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RNA thermodynamics play an important role in determining the two- and three-dimensional structures of RNA. Internal loops of the sequence 5'-GMNU/3'-UNMG are relatively unstable thermodynamically. Here, five duplexes with GU-flanked 2 × 2 nucleotide internal loops were structurally investigated to reveal determinants of their instability. The following internal loops were investigated: 5'-GCAU/3'-UACG, 5'-UUCG/3'-GCUU, 5'-GCUU/3'-UUCG, 5'-GUCU/3'-UCUG, and 5'-GCCU/3'-UCCG. Two-dimensional nuclear magnetic resonance spectra indicate the absence of GU wobble base pairing in 5'-GCUU/3'-UUCG, 5'-GUCU/3'-UCUG, and 5'-GCCU/3'-UCCG. The 5'-GCUU/3'-UUCG loop has an unusual conformation of the GU base pairs, in which U's O2 carbonyl forms a bifurcated hydrogen bond with G's amino and imino protons. The internal loop of 5'-GUCU/3'-UCUG displays a shifted configuration in which GC pairs flank a U-U pair and several U's are in fast exchange between positions inside and outside the helix. In contrast, 5'-GCAU/3'-UACG and 5'-UUCG/3'-GCUU both have the expected GU wobble base pairs flanking the internal loop. Evidently, GU base pairs flanking internal loops are more likely to display atypical structures relative to Watson-Crick base pairs flanking internal loops. This appears to be more likely when the G of the GU pair is 5' to the loop. Such unusual structures could serve as recognition elements for biological function and as benchmarks for structure prediction methods.

GU base pairs are important RNA structural motifs and often close loops. Accurate prediction of RNA structures relies upon understanding the interactions determining structure. The thermodynamics of some 2 × 2 nucleotide internal loops closed by GU pairs are not well understood. Here, several self-complementary oligonucleotide sequences expected to form duplexes with 2 × 2 nucleotide internal loops closed by GU pairs were investigated. Surprisingly, nuclear magnetic resonance revealed that many of the sequences exist in equilibrium between hairpin and duplex conformations. This equilibrium is not observed with loops closed by Watson-Crick pairs. To measure the thermodynamics of some 2 × 2 nucleotide internal loops closed by GU pairs, non-self-complementary sequences that preclude formation of hairpins were designed. The measured thermodynamics indicate that some internal loops closed by GU pairs are unusually unstable. This instability accounts for the observed equilibria between duplex and hairpin conformations. Moreover, it suggests that future three-dimensional structures of loops closed by GU pairs may reveal interactions that unexpectedly destabilize folding.

The prediction of RNA three-dimensional structure from sequence alone has been a long-standing goal. High-resolution, experimentally determined structures of simple noncanonical pairings and motifs are critical to the development of prediction programs. Here, we present the nuclear magnetic resonance structure of the (5'CCAGAAACGGAUGGA) 2 duplex, which contains an 8 × 8 nucleotide internal loop flanked by three Watson-Crick pairs on each side. The loop is comprised of a central 5'AC/3'CA nearest neighbor flanked by two 3RRs motifs, a known stable motif consisting of three consecutive sheared GA pairs. Hydrogen bonding patterns between base pairs in the loop, the all-atom root-mean-square deviation for the loop, and the deformation index were used to compare the structure to automated predictions by MC-sym, RNA FARFAR, and RNAComposer.

Knowledge of RNA structure is necessary to determine structure-function relationships and to facilitate design of potential therapeutics. RNA secondary structure prediction can be improved by applying constraints from nuclear magnetic resonance (NMR) experiments to a dynamic programming algorithm. Imino proton walks from NOESY spectra reveal double-stranded regions. Chemical shifts of protons in GH1, UH3, and UH5 of GU pairs, UH3, UH5, and AH2 of AU pairs, and GH1 of GC pairs were analyzed to identify constraints for the 5' to 3' directionality of base pairs in helices. The 5' to 3' directionality constraints were incorporated into an NMR-assisted prediction of secondary structure (NAPSS-CS) program. When it was tested on 18 structures, including nine pseudoknots, the sensitivity and positive predictive value were improved relative to those of three unrestrained programs. The prediction accuracy for the pseudoknots improved the most. The program also facilitates assignment of chemical shifts to individual nucleotides, a necessary step for determining three-dimensional structure.

