Non-nearest-neighbor dependence of the stability for RNA group II single-nucleotide bulge loops

Thirty-one RNA duplexes containing single-nucleotide bulge loops were optically melted in 1 M NaCl, and the thermodynamic parameters ΔH°, ΔS°, ΔG°(37), and T(M) for each sequence were determined. The bulge loops were of the group II variety, where the bulged nucleotide is identical to one of its nearest neighbors, leading to ambiguity as to the exact position of the bulge. The data were used to develop a model to predict the free energy of an RNA duplex containing a single-nucleotide bulge. The destabilization of the duplex by the bulge was primarily related to the stability of the stems adjacent to the bulge. Specifically, there was a direct correlation between the destabilization of the duplex and the stability of the less stable duplex stem. Since there is an ambiguity of the bulge position for group II bulges, several different stem combinations are possible. The destabilization of group II bulge loops is similar to the destabilization of group I bulge loops, if the second least stable stem is used to predict the influence of the group II bulge. In-line structure probing of the group II bulge loop embedded in a hairpin indicates that the bulged nucleotide is the one positioned farther from the hairpin loop.     

Non-nearest-neighbor dependence of stability for group III RNA single nucleotide bulge loops

Thirty-five RNA duplexes containing single nucleotide bulge loops were optically melted and the thermodynamic parameters for each duplex determined. The bulge loops were of the group III variety, where the bulged nucleotide is either a AG/U or CU/G, leading to ambiguity to the exact position and identity of the bulge. All possible group III bulge loops with Watson–Crick nearest-neighbors were examined. The data were used to develop a model to predict the free energy of an RNA duplex containing a group III single nucleotide bulge loop. The destabilization of the duplex by the group III bulge could be modeled so that the bulge nucleotide leads to the formation of the Watson–Crick base pair rather than the wobble base pair. The destabilization of an RNA duplex caused by the insertion of a group III bulge is primarily dependent upon non-nearest-neighbor interactions and was shown to be dependent upon the stability of second least stable stem of the duplex. In-line structure probing of group III bulge loops embedded in a hairpin indicated that the bulged nucleotide is the one positioned further from the hairpin loop irrespective of whether the resulting stem formed a Watson–Crick or wobble base pair. Fourteen RNA hairpins containing group III bulge loops, either 3′ or 5′ of the hairpin loop, were optically melted and the thermodynamic parameters determined. The model developed to predict the influence of group III bulge loops on the stability of duplex formation was extended to predict the influence of bulge loops on hairpin stability.     

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.     

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.     

Salt dependence of nucleic acid hairpin stability

Single-stranded junctions/loops are frequently occurring structural motifs in nucleic acid structures. Due to the polyanionic nature of the nucleic acid backbone, metal ions play a crucial role in the loop stability. Here we use the tightly bound ion theory, which can account for the possible ion correlation and ensemble (fluctuation) effects, to predict the ion-dependence of loop and stem-loop (hairpin) free energies. The predicted loop free energy is a function of the loop length, the loop end-to-end distance, and the ion (Na⁺ and Mg²⁺ in this study) concentrations. Based on the statistical mechanical calculations, we derive a set of empirical formulas for the loop thermodynamic parameters as functions of Na⁺ and Mg²⁺ concentrations. For three specific types of loops, namely, hairpin, bulge, and internal loops, the predicted free energies agree with the experimental data. Further applications of these empirical formulas to RNA and DNA hairpin stability lead to good agreements with the available experimental data. Our results indicate that the ion-dependent loop stability makes significant contribution to the overall ion-dependence of the hairpin stability.     

