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.     

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 characterization of single mismatches found in naturally occurring RNA

Many naturally occurring RNA structures contain single mismatches. However, the algorithms currently used to predict RNA structure from sequence rely on a minimal set of data for single mismatches, most of which occur rather infrequently in nature. As a result, several approximations and assumptions are used to predict the stability of RNA duplexes containing the most common single mismatches. Therefore, the relative frequency of single mismatches was determined by compiling and searching a database of 955 RNA secondary structures. Thermodynamic parameters for duplex formation, derived from optical melting experiments, are reported for 28 oligoribonucleotides containing frequently occurring single mismatches. These data were then combined with previous data to construct a dataset of 64 single mismatches, including the 30 most common in the database. Because of this increase in experimental thermodynamic parameters for single mismatches that occur frequently in nature, more accurate free energy calculations have resulted. To improve the prediction of the thermodynamic parameters for duplexes containing single mismatches that have not been experimentally measured, single mismatch-specific nearest neighbor parameters were derived. The free energy of an RNA duplex containing a single mismatch that has not been thermodynamically characterized can be calculated by: DeltaG degrees 37,single mismatch = DeltaG degrees 37,mismatch nt + DeltaG degrees 37,mismatch-NN interaction + DeltaG degrees 37,AU/GU. Here, DeltaG degrees 37,mismatch is -0.4, -2.1, and -0.3 kcal/mol for A.G, G.G, and U.U mismatches, respectively; DeltaG degrees 37,mismatch-NN interaction is 0.7, -0.5, 0.4, -0.4, and -1.0 kcal/mol for 5'YRR3'/3'RRY5', 5'RYY3'/3'YYR5', 5'YYR3'/3'RYY5', 5'YRY3'/3'RYR5', and 5'RRY3'/3'YYR5' mismatch-nearest neighbor combinations, respectively, when A and G are categorized as purines (R) and C and U are categorized as pyrimidines (Y); and DeltaG degrees 37,AU/GU is a penalty of 1.2 kcal/mol for replacing a G-C base pair with either an A-U or G-U base pair. Similar predictive models were also derived for DeltaH degrees single mismatch and DeltaS degrees single mismatch. These new predictive models, in conjunction with the reported thermodynamics for frequently occurring single mismatches, should allow for more accurate calculations of the free energy of RNA duplexes containing single mismatches and, furthermore, allow for improved prediction of secondary structure from sequence.     

Thermodynamics of RNA duplexes with tandem mismatches containing a uracil-uracil pair flanked by C.G/G.C or G.C/A.U closing base pairs

The thermodynamics governing the denaturation of RNA duplexes containing 8 bp and a central tandem mismatch or 10 bp were evaluated using UV absorbance melting curves. Each of the eight tandem mismatches that were examined had one U-U pair adjacent to another noncanonical base pair. They were examined in two different RNA duplex environments, one with the tandem mismatch closed by G.C base pairs and the other with G.C and A.U closing base pairs. The free energy increments (Delta Gdegrees(loop)) of the 2 x 2 loops were positive, and showed relatively small differences between the two closing base pair environments. Assuming temperature-independent enthalpy changes for the transitions, (Delta Gdegrees(loop)) for the 2 x 2 loops varied from 0.9 to 1.9 kcal/mol in 1 M Na(+) at 37 degrees C. Most values were within 0.8 kcal/mol of previously estimated values; however, a few sequences differed by 1.2-2.0 kcal/mol. Single strands employed to form the RNA duplexes exhibited small noncooperative absorbance increases with temperature or transitions indicative of partial self-complementary duplexes. One strand formed a partial self-complementary duplex that was more stable than the tandem mismatch duplexes it formed. Transitions of the RNA duplexes were analyzed using equations that included the coupled equilibrium of self-complementary duplex and non-self-complementary duplex denaturation. The average heat capacity change (DeltaC(p)) associated with the transitions of two RNA duplexes was estimated by plotting DeltaH degrees and DeltaS degrees evaluated at different strand concentrations as a function of T(m) and ln T(m), respectively. The average DeltaC(p) was 70 +/- 5 cal K(-)(1) (mol of base pairs)(-)(1). Consideration of this heat capacity change reduced the free energy of formation at 37 degrees C of the 10 bp control RNA duplexes by 0.3-0.6 kcal/mol, which may increase Delta Gdegrees(loop) values by similar amounts.     