Influenza A is a negative-sense RNA virus with an eight-segment genome. Some segments encode more than one polypeptide product, but how the virus accesses alternate internal open reading frames (ORFs) is not completely understood. In segment 2, ribosomal scanning produces two internal ORFs, PB1-F2 and N40. Here, chemical mapping reveals a Mg(2+)-dependent pseudoknot structure that includes the PB1-F2 and N40 start codons. The results suggest that interactions of the ribosome with the pseudoknot may affect the level of translation for PB1-F2 and N40.

Influenza A is an RNA virus with a genome of eight negative sense segments. Segment 7 mRNA contains a 3' splice site for alternative splicing to encode the essential M2 protein. On the basis of sequence alignment and chemical mapping experiments, the secondary structure surrounding the 3' splice site has an internal loop, adenine bulge, and hairpin loop when it is in the hairpin conformation that exposes the 3' splice site. We report structural features of a three-dimensional model of the hairpin derived from nuclear magnetic resonance spectra and simulated annealing with restrained molecular dynamics. Additional insight was provided by modeling based on (1)H chemical shifts. The internal loop containing the 3' splice site has a dynamic guanosine and a stable imino (cis Watson-Crick/Watson-Crick) GA pair. The adenine bulge also appears to be dynamic with the A either stacked in the stem or forming a base triple with a Watson-Crick GC pair. The hairpin loop is a GAAA tetraloop closed by an AC pair.

Influenza A virus utilizes RNA throughout infection. Little is known, however, about the roles of RNA structures. A previous bioinformatics survey predicted multiple regions of influenza A virus that are likely to generate evolutionarily conserved and stable RNA structures. One predicted conserved structure is in the pre-mRNA coding for essential proteins, M1 and M2. This structure starts 79 nucleotides downstream of the M2 mRNA 5' splice site. Here, a combination of biochemical structural mapping, mutagenesis, and NMR confirms the predicted three-way multibranch structure of this RNA. Imino proton NMR spectra reveal no change in secondary structure when 80 mM KCl is supplemented with 4 mM MgCl2. Optical melting curves in 1 M NaCl and in 100 mM KCl with 10 mM MgCl2 are very similar, with melting temperatures ∼14 °C higher than that for 100 mM KCl alone. These results provide a firm basis for designing experiments and potential therapeutics to test for function in cell culture.

The sequence dependence of RNA energetics is important for predicting RNA structure. Hairpins with C(n) loops are consistently less stable than hairpins with other loops, which suggests the structure of C(n) regions could be unusual in the "unfolded" state. For example, previous nuclear magnetic resonance (NMR) evidence suggested that polycytidylic acid forms a left-handed helix. In this study, UV melting experiments show that the hairpin formed by r(5'GGACCCCCGUCC) is less stable than r(5'GGACUUUUGUCC). NMR spectra for single-stranded C(4) oligonucleotide, mimicking the unfolded hairpin loop, are consistent with a right-handed A-form-like helix. Comparisons between NMR spectra and molecular dynamics (MD) simulations suggest that recent reparametrizations, parm99χ_YIL and parm99TOR, of the AMBER parm99 force field improve the agreement between structural features for C(4) determined by NMR and predicted by MD. Evidently, the force field revisions to parm99 improve the modeling of RNA energetics and therefore structure.

The RNA duplex, (5'GACGAGUGUCA)(2), has two conformations in equilibrium. The nuclear magnetic resonance solution structure reveals that the major conformation of the loop, 5'GAGU/3'UGAG, is novel and contains two unusual Watson-Crick/Hoogsteen GG pairs with G residues in the syn conformation, two A residues stacked on each other in the center of the helix with inverted sugars, and two bulged-out U residues. The structure provides a benchmark for testing approaches for predicting local RNA structure and a sequence that allows the design of a unique arrangement of functional groups and/or a conformational switch into nucleic acids.

Thermodynamic parameters for GU pairs are important for predicting the secondary structures of RNA and for finding genomic sequences that code for structured RNA. Optical melting curves were measured for 29 RNA duplexes with GU pairs to improve nearest neighbor parameters for predicting stabilities of helixes. The updated model eliminates a prior penalty assumed for terminal GU pairs. Six additional duplexes with the 5'GG/3'UU motif were added to the single representation in the previous database. This revises the ΔG°(37) for the 5'GG/3'UU motif from an unfavorable 0.5 kcal/mol to a favorable -0.2 kcal/mol. Similarly, the ΔG°(37) for the 5'UG/3'GU motif changes from 0.3 to -0.6 kcal/mol. The correlation coefficients between predicted and experimental ΔG°(37), ΔH°, and ΔS° for the expanded database are 0.95, 0.89, and 0.87, respectively. The results should improve predictions of RNA secondary structure.