Free energy of imperfect nucleic acid helices. II. Small hairpin loops

Physical studies of enzymically synthesized oligonucleotides of defined sequence are used to evaluate quantitatively the stability of small RNA hairpin loops and helices. The series (Ap)₄G(pC) _N(pU)₄, N = 4, 5 or 6, exists as monomolecular hairpin helices when N ≥ 5, and as imperfect dimer helices when N ≤ 4. In this size range, hairpin loops become more favorable (less destabilizing thermodynamically) as they increase in size from 3 to 4 to 5 unbonded nucleotides. Very small hairpin loops are particularly destabilizing; molecules whose base sequence would imply a hairpin loop of three nucleotides will generally exist with a loop of five, including a broken terminal base pair.Thermodynamic parameters for base pair and loop formation are calculated by a method which makes unnecessary the use of measured enthalpies of polynucleotide melting. Literature data on oligonucleotide double helices yield estimates of the free energy contribution from each of the six types of stacking interactions between three possible neighboring base pairs. The advantage of this approach is that the properties of oligonucleotides are used in predicting the stability of small RNA helices, avoiding the long extrapolation from the properties of high polymers.We provide Tables of temperature-dependent free energies that allow one to predict the stability and thermal transition temperature of many simple RNA secondary structures (applicable to ~1 m-Na⁺ concentration). As an example, we apply the rules to an isolated fragment of tRNA^Ser (yeast) (Coutts, 1971), whose properties were not used in calculating the free-energy parameters. The experimental melting temperature of 88 °C is predicted with an error margin of 5 deg. C.     

Stability of single-nucleotide bulge loops embedded in a GAAA RNA hairpin stem

Forty-six RNA hairpins containing combinations of 3' or 5' bulge loops and a 3' or 5' fluorescein label were optically melted in 1 M NaCl, and the thermodynamic parameters ΔH°, ΔS°, ΔG°(37), and T(M) for each hairpin were determined. The bulge loops were of the group I variety, in which the identity of the bulge is known, and the group II variety, in which the bulged nucleotide is identical to one of its nearest neighbors, leading to ambiguity as to the exact position of the bulge. The fluorescein label at either the 3' end or 5' end of the hairpin did not significantly influence the stability of the hairpin. As observed with bulge loops inserted into a duplex motif, the insertion of a bulge loop into the stem of a hairpin loop was destabilizing. The model developed to predict the influence of bulge loops on the stability of duplex formation was extended to predict the influence of bulge loops on hairpin stability. Specifically, the influence of the bulge is related to the stability of the hairpin stem distal from the hairpin loop.     

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.     

Studies of DNA dumbbells. VI. Analysis of optical melting curves of dumbbells with a sixteen-base pair duplex stem and end-loops of variable size and sequence

Optical melting curves of 22 DNA dumbbells with the 16-base pair duplex sequence 5′-G-C-A-T-C-A-T-C-G-A-T-G-A-T-G-C-3′ linked on both ends by single-strand loops of A_t or C_t sequences (˛ = 2, 3, 4, 6, 8, 10, 14), T_t sequences (˛ = 2, 3, 4, 6, 8, 10), and G_t sequences (t = 2, 4) were measured in phosphate buffered solvents containing 30, 70, and 120 mM Na⁺. For dumbbells with loops comprised of at least three nucleotides, stability is inversely proportional to end-loop size. Dumbbells with loops comprised of only two nucleotide bases generally have lower stabilities than dumbbells with three base nucleotide loops. Experimental melting curves were analyzed in terms of the numerically exact (multistate) statistical thermodynamic model of DNA dumbbell melting previously described (T. M. Paner, M. Amaratunga & A. S. Benight (1992), Biopolymers 32, 881). Theoretically calculated melting curves were fitted to experimental curves by simultaneously adjusting model parameters representing statistical weights of intramolecular hairpin loop and single-strand circle states. The systematically determined empirical parameters provided evaluations of the energetics of hairpin loop formation as a function of loop size, sequence, and salt environment. Values of the free energies of hairpin loop formation ΔG_loop(n > t) and single-strand circles, ΔG_cir(N) as a function of end-loop size, t = 2-14, circle size, N = 32 + 2_t, and loop sequence were obtained. These quantities were found to depend on end-loop size but not loop sequence. Their empirically determined values also varied with solvent ionic strength. Analytical expressions for the partition function Q(T) of the dumbbells were evaluated using the empirically evaluated best-fit loop parameters. From Q(T), the melting transition enthalpy ΔH, entropy ΔS, and free energy ΔG, were evaluated for the dumbbells as a function of end-loop size, sequence, and [Na⁺]. Since the multistate analysis is based on the numerically exact model, and considers a statistically significant number of theoretically possible partially melted states, it does not require prior assumptions regarding the nature of the melting transition, i.e., whether or not it occurs in a two-state manner. For comparison with the multistate analysis, thermodynamic transition parameters were also evaluated directly from experimental melting curves assuming a two-state transition and using the graphical van't Hoff analysis. Comparisons between results of the multistate and two-state analyses suggested dumbbells with loops comprised of six or fewer residues melted in a two-state manner, while the melting processes for dumbbells with larger end-loops deviate from two-state behavior.Dependence of thermodynamic parameters on[Na⁺] as a function of loop size suggests single-strand end-loops have different counterion binding properties than the melted circle. Results are compared with those obtained in an earlier study of dumbbells with the slightly different stem sequence 5'-G-C-A-T-A-G-A-T-G-A-G-A-A-T-G-C-3' linked on the ends by T loops (˛ = 2,3,4,6,8,10,14).© 1996 John Wiley &Sons, Inc.     