Structural characterization of a six-nucleotide RNA hairpin loop found in Escherichia coli, r(UUAAGU)

The binding region of the Escherichia coli S2 ribosomal protein contains a conserved UUAAGU hairpin loop. The structure of the hairpin formed by the oligomer r(GCGU4U5A6A7G8U9CGCA), which has an r(UUAAGU) hairpin loop, was determined by NMR and molecular modeling techniques as part of a study aimed at characterizing the structure and thermodynamics of RNA hairpin loops. Thermodynamic data obtained from melting curves for this RNA oligomer show that it forms a hairpin in solution with the following parameters: DeltaH degrees = -42.8 +/- 2.2 kcal/mol, DeltaS degrees = -127.6 +/- 6.5 eu, and DeltaG degrees (37) = -3.3 +/- 0.2 kcal/mol. Two-dimensional NOESY WATERGATE spectra show an NOE between U imino protons, which suggests that U4 and U9 form a hydrogen bonded U.U pair. The U5(H2') proton shows NOEs to both the A6(H8) proton and the A7(H8) proton, which is consistent with formation of a "U" turn between nucleotides U5 and A6. An NOE between the A7(H2) proton and the U9(H4') proton shows the proximity of the A7 base to the U9 sugar, which is consistent with the structure determined for the six-nucleotide loop. In addition to having a hydrogen-bonded U.U pair as the first mismatch and a U turn, the r(UUAAGU) loop has the G8 base protruding into the solvent. The solution structure of the r(UUAAGU) loop is essentially identical to the structure of an identical loop found in the crystal structure of the 30S ribosomal subunit where the guanine in the loop is involved in tertiary interactions with RNA bases from adjacent regions [Wimberly, B. T., Brodersen, D. E., Clemons, W. M., Morgan-Warren, R. J., Carter, A. P., Vonrhein, C., Hartsch, T., and Ramakrishnan, V. (2000) Nature 407, 327-339]. The similarity of the solution and solid-state structures of this hairpin loop suggests that formation of this hairpin may facilitate folding of 16S RNA.     

Dynamics and stability of GCAA tetraloops with 2-aminopurine and purine substitutions

The contributions of various interactions in the GGCGCAAGCC hairpin containing a GCAA tetraloop were studied by computer simulations using the substitutions of functional groups. The guanosine (G) in the first tetraloop position or in the C-G closing base pair was replaced by 2-aminopurine (AP), and the individual tetraloop's adenosines (A) were replaced by purine (PUR). These substitutions eliminated particular hydrogen bonds thought to stabilize the GCAA tetraloop. For each substitution, molecular dynamics (MD) simulations were carried out in an aqueous solution with sodium counterions, using the CHARMM27 force field. The MD simulations showed that the substitutions in the first (G-->AP) and the third (A-->PUR) position of the GCAA tetraloop did not significantly influence the conformation of the hairpin. A long-lived bridging water molecule observed in the GCAA loop was present in both modified loops. The substitutions made in the last loop position (A-->PUR) or in the C-G base pair closing the tetraloop (G-->AP) to some extent influenced the loop structure and dynamics. These loops did not display the long-lived bridging water molecules. When the second A in the GCAA loop was replaced by PUR, the first A in the loop was observed in the anti or in the syn orientation about the glycosyl bond. The G to AP substitution in C-G base pair led to a change of their arrangement from the Watson-Crick to wobble. The MD simulations of the hairpin with C-AP wobble closing base pair showed increased conformational dynamics of the hairpin. The changes of hairpin formation free energy associated with the substitutions of individual bases were calculated by the free energy perturbation method. Our theoretical estimates suggest a larger destabilization for the G to AP substitutions in GCAA loop than for the substitutions of individual A's by PUR, which is in accordance with experimental tendency. The calculations predicted a similar free energy change for G to AP substitutions in the GCAA tetraloop and in the C-G closing base pair.     

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 the histone mRNA hairpin required for cell cycle regulation of histone gene expression

Expression of replication-dependent histone genes requires a conserved hairpin RNA element in the 3' untranslated regions of poly(A)-less histone mRNAs. The 3' hairpin element is recognized by the hairpin-binding protein or stem-loop-binding protein (HBP/SLBP). This protein-RNA interaction is important for the endonucleolytic cleavage generating the mature mRNA 3' end. The 3' hairpin and presumably HBP/SLBP are also required for nucleocytoplasmic transport, translation, and stability of histone mRNAs. RNA 3' processing and mRNA stability are both regulated during the cell cycle. Here, we have determined the three-dimensional structure of a 24-mer RNA comprising a mammalian histone RNA hairpin using heteronuclear multidimensional NMR spectroscopy. The hairpin adopts a novel UUUC tetraloop conformation that is stabilized by base stacking involving the first and third loop uridines and a closing U-A base pair, and by hydrogen bonding between the first and third uridines in the tetraloop. The HBP interaction of hairpin RNA variants was analyzed in band shift experiments. Particularly important interactions for HBP recognition are mediated by the closing U-A base pair and the first and third loop uridines, whose Watson-Crick functional groups are exposed towards the major groove of the RNA hairpin. The results obtained provide novel structural insight into the interaction of the histone 3' hairpin with HBP, and thus the regulation of histone mRNA metabolism.     