Three-way multibranch loops (junctions) are common in RNA secondary structures. Computer algorithms such as RNAstructure and MFOLD do not consider the identity of unpaired nucleotides in multibranch loops when predicting secondary structure. There is limited experimental data, however, to parametrize this aspect of these algorithms. In this study, UV optical melting and a fluorescence competition assay are used to measure stabilities of multibranch loops containing up to five unpaired adenosines or uridines or a loop E motif. These results provide a test of our understanding of the factors affecting multibranch loop stability and provide revised parameters for predicting stability. The results should help to improve predictions of RNA secondary structure.

Microarrays with isoenergetic pentamer and hexamer 2'-O-methyl oligonucleotide probes with LNA (locked nucleic acid) and 2,6-diaminopurine substitutions were used to probe the binding sites on the RNase P RNA specificity domain of Bacillus subtilis. Unexpected binding patterns were revealed. Because of their enhanced binding free energies, isoenergetic probes can break short duplexes, merge adjacent loops, and/or induce refolding. This suggests new approaches to the rational design of short oligonucleotide therapeutics but limits the utility of microarrays for providing constraints for RNA structure determination. The microarray results are compared to results from chemical mapping experiments, which do provide constraints. Results from both types of experiments indicate that the RNase P RNA folds similarly in 1 M Na(+) and 10 mM Mg(2+).

Thermodynamic stabilities of 2 x 2 nucleotide tandem AG internal loops in RNA range from -1.3 to +3.4 kcal/mol at 37 degrees C and are not predicted well with a hydrogen-bonding model. To provide structural information to facilitate development of more sophisticated models for the sequence dependence of stability, we report the NMR solution structures of five RNA duplexes: (rGACGAGCGUCA)(2), (rGACUAGAGUCA)(2), (rGACAAGUGUCA)(2), (rGGUAGGCCA)(2), and (rGACGAGUGUCA)(2). The structures of these duplexes are compared to that of the previously solved (rGGCAGGCC)(2) (Wu, M., SantaLucia, J., Jr., and Turner, D. H. (1997) Biochemistry 36, 4449-4460). For loops bounded by Watson-Crick pairs, the AG and Watson-Crick pairs are all head-to-head imino-paired (cis Watson-Crick/Watson-Crick). The structures suggest that the sequence-dependent stability may reflect non-hydrogen-bonding interactions. Of the two loops bounded by G-U pairs, only the 5'UAGG/3'GGAU loop adopts canonical UG wobble pairing (cis Watson-Crick/Watson-Crick), with AG pairs that are only weakly imino-paired. Strikingly, the 5'GAGU/3'UGAG loop has two distinct duplex conformations, the major of which has both guanosine residues (G4 and G6 in (rGACGAGUGUCA)(2)) in a syn glycosidic bond conformation and forming a sheared GG pair (G4-G6*, GG trans Watson-Crick/Hoogsteen), both uracils (U7 and U7*) flipped out of the helix, and an AA pair (A5-A5*) in a dynamic or stacked conformation. These structures provide benchmarks for computational investigations into interactions responsible for the unexpected differences in loop free energies and structure.

RNA pseudoknots have important functions, and thermodynamic stability is a key to predicting pseudoknots in RNA sequences and to understanding their functions. Traditional methods, such as UV melting and differential scanning calorimetry, for measuring RNA thermodynamics are restricted to temperature ranges around the melting temperature for a pseudoknot. Here, we report RNA pseudoknot free energy changes at 37 degrees C measured by fluorescence competition assays. Sequence-dependent studies for the loop 1-stem 2 region reveal (1) the individual nearest-neighbor hydrogen bonding (INN-HB) model provides a reasonable estimate for the free energy change when a Watson-Crick base pair in stem 2 is changed, (2) the loop entropy can be estimated by a statistical polymer model, although some penalty for certain loop sequences is necessary, and (3) tertiary interactions can significantly stabilize pseudoknots and extending the length of stem 2 may alter tertiary interactions such that the INN-HB model does not predict the net effect of adding a base pair. The results can inform writing of algorithms for predicting and/or designing RNA secondary structures.

