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.     

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.     

Thermodynamic parameters for loop formation in RNA and DNA hairpin tetraloops.

We determined the melting temperatures (Tm) and thermodynamic parameters of 15 RNA and 19 DNA hairpins at 1 M NaCl, 0.01 M sodium phosphate, 0.1 mM EDTA, at pH 7. All these hairpins have loops of four bases, the most common loop size in 16S and 23S ribosomal RNAs. The RNA hairpins varied in loop sequence, loop-closing base pair (A.U, C.G, or G.C), base sequence of the stem, and stem size (four or five base pairs). The DNA hairpins varied in loop sequence, loop-closing base pair (C.G, or G.C), and base sequence of the four base-pair stem. Thermodynamic properties of a hairpin may be represented by nearest-neighbor interactions of the stem plus contributions from the loop. Thus, we obtained thermodynamic parameters for the formation of RNA and DNA tetraloops. For the tetraloops we studied, a free energy of loop formation (at 37 degrees C) of about +3 kcal/mol is most common for either RNA or DNA. There are extra stable loops with delta G degrees 37 near +1 kcal/mol, but the sequences are not necessarily the same for RNA and DNA. The closing base pair is also important; changing from C.G to G.C lowered the stability of several tetraloops in both RNA and DNA. These values will be useful in predicting RNA and DNA secondary structures.     

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.     

Circular dichroism studies of an oligodeoxyribonucleotide containing a hairpin loop made of a hexaethylene glycol chain: conformation and stability.

An oligodeoxyribonucleotide, d(GCTCACAAT-X-ATTGTGAGC), where X represents a hexaethylene glycol chain, was studied using circular dichroism spectroscopy. Its conformation and conformational stability were compared to those of compounds where X was replaced by four thymines and to the duplex of same sequence without loop. The compound with the hexaethylene glycol chain can form a hairpin looped structure as well as a bulged duplex structure. In both cases the duplex region of the oligodeoxyribonucleotide exhibits the same conformation. In similar conditions the oligodeoxyribonucleotide with a four thymines loop forms exclusively a hairpin structure. Comparison between the thermodynamic parameters (delta H, delta S, delta G) associated with the formation of the structure of the three compounds are presented. In the case of the compound with the hexaethylene glycol chain it is shown that the large increase in its melting temperature (by about 35 degrees C in our experimental conditions) when compared to the non looped structure is mainly due to the fact that its melting process is intramolecular (monomolecular) whereas the other one is bimolecular.     

Solution structure of the loop B domain from the hairpin ribozyme

The hairpin ribozyme is a small catalytic RNA with a unique two-domain structure. Here we present the solution structure of the loop B domain of the hairpin ribozyme, which contains most of the catalytically essential nucleotides. The 38-nucleotide domain contains a 16-nucleotide internal loop that forms one of the largest non-Watson-Crick segments of base pairing thus far determined by either NMR or crystallography. Since the solution structure of the smaller loop A domain has been previously solved, an NMR structure-based model of the 22,000 Mr hairpin ribozyme-substrate open complex emerges by joining the two domain structures. Strikingly, catalytically essential functional groups for the loop B domain are concentrated within an expanded minor groove, presenting a clear docking surface for the loop A domain.     

A specific RNA hairpin loop structure binds the RNA recognition motifs of the Drosophila SR protein B52.

B52, also known as SRp55, is a member of the Drosophila melanogaster SR protein family, a group of nuclear proteins that are both essential splicing factors and specific splicing regulators. Like most SR proteins, B52 contains two RNA recognition motifs in the N terminus and a C-terminal domain rich in serine-arginine dipeptide repeats. Since B52 is an essential protein and is expected to play a role in splicing a subset of Drosophila pre-mRNAs, its function is likely to be mediated by specific interactions with RNA. To investigate the RNA-binding specificity of B52, we isolated B52-binding RNAs by selection and amplification from a pool of random RNA sequences by using full-length B52 protein as the target. These RNAs contained a conserved consensus motif that constitutes the core of a secondary structural element predicted by energy minimization. Deletion and substitution mutations defined the B52-binding site on these RNAs as a hairpin loop structure covering about 20 nucleotides, which was confirmed by structure-specific enzymatic probing. Finally, we demonstrated that both RNA recognition motifs of B52 are required for RNA binding, while the RS domain is not involved in this interaction.     

Solution structure of an RNA internal loop with three consecutive sheared GA pairs

Internal loops in RNA are important for folding and function. Many folding motifs are internal loops containing GA base pairs, which are usually thermodynamically stabilizing, i.e., contribute favorable free energy to folding. Understanding the sequence dependence of folding stability and structure in terms of molecular interactions, such as hydrogen bonding and base stacking, will provide a foundation for predicting stability and structure. Here, we report the NMR structure of the oligonucleotide duplex, 5'GGUGGAGGCU3'/3'PCCGAAGCCG5' (P = purine), containing an unusually stable and relatively abundant internal loop, 5'GGA3'/3'AAG5'. This loop contains three consecutive sheared GA pairs (trans Hoogsteen/Sugar edge AG) with separate stacks of three G's and three A's in a row. The thermodynamic consequences of various nucleotide substitutions are also reported. Significant destabilization of approximately 2 kcal/mol at 37 degrees C is found for substitution of the middle GA with AA to form 5'GAA3'/3'AAG5'. This destabilization correlates with a unique base stacking and hydrogen-bonding network within the 5'GGA3'/3'AAG5' loop. Interestingly, the motifs, 5'UG3'/3'GA5' and 5'UG3'/3'AA5', have stability similar to 5'CG3'/3'GA5' even though UG and UA pairs are usually less stable than CG pairs. Consecutive sheared GA pairs in the 5'GGA3'/3'AAG5' loop are preorganized for potential tertiary interactions and ligand binding.     