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.     

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.     

Dynamics and Stability of GCAA Tetraloops with 2-Aminopurine and Purine Substitutions

Abstract

The contributions of various interactions in the GGCGCAAGCC hairpin containing a GCAA tetraloop were studied by computer simulations using the substitutions of functional groups. The guanosine (G) in the first tetraloop position or in the C-G closing base pair was replaced by 2-aminopurine (AP), and the individual tetraloop's adenosines (A) were replaced by purine (PUR). These substitutions eliminated particular hydrogen bonds thought to stabilize the GCAA tetraloop. For each substitution, molecular dynamics (MD) simulations were carried out in an aqueous solution with sodium counterions, using the CHARMM27 force field. The MD simulations showed that the substitutions in the first (G→AP) and the third (A→PUR) position of the GCAA tetraloop did not significantly influence the conformation of the hairpin. A long-lived bridging water molecule observed in the GCAA loop was present in both modified loops. The substitutions made in the last loop position (A→PUR) or in the C-G base pair closing the tetraloop (G→AP) to some extent influenced the loop structure and dynamics. These loops did not display the long- lived bridging water molecules. When the second A in the GCAA loop was replaced by PUR, the first A in the loop was observed in the anti or in the syn orientation about the gly- cosyl bond. The G to AP substitution in C-G base pair led to a change of their arrangement from the Watson-Crick to wobble. The MD simulations of the hairpin with C-AP wobble closing base pair showed increased conformational dynamics of the hairpin. The changes of hairpin formation free energy associated with the substitutions of individual bases were calculated by the free energy perturbation method. Our theoretical estimates suggest a larger destabilization for the G to AP substitutions in GCAA loop than for the substitutions of individual A's by PUR, which is in accordance with experimental tendency. The calculations predicted a similar free energy change for G to AP substitutions in the GCAA tetraloop and in the C-G closing base pair.     

Thermodynamics of RNA internal loops with a guanosine-guanosine pair adjacent to another noncanonical pair

Thermodynamic parameters measured by optical melting are reported for formation of RNA duplexes containing tandem noncanonical pairs with at least one guanosine-guanosine (GG) pair. For selected sequences, imino proton NMR provides evidence that the desired duplex forms and that the structure of a GG pair adjacent to a noncanonical pair depends on context. A GG pair next to a different noncanonical pair is more stable than expected from measurements of adjacent GG pairs. This is likely due to an unfavorable stacking interaction between adjacent GG pairs, where areas of high negative charge probably overlap. The results suggest a model where tandem noncanonical pairs closed by two GC pairs are assigned the following free energy increments at 37 degrees C: 0.8 kcal/mol for adjacent GG pairs, 1.0 kcal/mol for GG next to UU, and -0.3 kcal/mol for all others. These values are adjusted by 0.65 kcal/mol for each closing AU pair.     

The effect of base sequence on the stability of RNA and DNA single base bulges

Forty-eight RNA duplexes were constructed that contained all common single base bulges at six different locations. The stabilities of the RNAs were determined by temperature gradient gel electrophoresis (TGGE). The relative stability of a single base bulge was dependent on both base identity and the nearest neighbor context. The single base bulges were placed into two categories. A bulged base with no identical neighboring base was defined as a Group I base bulge. Group II-bulged bases had at least one neighboring base identical to it. Group II bulges were generally more stable than Group I bulges in the same nearest neighbor environments. This indicates that position degeneracy of an unpaired base enhances stability. Differences in the mobility transition temperatures between the RNA fragments with bulges and the completely base-paired reference RNAs were related to free energy differences. Simple models for estimating the free energy contribution of single base bulges were evaluated from the free energy difference data. The contribution of a Group I bulge 5'-(XNZ)-3'.5'-(Z'-X')-3' where N is the unpaired base and X.X' and Z.Z' the neighboring base pairs, could be well-represented (+/-0.34 kcal/mol) by the equation, DeltaG((X)(N)()(Z))(.)((Z)(')(-)(X)(')()) = 3.11 + 0. 40DeltaG(s)()((XZ))(.)((Z)(')(X)(')()). DeltaG(s)()((XZ))(. )((Z)(')(X)(')()) is the stacking energy of the closing base pair doublet. By adding a constant term, delta = -0.3 kcal/mol, to the right side of the above equation, free energies of Group II bulges could also be predicted with the same accuracy. The term delta represents the stabilizing effect due to position degeneracy. A similar equation/model was applied to previous data from 32 DNA fragments with single base bulges. It predicted the free energy differences with a similar standard deviation.     