Hybridization to RNA is important for many applications, including antisense therapeutics, RNA interference, and microarray screening. Similar thermodynamic stabilities of A-U and G-U base pairs result in difficulties in selective binding to RNA. Moreover, A-U pairs are weaker than G-C pairs so that binding is sometimes weak when many A-U pairs are present. It is known, however, that replacement of uridine with 2-thiouridine significantly improves binding and selectivity. To test for additional improvement of binding and of the specificity for binding A over G, LNA-2-thiouridine was synthesized for the first time and incorporated into many LNA-2'-O-methyl-RNA/RNA duplexes. UV melting was used to measure the thermodynamic effect of replacing 2'-O-methyluridine with 2'-O-methyl-2-thiouridine or LNA-2-thiouridine. The 2-thiouridine usually enhances binding and selectivity. Selectivity is optimized when a single 2-thiouridine is placed at an internal position in a duplex.

RNA internal loops are often important sites for folding and function. Residues in internal loops can have pKa values shifted close to neutral pH because of the local structural environment. A series of RNA internal loops were studied at different pH by UV absorbance versus temperature melting experiments and imino proton nuclear magnetic resonance (NMR). A stabilizing CA pair forms at pH 7 in the CG/AA and CA/AA nearest neighbors when the CA pair is the first noncanonical pair (loop-terminal pair) in 3 x 3 nucleotide and larger size-symmetric internal loops. These CG/AA and CA/AA nearest neighbors, with CA adjacent to a closing Watson-Crick pair, are further stabilized when the pH is lowered from 7 to 5.5. The results are consistent with a significantly larger fraction (from approximately 20% at pH 7 to approximately 90% at pH 5.5) of adenines being protonated at the N1 position to form stabilizing wobble CA+ pairs adjacent to a sheared GA or AA pair. The noncanonical pair adjacent to the GA pair in CG/AA can either stabilize or destabilize the loop, consistent with the sequence-dependent thermodynamics of GA pairs. No significant pH-dependent stabilization is found for most of the other nearest neighbor combinations involving CA pairs (e.g., CA/AG and AG/CA), which is consistent with the formation of various nonwobble pairs observed in different local sequence contexts in crystal and NMR structures. A revised free-energy model, including stabilization by wobble CA+ pairs, is derived for predicting stabilities of medium-size RNA internal loops.

Locked nucleic acids (LNA) considerably enhance the thermodynamic stability of DNA and RNA duplexes. We report the thermodynamic stabilities of LNA-2'-O-methyl RNA/RNA duplexes designed to provide insight into the contributions of stacking and hydrogen bonding interactions to the enhanced stability. The results show that hydrogen bonding of LNA nucleotides is similar to that of 2'-O-methyl RNA nucleotides, whereas the 3'-stacking interactions are on average approximately 0.7 kcal/mol more favorable at 37 degrees C than for 2'-O-methyl or RNA nucleotides. Moreover, NMR spectra suggest helical preorganization of the single-stranded tetramer, C(L)A(M)A(L)U(M), probably due to restriction of some torsion angles. Thus, enhanced stacking interactions and helical preorganization of single-stranded oligonucleotides contribute to the extraordinary stabilization of duplexes by LNA nucleotides.

To facilitate design of short isoenergetic hybridization probes for RNA, we report the influence of adding 5'- or 3'-terminal 2'-O-methylguanosine (GM), LNA-guanosine (GL), or 3'-terminal pyrene pseudo-nucleotide (PPN) on the thermodynamic stability of 2'-O-methyl-RNA/RNA (2'-O-Me-RNA/RNA) duplexes with sequences 5'CMGMGMCMAM/3'AAXGCCGUXAA, where X is A, C, G, or U. A 3'-terminal GM or GL added to the 2'-O-Me-RNA strand to form a G-A, G-G or G-U mismatch enhances thermodynamic stability (DeltaDeltaG degrees 37) of the 2'-O-Me-RNA/RNA duplexes on average by 0.7 and 1.5 kcal/mol, respectively. A 3'-terminal GM or GL in a GM-C or GL-C pair stabilizes the 2'-O-Me-RNA/RNA duplex by 2.6 and 3.4 kcal/mol, respectively. A 5'-terminal GM or GL in a G-A or G-G mismatch provided less stabilization in comparison with a 3'-terminal G-A or G-G mismatch, but more stabilization in a G-C or G-U pair. In contrast to guanosine derivatives, pyrene residue (P) as PPN at the 3'-terminal position enhances thermodynamic stability of the 2'-O-Me-RNA/RNA duplexes on average by 2.3 +/- 0.1 kcal/mol, relatively independent of the type of ribonucleotide placed in the opposite strand. The thermodynamic data can be applied to design 2'-O-Me-RNA/RNA duplexes with enhanced thermodynamic stability that is also sequence independent. This is useful for design of hybridization probes to interrogate RNA structure and/or expression by microarray and other methods.

