Perturbation of DNA hairpins containing the EcoRI recognition site by hairpin loops of varying size and composition: physical (NMR and UV) and enzymatic (EcoRI) studies.

We have investigated loop-induced structural perturbation of the stem structure in hairpins d(GAATTCXnGAATTC) (X = A, T and n = 3, 4, 5 and 6) that contain an EcoRI restriction site in close proximity to the hairpin loop. Oligonucleotides containing either a T3 or a A3 loop were not hydrolyzed by the restriction enzyme and also showed only weak binding to EcoRI in the absence of the cofactor Mg2+. In contrast, hairpins with larger loops are hydrolyzed by the enzyme at the scission site next to the loop although the substrate with a A4 loop is significantly more resistant than the oligonucleotide containing a T4 loop. The hairpin structures with 3 loop residues were found to be thermally most stable while larger hairpin loops resulted in structures with lower melting temperatures. The T-loop hairpins are thermally more stable than the hairpins containing the same number of A residues in the loop. As judged from proton NMR spectroscopy and the thermodynamic data, the base pair closest to the hairpin loop did form in all cases studied. The hairpin loops did, however, affect the conformation of the stem structure of the hairpins. From 31P and 1H NMR spectroscopy we conclude that the perturbation of the stem structure is stronger for smaller hairpin loops and that the extent of the perturbation is limited to 2-3 base pairs for hairpins with T3 or A4 loops. Our results demonstrate that hairpin loops modulate the conformation of the stem residues close to the loop and that this in turn reduces the substrate activity for DNA sequence specific proteins.     

Stable formation of a pyrimidine-rich loop hairpin in a cruciform promoter.

We have determined the solution structure of a TCC-loop hairpin in the cruciform promoter for the bacteriophage N4 virion RNA polymerase (N4 vRNAP). This hairpin and its complementary GGA-loop hairpin are extruded at physiological superhelical density and are required for vRNAP recognition. Contrary to its complementary GGA-loop, the three pyrimidines in the TCC-loop are all unpaired. However, with the help of two juxtaposed stem Watson-Crick G.C base-pairs, each nucleotide in the loop employs a special method to stabilize the hairpin structure. The resulting structures display extensive loop base-stacking rearrangement yet minor backbone distortion, which is largely accomplished through some loop zeta and alpha torsional angle changes. Consistent with the structural studies, UV melting of the GAAGCTCCGCTTC hairpin revealed a higher melting temperature (66 degrees C) than that of the GAACGTCCCGTTC hairpin (58 degrees C) with reversed stem G.C base-pairs, indicating significant contribution from the extra three loop-stem H-bonds. Thermodynamic parameters DeltaG degrees 25of the GAAGCTCCGCTTC hairpin and its complementary GAAGCGGAGCTTC hairpin are -4.1 and -4. 3 kcal/mol respectively, indicating approximately equal contribution of each hairpin to the cruciform formation of the N4 virion RNA polymerase promoter. No significant loop dynamics in the microsecond to millisecond NMR time-scale was observed, and the abundant well-defined exchangeable and non-exchangeable proton NOEs allowed us to efficiently determine a well-converged family for the final structures of the TCC-loop hairpin.     

Complexes of DNA hairpins and a single-stranded oligonucleotide detected by affinity chromatography and mung bean nuclease cleavage

The emergence of a simple translation device consisting of an assembler strand (primordial mRNA) and RNA hairpins (primordial tRNA) is presumed to be an important step leading to the origin of life. The assumption of a non-enzymatic interaction of primordial tRNA and mRNA is experimentally approached. DNA hairpins containing five or more adenosine residues in the loop are able to bind to complementary oligonucleotides covalently bound to cellulose. The exact number of base pairs formed between the hairpins and the assembler strand is determined by two methods applied to DNA hairpin/assembler complexes. The melting temperature of a complex is measured and the cleavage pattern by nuclease from mung bean is determined. The loop of the smallest hairpin able to bind consists of five adenosine residues and only three base pairs are formed. This supports the idea of a primordial recognition similar to the contemporary codon-anticodon interaction.     