A test of the model to predict unusually stable RNA hairpin loop stability

To investigate the accuracy of a model [Giese et al., 1998, Biochemistry37:1094-1100 and Mathews et al., 1999, JMol Biol 288:911-940] that predicts the stability of RNA hairpin loops, optical melting studies were conducted on sets of hairpins previously determined to have unusually stable thermodynamic parameters. Included were the tetraloops GNRA and UNCG (where N is any nucleotide and R is a purine), hexaloops with UU first mismatches, and the hairpin loop of the iron responsive element, CAGUGC. The experimental values for the GNRA loops are in excellent agreement (deltaG degrees 37 within 0.2 kcal/mol and melting temperature (TM) within 4 degrees C) with the values predicted by the model. When the UNCG hairpin loops are treated as tetraloops, and a bonus of 0.8 kcal/mol included in the prediction to account for the extra stable first mismatch (UG), the measured and predicted values are also in good agreement (deltaG degrees 37 within 0.7 kcal/mol and TM within 3 degrees C). Six hairpins with unusually stable UU first mismatches also gave good agreement with the predictions (deltaG degrees 37 within 0.5 kcal/mol and TM within 8 degrees C), except for hairpins closed by wobble base pairs. For these hairpins, exclusion of the additional stabilization term for UU first mismatches improved the prediction (AG degrees 37 within 0.1 kcal/mol and TM within 3 degrees C). Hairpins with the iron-responsive element loop were not predicted well by the model, as measured deltaG degrees 37 values were at least 1 kcal/mol greater than predicted.     

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.     

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 conformation of tuftsin.

Tuftsin, a natural linear tetrapeptide (Thr-Lys-Pro-Arg) of potential antitumor activity, has been studied in DMSO-d6 solution by 2D NMR spectroscopy. 1H and 13C spectra show the presence of two families of conformations characterized by a trans or cis Lys-Pro bond, respectively. The family of conformers containing the cis peptide bond is a mixture of extended structures as expected for a short linear peptide. On the contrary, the trans isomer appears to be a rigid, folded conformer, as indicated by crucial NOEs and by the exceptionally low temperature coefficient of Arg NH. Analysis of the solution data by means of energy calculations leads to a unique structure, characterized by a Lys-Pro inverse gamma-turn.     

Solution conformation of a bulged adenosine base in an RNA duplex by relaxation matrix refinement

Bulges are common structural motifs in RNA secondary structure and are thought to play important roles in RNA-protein and RNA-drug interactions. Adenosine bases are the most commonly occurring unpaired base in double helical RNA secondary structures. The solution conformation and dynamics of a 25-nucleotide RNA duplex containing an unpaired adenosine, r(GGCAGAGUGCCGC): r(GCGGCACCUGCC) have been studied by NMR spectroscopy and MORASS iterative relaxation matrix structural refinement. The results show that the bulged adenosine residue stacks into the RNA duplex with little perturbation around the bulged region. Most of the bases in the RNA duplex adopt C(3)'-endo conformation, exhibiting the N-type sugar pucker as found in the A form helices. The sugars of the bulged residue and the 5' flanking residue to it are found to exhibit C(2)'-endo conformation. None of the residues are in syn conformation.     

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.     

Triplex-forming oligonucleotides trigger conformation changes of a target hairpin sequence.

We used a DNA duplex formed between the 5' end of a 69mer (69T) and an 11mer (OL7) as a substrate for BamHI. The former oligonucleotide folds into a hairpin structure, the stem of which contains a stretch of pyrimidines in one strand and consequently a stretch of purines in the other strand. The oligomer 69T was used as a target for complementary oligodeoxypyrimidines made of 10 nt (OL1), 16 nt (OL5) or 26 nt (OL2) which can engage the same 10 pyrimidine-purine-pyrimidine triplets with the 69T hairpin stem. Although the binding site of OL7 did not overlap that of OL1, OL2 or OL5, the BamHI activity on 69T-OL7 complexes was drastically modified in the presence of these triplex-forming oligomers: OL1 abolished the cleavage by BamHI whereas OL5 and OL2 strongly increased it. Using footprinting assays and point-mutated oligonucleotides we demonstrated that these variations were due to different conformations of the 69T-OL7 complex induced by the binding of oligomers OL1, OL2 or OL5. Therefore, oligonucleotides can act as structural switchers, offering one additional mode for modulating gene expression.