The structure of the L3 loop from the hepatitis delta virus ribozyme: a syn cytidine.

The structure of the L3 central hairpin loop isolated from the antigenomic sequence of the hepatitis delta virus ribozyme with the P2 and P3 stems from the ribozyme stacked on top of the loop has been determined by NMR spectroscopy. The 26 nt stem-loop structure contains nine base pairs and a 7 nt loop (5'-UCCUCGC-3'). This hairpin loop is critical for efficient catalysis in the intact ribozyme. The structure was determined using homonuclear and heteronuclear NMR techniques on non-labeled and15N-labeled RNA oligonucleotides. The overall root mean square deviation for the structure was 1.15 A (+/- 0.28 A) for the loop and the closing C.G base pair and 0.90 A (+/- 0.18 A) for the loop and the closing C.G base pair but without the lone purine in the loop, which is not well defined in the structure. The structure indicates a U.C base pair between the nucleotides on the 5'- and 3'-ends of the loop. This base pair is formed with a single hydrogen bond involving the cytosine exocyclic amino proton and the carbonyl O4 of the uracil. The most unexpected finding in the loop is a syn cytidine. While not unprecedented, syn pyrimidines are highly unusual. This one can be confidently established by intranucleotide distances between the ribose and the base determined by NMR spectroscopy. A similar study of the structure of this loop showed a somewhat different three-dimensional structure. A discussion of differences in the two structures, as well as possible sites of interaction with the cleavage site, will be presented.     

Targeting degradation of RNA by RNase H using DNA hairpins

RNase H degradation of two 15 nt RNA target sites was examined in the presence of hairpin DNAs with a 5 nt loop and a 10 bp stem or single-stranded 15 nt DNAs. One target site was a segment of a 79 nt RNA, and the other was part of a 53 nt RNA. Secondary structure predictions indicate that the 53 nt RNA target site is entirely single stranded, while a portion of the 79 nt RNA target site forms an intramolecular duplex. Less RNase H and DNA were needed to cleave the 53 nt RNA target site than the less accessible 79 nt RNA site. The hairpin DNAs had their 5 nt loop and 3' side of the stem fully complementary to the target sites or had sequence changes that produced one to nine mismatched pairs. T(m) values ranged from 57 to 80 degrees C. The stability of the hairpin DNAs relative to the stability of their corresponding RNA-DNA hybrids influenced the extent of RNase H degradation at 37 degrees C. Under the assay conditions employed, the amount of degradation directed by the hairpin DNAs was correlated with their predicted DeltaG(o) (37) of binding to the RNA targets. A DNA hairpin with one mismatch to the target site of the 79 nt RNA did not induce degradation under conditions where fully complementary DNA hairpins produced 50-80% degradation. The in vitro results indicate that DNA hairpins can enhance the stringency of RNase H targeted degradation of the RNA sites.     

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.     

Stability of 3' double nucleotide overhangs that model the 3' ends of siRNA

Thermodynamic parameters are reported for duplex formation in 1 M NaCl for 16 RNA sequences, each containing a core tetramer duplex, GGCC, and a 3' overhang consisting of two bases. The results indicate additional double-helical stability is conferred by the double 3' terminal overhang relative to the single 3' terminal overhang. A nearest-neighbor analysis of the data indicates that the free energy contribution at 37 degrees C of the second base in the double 3' terminal overhang varies from 0 to 0.7 kcal/mol. The second base in the 3' double overhang can contribute nearly the same stability to a duplex as a base pair or a 3' dangling overhang. Stability contribution of a dangling base, two nucleotides removed from the 3' end of a duplex, is dependent upon both the identity of the base as well as that of the dangling base that it neighbors. A second dangling base only increases the stability of the duplex when it is neighboring a 3' purine dangling nucleotide. Furthermore, a second dangling pyrimidine provides a greater contribution to duplex stability than a purine. A nearest-neighbor model was developed to predict the influence of 3' double overhang on the stability of duplex formation. The model improves the prediction of free energy and melting temperature when tested against six sequences with different core duplexes.     