Use of ultra stable UNCG tetraloop hairpins to fold RNA structures: thermodynamic and spectroscopic applications.

RNA molecules of > 20 nucleotides have been the focus of numerous recent NMR structural studies. Several investigators have used the UNCG family of hairpins to ensure proper folding. We show that th UUCG hairpin has a minimum requirement of a two base-pair stem. Hairpins with a CG loop closing base pair and an initial 5'CG or 5'GC base pair have a melting temperature approximately 55 degrees C in 10 mM sodium phosphate. The high stability of even such small hairpins suggests that the hairpin can serve as a nucleation site for folding. For high resolution NMR work, the UNCG loop family (UACG in particular) provides excellent spectroscopic markers in one-dimensional exchangeable spectra, in two-dimensional COSY spectra and in NOESY spectra that clearly define it as forming a hairpin. This allows straightforward initiation of chemical shift assignments.     

Protonatable hairpins are conserved in the 5'-untranslated region of tymovirus RNAs.

The secondary structures of the 5'-untranslated region (5'-UTR) of five different tymoviruses have been determined by structure probing, computer prediction and sequence comparison. Despite large sequence differences, there are remarkable similarities in the secondary structure. In all viruses two or four hairpins are found, most of which contain a symmetrical internal loop consisting of adjacent C-C or C-A mismatches. Since it is known that such mismatches can be protonated and protonated cytosines play an important role in RNA-protein interactions in tymoviral virions, the influence of pH on the conformation of the internal loop was studied. UV melting experiments and 1-dimensional proton NMR at varying pH values and salt concentrations confirm that the hairpins can be protonated under relatively mild conditions. The hairpin found in the 5'-UTR of erysimum latent virus, which has an asymmetrical internal loop consisting of cytosines and uridines, shows comparable behaviour. It is concluded that all tymoviral RNAs contain protonatable hairpins in the 5'-UTR. Binding experiments with empty viral capsids, however, do not yet establish a role in capsid protein binding.     

Effects of Locked Nucleic Acid Substitutions on the Stability of Oligonucleotide Hairpins

An understanding of the stability of nucleic acid folding is critical for applications involving RNA viruses, small molecule–RNA binding, and therapeutics, for example. To explore factors that affect this stability, hairpins made from oligonucleotides containing both a GAAA tetraloop and three to five complements in the stem have been used as models where locked nucleic acids (LNAs) have been substituted into the sequence. UV spectroscopy was used to obtain melting curves in 20% by volume formamide, and the enthalpies and entropies of melting were determined. Although LNA substitutions typically increase the stability of a hybrid, we have found a decrease in stability for DNA and RNA GAAA hairpins when LNA is substituted into the loop. Tetraloops synthesized from natural bases show higher enthalpies and entropies of melting compared to the LNA substituted sequences indicating that LNA substitutions can destabilize a hairpin but stabilize the corresponding double stranded structure.     

RNA pseudoknots. Stability and loop size requirements.

The effects of ionic conditions, loop size and loop sequence on the formation of pseudoknots by RNA oligonucleotides have been investigated using biochemical and biophysical methods. An oligonucleotide with the sequence 5' GCGAUUUCUGACCGCUUUUUUGUCAG 3' and oligonucleotides with variations in the sequences of the two loop regions, denoted by bold face type, were studied. Each sequence with the potential to form a pseudoknot can also form two stable hairpins. The pseudoknot structure is stabilized relative to the hairpins by addition of Mg2+. Even in the presence of Mg2+, the pseudoknots formed by the sequences investigated are only marginally more stable (1.5 to 2 kcal mol-1 in free energy at 37 degrees C) than either of the constituent hairpins. The pseudoknot structure is the stable conformation in the presence of Mg2+ when the first loop region is at least three nucleotides and the second is at least four nucleotides. Further deletion of nucleotides from the loop regions stabilizes possible hairpin structures relative to the pseudoknot and equilibria among secondary and tertiary structures result.     

