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.     

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.     

High-resolution NMR structure of an RNA model system: the 14-mer cUUCGg tetraloop hairpin RNA

We present a high-resolution nuclear magnetic resonance (NMR) solution structure of a 14-mer RNA hairpin capped by cUUCGg tetraloop. This short and very stable RNA presents an important model system for the study of RNA structure and dynamics using NMR spectroscopy, molecular dynamics (MD) simulations and RNA force-field development. The extraordinary high precision of the structure (root mean square deviation of 0.3 Å) could be achieved by measuring and incorporating all currently accessible NMR parameters, including distances derived from nuclear Overhauser effect (NOE) intensities, torsion-angle dependent homonuclear and heteronuclear scalar coupling constants, projection-angle-dependent cross-correlated relaxation rates and residual dipolar couplings. The structure calculations were performed with the program CNS using the ARIA setup and protocols. The structure quality was further improved by a final refinement in explicit water using OPLS force field parameters for non-bonded interactions and charges. In addition, the 2′-hydroxyl groups have been assigned and their conformation has been analyzed based on NOE contacts. The structure currently defines a benchmark for the precision and accuracy amenable to RNA structure determination by NMR spectroscopy. Here, we discuss the impact of various NMR restraints on structure quality and discuss in detail the dynamics of this system as previously determined.     

Structure and dynamics of phosphate linkages and sugars in an abasic hexaloop RNA hairpin

Hairpins containing hexaloops are well represented among the diverse conformations adopted by the RNA molecules. To investigate the intrinsic properties of a backbone submitted to a hexaloop fold, we present here a molecular dynamics study of an abasic hexaloop closed by an A-form 6 basepair stem. The analysis of the 23 ns trajectory made in explicit solvent shows that both the sugars and the torsion angles in the loop undergo numerous conformational transitions. The south sugars, although not in a majority, are the major actors of the loop stretching. The five torsion angles, epsilon, zeta, alpha, beta, and gamma, are unequally variable, and only zeta and alpha exhibit trimodal distributions. The analysis of the phosphate linkages in terms of epsilonzeta'-alpha'-beta'-gamma-combinations allows us to define five conformational families, each one composed of one major substate in equilibrium with several less populated ones. The transitions between the substates within a family follow specific pathways involving the angles epsilon, zeta, and alpha. Thus, this work reveals that the backbone conformational space is both reduced and ordered even in a hexaloop devoid of bases.     

Structure of a U.U pair within a conserved ribosomal RNA hairpin.

A conserved hairpin corresponding to nt 1057-1081 of large subunit rRNA (Escherichia coli numbering) is part of a domain targeted by antibiotics and ribosomal protein L11. The stem of the hairpin contains a U.U juxtaposition, found as either U.U or U.C in virtually all rRNA sequences. This hairpin has been synthesized and most of the aromatic and sugar protons were assigned by two-dimensional proton NMR. Distances and sugar puckers deduced from the NMR data were combined with restrained molecular dynamics calculations to deduce structural features of the hairpin. The two U residues are stacked in the helix, form one NH3-O4 hydrogen bond and require an extended backbone conformation (trans alpha and gamma) at one of the U nucleotides. The hairpin loop, UAGAAGC closed by a U-A pair, is the same size as tRNA anticodon loops, but not as well ordered.     

Characterization of RNA hairpin loop stability.

Fifteen RNA hairpins that share the same stem sequence and have homopolymer loops of A, C and U residues which vary in length from three to nine nucleotides were synthesized and their thermal stabilities determined. Tm varies as a function of loop size but is almost independent of loop composition. Loops of four or five nucleotides are found to be the most stable loop size. This is consistent with the observation that four-membered loops are the most prevalent loop size in 16S-like RNAs. The contribution of each loop to hairpin stability was calculated by subtracting the known contribution of the helical stem. These data should be useful for predicting the stability of other hairpins.     

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.     

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.     

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.     

A phenanthrene modified RNA hairpin

The influence of hairpin loop replacement with the phenanthrene moiety in RNA was investigated. The stability of this novel structure was compared to a hairpin with a U(4) loop, an extra stable tetra-loop (UUCG), and an analogous phenanthrene modified DNA hairpin. Thermal denaturation experiments and CD spectra were used to study the structure and stability of the modified hairpin.     