A semiflexible polymer model applied to loop formation in DNA hairpins

A statistical mechanical "zipper" model is applied to describe the equilibrium melting of short DNA hairpins with poly(dT) loops ranging from 4 to 12 bases in the loop. The free energy of loop formation is expressed in terms of the persistence length of the chain. This method provides a new measurement of the persistence length of single-stranded DNA, which is found to be approximately 1.4 nm for poly(dT) strands in 100 mM NaCl. The free energy of the hairpin relative to the random coil state is found to scale with the loop size with an apparent exponent of > or = 7, much larger than the exponent of approximately 1.5-1.8 expected from considerations of loop entropy alone. This result indicates a strong dependence of the excess stability of the hairpins, from stacking interactions of the bases within the loop, on the size of the loop. We interpret this excess stability as arising from favorable hydrophobic interactions among the bases in tight loops and which diminish as the loops get larger. Free energy profiles along a generalized reaction coordinate are calculated from the equilibrium zipper model. The transition state for hairpin formation is identified as an ensemble of looped conformations with one basepair closing the loop, and with a lower enthalpy than the random coil state. The equilibrium model predicts apparent activation energy of approximately -11 kcal/mol for the hairpin closing step, in remarkable agreement with the value obtained from kinetics measurements.     

Isolation and characterization of a family of stable RNA tetraloops with the motif YNMG that participate in tertiary interactions

RNA is known to fold into a variety of structural elements, many of which have sufficient sequence complexity to make the thermodynamic study of each possible variant impractical. We previously reported a method for isolating stable and unstable RNA sequences from combinatorial libraries using temperature gradient gel electrophoresis (TGGE). This method was used herein to analyze a six-nucleotide RNA hairpin loop library. Three rounds of in vitro selection were performed using TGGE, and unusually stable RNAs were identified by cloning and sequencing. Known stable tetraloops were found, including sequences belonging to the UNCG motif closed by a CG base pair, and the CUUG motif closed by a GC base pair. In addition, unknown tetraloops were found that were nearly as stable as cUNCGg, including sequences related through substitution of the U with a C (Y), the C with an A (M), or both. These substitutions allow hydrogen bonding and stacking interactions in the UNCG loop to be maintained. Thermodynamic analysis of YNMG and variant loops confirmed optimal stability with Y at position 1 and M at position 3. Similarity in structure and stability among YNMG loops was further supported by deoxyribose substitution, CD, and NMR experiments. A conserved tertiary interaction in 16S rRNA exists between a YAMG loop at position 343 and two adenines in the loop at position 159 (Escherichia coli numbering). NMR and functional group substitution experiments suggest that YNAG loops in particular have enhanced flexibility, which allows the tertiary interaction to be maintained with diverse loop sequences at position 159. Taken together, these results support the existence of an extended family of UNCG-like tetraloops with the motif cYNMGg that are thermodynamically stable and structurally similar and can engage in tertiary interactions in large RNA molecules.     