Conformational and thermodynamic effects of naturally occurring base methylations in a ribosomal RNA hairpin of Bacillus stearothermophilus.

The 3'-terminal colicin fragments of 16S ribosomal RNA were isolated from Bacillus stearothermophilus and from its kasugamycin-resistant (ksgA) derivative lacking N6-dimethylation of the two adjacent adenosines in a hairpin loop. The fragment from the ksgA strain still contains a naturally occurring N2-methylguanosine in the loop. An RNA molecule resembling the B. stearothermophilus colicin fragment but without modified nucleosides was synthesized in vitro using a DNA template and bacteriophage T7 RNA polymerase. Proton-NMR spectra of the RNAs were recorded at 500 MHz. The imino-proton resonances of base-paired G and U residues could be assigned on the basis of previous NMR studies of the colicin fragment of Escherichia coli and by a combination of methylation-induced shifts and thermal melting of base pairs. The assignments were partly confirmed by NOE measurements. Adenosine dimethylation in the loop has a distinct conformational effect on the base pairs adjoining the loop. The thermal denaturation melting curve of the enzymatically synthesized RNA fragment was also determined and the transition midpoint (tm) was found to be 73 degrees C at 15 mM Na+. A comparison with previously determined thermodynamic parameters for various colicin fragments demonstrates that base methylations in the loop lead to a relatively strong destabilization of the hairpin helix. In terms of free energy the positive contribution of the methylations are in the order of the deletion of one base pair from the stem. Other data show that recently published free-energy parameters do not apply for certain RNA hairpins.     

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.     

Role of loop residues and cations on the formation and stability of dimeric DNA G-quadruplexes

Formation of guanine-quadruplexes by four DNA oligonucleotides with common sequence dG4-loop-dG4 has been studied by a combination of NMR and UV spectroscopy. The loops consisted of 1',2'-dideoxyribose, propanediol, hexaethylene glycol, and thymine residues. The comparison of data on modified and parent oligonucleotides gave insight into the role of loop residues on formation and stability of dimeric G-quadruplexes. All modified oligonucleotides fold into dimeric fold-back G-quadruplexes in the presence of sodium ions. Multiple structures form in the presence of potassium and ammonium ions, which is in contrast to the parent oligonucleotide with dT4 loop. 15N-filtered 1H NMR spectra demonstrate that all studied G-quadruplexes exhibit three 15NH4(+) ion binding sites. Topology of intermolecular G-quadruplexes was evaluated by NMR measurements and diffusion experiments. The spherical, prolate-ellipsoid and symmetric cylinder models were used to interpret experimental translational diffusion constants in terms of diameters and lengths of unfolded oligonucleotides and their respective G-quadruplexes. UV melting and annealing curves show that oligonucleotides with non-nucleosidic loop residues fold faster, exhibit no hysteresis, and are less stable than dimeric d(G4T4G4)2 which can be attributed to the absence of H-bonds, stacking between loop residues and the outer G-quartets as well as cation-pi interactions. Oligonucleotide consisting of hexaethylene glycol linkage with only two phosphate groups in the loop exhibits higher melting temperature and more negative deltaH(o) and deltaG(o) values than oligonucleotides with four 1',2'-dideoxyribose or propanediol residues.     

Thermal stability, structural features, and B-to-Z transition in DNA tetraloop hairpins as determined by optical spectroscopy in d(CG)(3)T(4)(CG)(3) and d(CG)(3)A(4)(CG)(3) oligodeoxynucleotides