Rapid one-step construction of hairpin RNA

Hairpin RNA (hpRNA) is commonly used for gene-function exploration and gene engineering. In this study, a novel method was developed to construct intron-containing hairpin RNA (ihpRNA) rapidly and efficiently based on Overlap Extension PCR (OE-PCR). This method, Mixed One-step OE-PCR (MOOE-PCR), can amplify two inverted repeats of DNA fragments and a spliceable intron in parallel, and then assemble them to generate ihpRNA constructs in the same tube without the purification of intermediate products. This method required a PCR process of 38-40 cycles and ordinary PCR reagents. A total of 10 ihpRNA constructs were amplified successfully using this method, with the stems ranging from 50 bp to 484 bp in length. Our results suggest that this novel method is a useful strategy for constructing ihpRNA.     

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.     

Construction of equalized short hairpin RNA library from human brain cDNA

Short hairpin RNA (shRNA) library is a powerful new tool for high-throughput loss-of-function genetic screens in mammalian cells. An shRNA library can be constructed from synthetic oligonucleotides or enzymatically cleaved natural cDNA. Here, we describe a new method for constructing equalized shRNA libraries from cDNA. First, enzymatically digested cDNA fragments are equalized by a suppression PCR-based method modified from suppression subtractive hybridization. The efficiency of equalization was confirmed by quantitative real-time PCR. The fragments are then converted into an shRNA library by a series of enzymatic treatments. With this new technology, we constructed a library from human brain cDNA. Sequence analysis showed that most of the randomly selected clones had inverted repeat sequences converted from different cDNA. After transfecting HEK 293T cells and detecting gene expression, three out of eight clones were demonstrated to significantly inhibit their target genes.     

Dynamics of the RNA hairpin GNRA tetraloop

The dynamics of RNA hairpin tetraloops of the GNRA type [sequence G- any ribonucleotide (N)-purine (R)-A] was analyzed by fluorescence spectroscopy and by fluorescence-detected temperature-jump relaxation, using RNA oligomers with 2-aminopurine (2AP) substituted in two different positions of the loop sequence, Gp2APpApA (HP1) and GpAp2APpA (HP2), as indicator. The fluorescence of HP1 is much higher than that of HP2, indicating a lower degree of 2AP-stacking in HP1. Addition of Mg(2+) or Ca(2+) ions leads to an increase of fluorescence in HP1, whereas a decrease of fluorescence is observed in HP2. In both cases at least two ion-binding equilibria are required to fit titration data. T-jump experiments using fluorescence detection show a relaxation process with a time constant of 22 micros for HP1, whereas two relaxation processes with time constants 5 and 41 micros, are found for HP2. These results clearly demonstrate the existence of more than the single conformation state detected by NMR analysis. The T-jump amplitudes decrease with increasing bivalent ion concentration, indicating that one of the states is favored in the presence of bivalent ions. The loop relaxation processes are slower than standard stacking processes, probably because of activation barriers imposed by a restricted mobility of loop residues, and are assigned to a stacking rearrangement, probably between the 5' and the 3'-side. A similar process has been observed previously for the anticodon loop of tRNA(Phe). The rate constants of the transition are in the range of 10(4) s(-1) in the case of HP1. The data demonstrate the existence of structures that are not resolved by standard NMR because of fast exchange and are not found by X-ray analysis because of restrictions by crystal packing.     

RNA hairpin loop stability depends on closing base pair.

Thermodynamic parameters are reported for hairpin formation in 1 M NaCl by RNA sequences of the type GGXAUAAUAYCC, where X and Y are CG, GC, AU, UA, GU, or UG. A nearest neighbor analysis of the data indicates the free energy change for loop formation at 37 degrees C, delta degrees Gl,37, averages 3.4 kcal/mol for hairpin loops closed with C.G, G.C, and G.U pairs. In contrast, delta G degree l,37 averages 4.6 kcal/mol for loops closed with A.U, U.A, or U.G pairs. Thus the stability of an RNA hairpin depends on the closing base pair. The hairpin with a GA mismatch that is formed by GGCGUAAUAGCC is more stable than the corresponding hairpin with an AA mismatch. Thus hairpin stability also depends on loop sequence. These effects are not included in current algorithms for prediction of RNA structure from sequence.