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     

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.     

Sequence dependence of the stability of RNA hairpin molecules with six nucleotide loops

Thermodynamic parameters are reported for hairpin formation in 1 M NaCl by RNA sequence of the types GCGXUAAUYCGC and GGUXUAAUYACC with Watson-Crick loop closure, where XY is the set of 10 possible mismatch base pairs. A nearest-neighbor analysis of the data indicates the free energy of loop formation at 37 degrees C varies from 3.1 to 5.1 kcal/mol. These results agree with the model previously developed [Vecenie, C. J., and Serra, M. J. (2004) Biochemistry 43, 11813] to predict the stability of RNA hairpin loops: DeltaG degrees (37L(n) = DeltaG degrees (37i(n) + DeltaG degrees (37MM) - 0.8 (if first mismatch is GA or UU) - 0.8 (if first mismatch is GG and loop is closed on the 5' side by a purine). Here, DeltaG degrees (37i(n) is the free energy for initiating a loop of n nucleotides, and DeltaG degrees (37MM) is the free energy for the interaction of the first mismatch with the closing base pair. Thermodynamic parameters are also reported for hairpin formation in 1 M NaCl by RNA sequence of the types GACGXUAAUYUGUC and GGUXUAAUYGCC with GU base pair closure, where XY is the set of 10 possible mismatch base pairs. A nearest-neighbor analysis of the data indicates the free energy of loop formation at 37 degrees C varies from 3.6 to 5.3 kcal/mol. These results allow the development of a model for predicting the stability of hairpin loops closed by GU base pairs. DeltaG degrees (37L(n) (kcal/mol) = DeltaG degrees (37i(n) - 0.8 (if the first mismatch is GA) - 0.8 (if the first mismatch is GG and the loop is closed on the 5' side by a purine). Note that for these hairpins, the stability of the loops does not depend on DeltaG degrees (37MM). For hairpin loops closed by GU base pairs, the DeltaG degrees (37i(n) values, when n = 4, 5, 6, 7, and 8, are 4.9, 5.0, 4.6, 5.0, and 4.8 kcal/mol, respectively. The model gives good agreement when tested against six naturally occurring hairpin sequences. Thermodynamic values for terminal mismatches adjacent to GC, GU, and UG base pairs are also reported.     

Role of the closing base pair for d(GCA) hairpin stability: free energy analysis and folding simulations

Hairpin loops belong to the most important structural motifs in folded nucleic acids. The d(GNA) sequence in DNA can form very stable trinucleotide hairpin loops depending, however, strongly on the closing base pair. Replica-exchange molecular dynamics (REMD) were employed to study hairpin folding of two DNA sequences, d(gcGCAgc) and d(cgGCAcg), with the same central loop motif but different closing base pairs starting from single-stranded structures. In both cases, conformations of the most populated conformational cluster at the lowest temperature showed close agreement with available experimental structures. For the loop sequence with the less stable G:C closing base pair, an alternative loop topology accumulated as second most populated conformational state indicating a possible loop structural heterogeneity. Comparative-free energy simulations on induced loop unfolding indicated higher stability of the loop with a C:G closing base pair by ~3 kcal mol(-1) (compared to a G:C closing base pair) in very good agreement with experiment. The comparative energetic analysis of sampled unfolded, intermediate and folded conformational states identified electrostatic and packing interactions as the main contributions to the closing base pair dependence of the d(GCA) loop stability.     

Structural characterization of d(CAACCCGTTG) and d(CAACGGGTTG) mini-hairpin loops by heteronuclear NMR: the effects of purines versus pyrimidines in DNA hairpins.