NMR and CD data have previously shown the formation of the T(4) tetraloop hairpin in aqueous solutions, as well as the possibility of the B-to-Z transition in its stem in high salt concentration conditions. It has been shown that the stem B-to-Z transition in T(4) hairpins leads to S (south)- to N (north)-type conformational changes in the loop sugars, as well as anti to syn orientations in the loop bases. In this article, we have compared by means of UV absorption, CD, Raman, and Fourier transform infrared (FTIR), the thermodynamic and structural properties of the T(4) and A(4) tetraloop hairpins formed in 5'-d(CGCGCG-TTTT-CGCGCG)-3' and 5'-d(CGCGCG-AAAA-CGCGCG)-3', respectively. In presence of 5M NaClO(4), a complete B-to-Z transition of the stems is first proved by CD spectra. UV melting profiles are consistent with a higher thermal stability of the T(4) hairpin compared to the A(4) hairpin. Order-to-disorder transition of both hairpins has also been analyzed by means of Raman spectra recorded as a function of temperature. A clear Z-to-B transition of the stem has been confirmed in the T(4) hairpin, and not in the A(4) hairpin. With a right-handed stem, Raman and FTIR spectra have confirmed the C2'-endo/anti conformation for all the T(4) loop nucleosides. With a left-handed stem, a part of the T(4) loop sugars adopt a N-type (C3'-endo) conformation, and the C3'-endo/syn conformation seems to be the preferred one for the dA residues involved in the A(4) tetraloop.     

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.     

Binding of DNA hairpins to an assembler-strand as part of a primordial translation device

A crucial event in the process leading to the origin of life is the emergence of a simple translation device. To approach experimental realization of this device the binding ability of short DNA hairpins to complementary oligonucleotides fixed on a solid support was investigated. The binding is achieved by base pairing between the loop nucleotides of the hairpins containing different numbers of adenosine residues and oligothymidylates covalently linked to cellulose. The loop has to consist of at least five nucleotides to achieve binding. The exact number of established base pairs was determined in two ways. First, the elution temperatures of hairpins and those of oligoadenylates which had the length of the loop were compared. Secondly, the architecture of the loop was analyzed by means of the single-strand-specific nuclease from mung bean acting as structural probe. Onlyn-2 of n loop nucleotides of a hairpin are able to form base pairs. Therefore, a strong evidence for the formation of a triplet of base pairs between primeval tRNA and mRNA sufficient to stabilize the complex enzyme-free is given.     

Enhanced loop DNA folding induced by thymine-CH3 group contact and perpendicular guanine-thymine interaction

A remarkable stabilizing effect induced by T-CH₃ group and perpendicular guanine–thymine interactions in the DNA loop conformation has been demonstrated for the d(TTTG) loop structure using UV melting, high resolution NMR, distance geometry, and molecular dynamics studies. Contrary to the previously published d(TTCG) sequence that exhibits no specific inter-nucleotide interaction, we have found that d(TTTG), which differs only by one nucleotide with the d(TTCG) sequence (C7 T7), forms a rather stable and well-defined loop structure. Two characteristic structural features account for the stabilization of an otherwise flexible loop structure; the second loop T (T6) residue folds into the minor groove and engages in perpendicular interaction with the G8-NH₂, while the third loop T (T7) residue stacks well upon the closing T5G8 wobble base pair and exhibits good contacts with many of the loop T5 and T6 sugar protons, which may form a hydrophobic core in the loop region. The importance of the bulky T7-CH₃ was also proved by the UV melting study; while d(TTCG) hairpin exhibits a lower melting point (74.5°C ) than d(TTTG) hairpin (80.5°C ), d(TT^5–methylCG) hairpin resumes the same higher melting point (80°C ). Similarly, the fact that the melting temperature (74°C ) of d(TTTI) is lower than that of d(TTTG) indicates the critical role played by the G8-NH₂ group. Our structural studies of the d(TTTG) loop indicate that DNA and RNA use a different strategy to establish stable tertiary folds. Comparison with several other pyrimidine-rich loop hairpins suggests that different minor-groove folding modes exist for the folding thymidine residue.     