Thermodynamic parameters for an expanded nearest-neighbor model for the formation of RNA duplexes with single nucleotide bulges

Thirty-four RNA duplexes containing single nucleotide bulges were optically melted, and the thermodynamic parameters deltaH degrees, deltaS degrees, deltaG degrees (37), and T(M) for each sequence were determined. Data from this study were combined with data from previous thermodynamic data [Longfellow, C. E., Kierzek, R., and Turner, D. H. (1990) Biochemistry 29, 278-85] to develop a model that will more accurately predict the free energy of an RNA duplex containing a single nucleotide bulge. Differences between purine and pyrimidine bulges as well as differences between Group I duplexes, those in which the bulge is not identical to either neighboring nucleotide, and Group II duplexes, those in which the bulge is identical to at least one neighboring nucleotide, were considered. The length of the duplex, non-nearest-neighbor effects, and bulge location were also examined. A model was developed which divides sequences into two groups: those with pyrimidine bulges and those with purine bulges. The proposed model for pyrimidine bulges predicts deltaG degrees (37,bulge) = 3.9 kcal/mol + 0.10deltaG degrees (37,nn) + beta, while the model for purine bulges predicts deltaG degrees (37,bulge) = 3.3 kcal/mol - 0.30deltaG degrees (37,nn) + beta, where beta has a value of 0.0 and -0.8 kcal/mol for Group I and Group II sequences, respectively, and deltaG degrees (37,nn) is the nearest-neighbor free energy of the base pairs surrounding the bulge. The conformation of bulge loops present in rRNA was examined. Three distinct families of structures were identified. The bulge loop was either extrahelical, intercalated, or in a "side-step" conformation.     

Non-nearest-neighbor dependence of the stability for RNA bulge loops based on the complete set of group I single-nucleotide bulge loops

Fifty-nine RNA duplexes containing single-nucleotide bulge loops were optically melted in 1 M NaCl, and the thermodynamic parameters DeltaH degrees, DeltaS degrees, DeltaG 37 degrees, and TM for each sequence were determined. Sequences from this study were combined with sequences from previous studies [Longfellow, C. E., et al. (1990) Biochemistry 29, 278-285; Znosko, B. M., et al. (2002) Biochemistry 41, 10406-10417], thus examining all possible group I single-nucleotide bulge loop and nearest-neighbor sequence combinations. The free energy increments at 37 degrees C for the introduction of a group I single-nucleotide bulge loop range between 1.3 and 5.2 kcal/mol. The combined data were used to develop a model for predicting the free energy of a RNA duplex containing a single-nucleotide bulge. For bulge loops with adjacent Watson-Crick base pairs, neither the identity of the bulge nor the nearest-neighbor base pairs had an effect on the influence of the bulge loop on duplex stability. The proposed model for prediction of the stability of a duplex containing a bulged nucleotide was primarily affected by non-nearest-neighbor interactions. The destabilization of the duplex by the bulge was 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. The stability of a duplex containing a bulged nucleotide adjacent to a wobble base pair also was primarily affected by non-nearest-neighbor interactions. Again, there was a direct correlation between the destabilization of the duplex and the stability of the less stable duplex stem. However, when one or both of the bulge nearest neighbors was a wobble base pair, the free energy increment for insertion of a bulge loop is dependent upon the position and orientation of the wobble base pair relative the bulged nucleotide. Bulge sequences of the type ((5'UBX)(3'GY)), ((5'GBG)(3'UU)) and ((5'UBU)(3'GG)) are less destabilizing by 0.6 kcal/mol, and bulge sequences of the type ((5'GBX)(3'UY)) and ((5'XBU)(3'YG)) are more destabilizing by 0.4 kcal/mol than bulge loops adjacent to Watson-Crick base pairs.     

The structure of the isolated, central hairpin of the HDV antigenomic ribozyme: novel structural features and similarity of the loop in the ribozyme and free in solution.

The structure of an RNA hairpin containing a seven-nucleotide loop that is present in the self-cleaving sequence of hepatitis delta virus antigenomic RNA was determined by high resolution NMR spectroscopy. The loop, which is composed of only one purine and six pyrimidines, has a suprisingly stable structure, mainly supported by sugar hydroxyl hydrogen bonds and base-base and base-phosphate stacking interactions. Compared with the structurally well-determined, seven-membered anticodon loop in tRNA, the sharp turn which affects the required 180 degrees change in direction of the sugar-phosphate backbone in the loop is shifted one nucleotide in the 3' direction. This change in direction can be characterized as a reversed U-turn. It is expected that the reversed U-turn may be found frequently in other molecules as well. There is evidence for a new non-Watson-Crick UC base pair formed between the first and the last residue in the loop, while most of the other bases in the loop are pointing outwards making them accessible to solvent. From chemical modification, mutational and photocrosslinking studies, a similar picture develops for the structure of the hairpin in the active ribozyme indicating that the loop structure in the isolated hairpin and in the ribozyme is very similar.