The DNA decamers, d(CAACCCGTTG) and d(CAACGGGTTG) were studied in solution by proton and heteronuclear NMR. Under appropriate conditions of pH, temperature, salt concentration and DNA concentration, both decamers form hairpin conformations with similar stabilities [Avizonis and Kearns (1995) Biopolymers, 35, 187-200]. Both decamers adopt mini-hairpin loops, where the first and last four nucleotides are involved in Watson-Crick hydrogen bonding and the central two nucleotides, CC or GG respectively, form the loop. Through the use of proton-proton, proton-phosphorus and natural abundance proton-carbon NMR experiments, backbone torsion angles (beta, gamma and epsilon), sugar puckers and interproton distances were measured. The nucleotides forming the loops of these decamers were found to stack upon one another in an L1 type of loop conformation. Both show gamma tr and unusual beta torsion angles in the loop-closing nucleotide G7, as expected for mini-hairpin loop formation. Our results indicate that the beta and epsilon torsion angles of the fifth and sixth nucleotides that form the loop and the loop-closing nucleotide G7 are not in the standard trans conformation as found in B-DNA. Although the loop structures calculated from NMR-derived constraints are not well defined, the stacking of the bases in the two different hairpins is different. This difference in the base stacking of the loop may provide an explanation as to why the cytosine-containing hairpin is thermodynamically more stable than the guanine-containing hairpin.     

Structural analyses of mammalian ribosomal ribonucleic acid and its precursors. Nucleotide sequence of ribosomal 5.8 S ribonucleic acid.

The nucleotide sequence of ribosomal 5.8 S RNA (also known as 7 S or 5.5 S rRNA) from Novikoff hepatoma ascites cells has been determined to be (see article). Estimations of the secondary structure based upon maximized base pairing and the fragments of partial ribonuclease digestion indicate that there may be five base-paired regions in the molecule, three forming a folding of the termini and two forming secondary hairpin loops. The sequence of Novikoff hepatoma 5.8 S rRNA is about 75% homologous with that of yeast 5.8 S rRNA (Rubin, G.M. (1973) J. Biol. Chem. 248, 3860-3875) and similar models for secondary structure are proposed. Both models contain a very stable G-C rich hairpin loop (residues 116 to 138), a less stable A-U-rich hairpin loop (residues 64 to 91) and two symmetrical bulges (residues 15 to 25 and 40 to 44).     

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.     

Thermal stability of RNA hairpins containing a four-membered loop and a bulge nucleotide

Fourteen RNA hairpins containing a four-membered loop and a bulge nucleotide were synthesized and their thermal stabilities determined. The combined contribution of a four-membered loop and bulge A to the free energy of a hairpin is calculated to be 9.3 kcal/mol at 37 degrees C and successfully predicts the stability of an independent RNA hairpin. The introduction of a bulge nucleotide to the helical stem of an RNA hairpin destabilizes the molecule in a sequence-dependent manner. The individual thermodynamic contributions of a four-membered loop and bulge A, G, and U residues to the stability of an RNA hairpin loop are presented.     

A thermodynamic study of unusually stable RNA and DNA hairpins.

About 70% of the RNA tetra-loop sequences identified in ribosomal RNAs from different organisms fall into either (UNCG) or (GNRA) families (where N = A, C, G, or U; and R = A or G). RNA hairpins with these loop sequences form unusually stable tetra-loop structures. We have studied the RNA hairpin GGAC(UUCG)GUCC and several sequence variants to determine the effect of changing the loop sequence and the loop-closing base pair on the thermodynamic stability of (UNCG) tetra-loops. The hairpin GGAG(CUUG)CUCC with the conserved loop G(CUUG)C was also unusually stable. We have determined melting temperatures (Tm), and obtained thermodynamic parameters for DNA hairpins with sequences analogous to stable RNA hairpins with (UNCG), C(GNRA)G, C(GAUA)G, and G(CUUG)C loops. DNA hairpins with (TTCG), (dUdUCG), and related sequences in the loop, unlike their RNA counterparts, did not form unusually stable hairpins. However, DNA hairpins with the consensus loop sequence C(GNRA)G were very stable compared to hairpins with C(TTTT)G or C(AAAA)G loops. The C(GATA)G and G(CTTG)C loops were also extra stable. The relative stabilities of the unusually stable DNA hairpins are similar to those observed for their RNA analogs.