Structural equilibria in RNA as revealed by 19F NMR

We have incorporated 5-fluorouridine into several sites within a 19-mer RNA modelled on the translational operator of the MS2 bacteriophage. The 19F NMR spectra demonstrate the different chemical shifts of helical and loop fluorouridines of the hairpin secondary structure. Addition of salt gives rise to a species in which the loop fluorouridine gains the chemical shift of its helical counterparts, due to the formation of the alternative bi-molecular duplex form. This is supported by UV thermal melting behaviour which becomes highly dependent on the RNA concentration. Distinct 19F NMR signals for duplex and hairpin forms allow the duplex-hairpin equilibrium constant to be determined under a range of conditions, enabling thermodynamic characterisation and its salt dependence to be determined. Mg2+ also promotes duplex formation, but more strongly than Na+, such that at 25 degrees C, 10 mM MgCl2 has a comparable duplex-promoting effect to 300 mM NaCl. A similar effect is observed with Sr2+, but not Ca2+ or Ba2+. Additional hairpin species are observed in the presence of Na+ as well as Mg2+, Ca2+, Sr2+ and Ba2+ ions. The overall, ensemble average, hairpin conformation is therefore salt-dependent. Electrostatic considerations are thus involved in the balance between different hairpin conformers as well as the duplex-hairpin equilibrium. The data presented here demonstrate that 19F NMR is a powerful tool for the study of conformational heterogeneity in RNA, which is particularly important for probing the effects of metal ions on RNA structure. The thermodynamic characterisation of duplex-hairpin equilibria will also be valuable in the development of theoretical models of nucleic acid structure.     

Tandem GA residues on opposite sides of the loop in molecular beacon-like DNA hairpins compact the loop and increase hairpin stability

The free solution electrophoretic mobilities and thermal stabilities of hairpins formed by two complementary 26-nucleotide oligomers have been measured by capillary electrophoresis. The oligomers are predicted to form molecular beacon-like hairpins with 5 bp stems and 16 nucleotides in the loop. One hairpin, called hairpin2 (hp2), migrates with a relatively fast free solution mobility and exhibits melting temperatures that are reasonably well predicted by the popular structure-prediction program Mfold. Its complement, called hairpin1 (hp1), migrates with a slower free solution mobility and forms a stable hairpin only in solutions containing ≥200 mM Na(+). The melting temperatures observed for hp1 are ~18 °C lower than those observed for hp2 and ~20 °C lower than those predicted by Mfold. The greater thermal stability of hp2 is due to the presence of tandem GA residues on opposite sides of the loop. If the corresponding TC residues in the hp1 loop are replaced by tandem GA residues, the melting temperatures of the modified hairpin are close to those observed for hp2. Eliminating the tandem GA residues in the hp2 loop significantly decreases the thermal stability of hp2. If the loops are replaced by a loop of 16 thymine residues, the free solution mobilities and thermal stabilities of the T-loop hairpin are equal to those observed for hp1. Hence, the loop of hp1 appears to be relatively unstructured, with few base-base stacking interactions. Interactions between tandem GA residues on opposite sides of the hp2 loop appear to compact the loop and increase hairpin stability.     

Asymmetric structure of five and six membered DNA hairpin loops

The tertiary structure of nucleic acid hairpins was elucidated by means of the accessibility of the single-strand-specific nuclease from mung bean. This molecular probe has proven especially useful in determining details of the structural arrangement of the nucleotides within a loop. In this study 3'-labeling is introduced to complement previously used 5'-labeling in order to assess and to exclude possible artifacts of the method. Both labeling procedures result in mutually consistent cleavage patterns. Therefore, methodological artifacts can be excluded and the potential of the nuclease as structural probe is increased. DNA hairpins with five and six membered loops reveal an asymmetric loop structure with a sharp bend of the phosphate-ribose backbone between the second and third nucleotide on the 3'-side of a loop. These hairpin structures differ from smaller loops with 3 or 4 members, which reveal this type of bend between the first and second 3' nucleotide, and resemble with respect to the asymmetry anticodon loops of tRNA.Abbreviations The hairpin oligonucleotides are indicated by hp hairpin followed by the loop sequence, starting at the 5'-end, in parenthesis; d for deoxy is omitted for clarity     

Studies on the structure and stabilizing factor of the CUUCGG hairpin RNA using chemically synthesized oligonucleotides.

