salt and solvent compensate each other, indicating the dynamic nature of interactions of the environment with the nucleic acid system and the coupling between these two components.     

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 of the UGAGAU hexaloop that braces Bacillus RNase P for action

Long-range interactions involving the P5.1 hairpin of Bacillus RNase P RNA are thought to form a structural truss to support RNA folding and activity. We determined the structure of this element by NMR and refined the structure using residual dipolar couplings from a sample weakly oriented in a dilute liquid crystalline mixture of polyethylene glycol and hexanol. Dipolar coupling refinement improved the global precision of the structure from 1.5 to 1.2 A (to the mean), revised the bend angle between segments of the P5.1 stem and corroborated the structure of the loop region. The UGAGAU hexaloop of P5.1 contains two stacks of bases on opposite sides of the loop, distinguishing it from GNRA tetraloops. The unusual conformation of the juxtaposed uracil residues within the hexaloop may explain their requirement in transactivation assays.