A tridecaribonucleotide, r-UGAGCUUCGGCUC, and two analogues r(UGAGC)d(UUCG)r(GCUC) and r-UGAGCUUCIGCUC, which form a hairpin structure with a four-base-paired stem and a UUCG loop, were synthesized by the solid-phase phosphoramidite method. Properties of these three oligomers and d-TGAGCTTCGGCTC, the DNA analogue, were studied by UV, CD and NMR spectroscopy. The melting temperature (Tm) data suggest that the 2'-hydroxy1 groups and the 2-amino group of guanosine in the loop (9G) stabilize the CUUCGG hairpin which is known to have an unusually high Tm. NMR studies show that this 9G takes a syn conformation and the phosphodiester backbone has a turn at 9G-10G which is a junction of the stem and loop.     

Analysis of melting transitions of the DNA hairpins formed from the oligomer sequences d[GGATAC(X)4GTATCC] (X = A, T, G, C)

Optical melting transitions of the short DNA hairpins formed from the self-complementary DNA oligomers d[GGATACX4GTATCC] where X = A, T, G, or C measured in 100 mM NaCl are presented. A significant dependence of the melting transitions on loop sequence is observed and transition temperatures, tm, of the hairpins vary from 58.3 degrees C for the T4 loop hairpin to 55.3 degrees C for the A4 loop. A nearest-neighbor sequence-dependent theoretical algorithm for calculating melting curves of DNA hairpins is presented and employed to analyze the experimental melting transitions. Experimental melting curves were fit by adjustment of a single theoretical parameter, Fend(n), the weighting function for a hairpin loop comprised of n single-strand bases. Empirically determined values of Fend(n) provide an evaluation of the free-energy of hairpin loop formation and stability. Effects of heterogeneous nearest-neighbor sequence interactions in the duplex stem on hairpin loop formation were investigated by evaluating Fend(n) in individual fitting procedures using two of the published sets of nearest-neighbor stacking interactions in DNA evaluated in 100 mM NaCl and given by Wartell and Benight, 1985. In all cases, evaluated values of Fend(n) were obtained that provided exact theoretical predictions of the experimental transitions. Results of the evaluations indicate: (1) Evaluated free-energies of hairpin loop formation are only slightly dependent on loop sequences examined. At the transition temperature, Tm, the free-energy of forming a loop of four bases is approximately equal for T4, G4, or C4 loops and varies from 3.9 to 4.8 kcal/mole depending on the set of nearest-neighbor interactions employed in the evaluations. This result suggests, in light of the observed differences in stability between the T4, G4, and C4 loop hairpins, that sequence-dependent interactions between base residues of the loop are most likely not the source of the enhanced stability of a T4 loop.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparison between CUUG and UUCG tetraloops: thermodynamic stability and structural features analyzed by UV absorption and vibrational spectroscopy

CUUG loop is one of the most frequently occurring tetraloops in bacterial 16S rRNA. This tetraloop has a high thermodynamic stability as proved by previous UV absorption and NMR experiments. Here, we present our results concerning the thermodynamic and structural features of the 10mer 5′-r(GCG-CUUG-CGC)-3′, forming a highly stable CUUG tetraloop hairpin in aqueous solution, by means of several optical techniques (UV and FT-IR absorption, Raman scattering). UV melting profile of this decamer provides a high melting temperature (60.7°C). A set of Raman spectra recorded at different temperatures allowed us to analyze the order-to-disorder (hairpin-to-random coil) transition. Assignment of vibrational markers led us to confirm the particular nucleoside conformation, and to get information on the base stacking and base pairing in the hairpin structure. Moreover, comparison of the data obtained from two highly stable CUUG and UUCG tetraloops containing the same nucleotides but in a different order permitted an overall discussion of their structural features on the basis of Raman marker evidences.