NMR studies provide insights into structural features of internal loops. These insights can be combined with thermodynamic studies to generate models for predicting structure and energetics. The tandem mismatch internal loop, 5'GUGG3'(3'CUAC5'), has been studied by NMR. The NMR structure reveals an internal loop with no hydrogen bonding between the loop bases and with the G in the AG mismatch flipped out of the helix. The sequence of this internal loop is highly conserved in rRNA. The loop is located in the large ribosomal subunit and is part of a conserved 58-nt fragment that is the binding domain of ribosomal protein L11. Structural comparisons between variants of this internal loop in crystal structures of the 58-nt domain complexed with L11 protein and of the large ribosomal subunit (LSU) suggest that this thermodynamically destabilizing internal loop is partially preorganized for tertiary interactions and for binding L11. A model for predicting the base pairing and free energy of 2 x 2 nucleotide internal loops with a purine-purine mismatch next to a pyrimidine-pyrimidine mismatch is proposed on the basis of the present NMR structure and previously reported thermodynamics.

The NMR structures of [see text] and [see text] are reported. The internal loop, [see text], is about 2 kcal/mol more stable than [see text] at 37 degrees C. The duplexes assemble into similar global folds characterized by the formation of tandem sheared GA pairs. The different stabilities of the loops are accompanied by differences in the local structure of the closing GU pairs. In the [see text] internal loop, the GU pairs form canonical wobble configurations with two hydrogen bonds, whereas in [see text], the GU pairs form a single hydrogen bond involving the amino group, GH22, and the carbonyl group, UO4. This pairing is similar to the GU closing pair of the 690 hairpin loop found in E. coli 16S rRNA. The [see text] and [see text] structures reveal how the subtle interplay between stacking and hydrogen bonding determines sequence dependent conformation and thermodynamic stability. Thus, this work provides structural and thermodynamic benchmarks for theoreticians in the ongoing effort to understand the sequence dependence of RNA physicochemical properties.

Internal loops play an important role in structure and folding of RNA and in recognition of RNA by other molecules such as proteins and ligands. An understanding of internal loops with propensities to form a particular structure will help predict RNA structure, recognition, and function. The structures of internal loops 5' 1009CUAAG1013 3'/3' 1168GAAGC1164 5' and 5' 998CUAAG1002 3'/3' 1157GAAGC1153 5' from helix 40 of the large subunit rRNA in Deinococcus radiodurans and Escherichia coli, respectively, are phylogenetically conserved, suggesting functional relevance. The energetics and NMR solution structure of the loop were determined in the duplex 5' 1GGCUAAGAC9 3'/3' 18CCGAAGCUG10 5'. The internal loop forms a different structure in solution and in the crystal structures of the ribosomal subunits. In particular, the crystal structures have a bulged out adenine at the equivalent of position A15 and a reverse Hoogsteen UA pair (trans Watson-Crick/Hoogsteen UA) at the equivalent of U4 and A14, whereas the solution structure has a single hydrogen bond UA pair (cis Watson-Crick/sugar edge A15U4) between U4 and A15 and a sheared AA pair (trans Hoogsteen/sugar edge A14A5) between A5 and A14. There is cross-strand stacking between A6 and A14 (A6/A14/A15 stacking pattern) in the NMR structure. All three structures have a sheared GA pair (trans Hoogsteen/sugar edge A6G13) at the equivalent of A6 and G13. The internal loop has contacts with ribosomal protein L20 and other parts of the RNA in the crystal structures. These contacts presumably provide the free energy to rearrange the base pairing in the loop. Evidently, molecular recognition of this internal loop involves induced fit binding, which could confer several advantages. The predicted thermodynamic stability of the loop agrees with the experimental value, even though the thermodynamic model assumes a Watson-Crick UA pair.

A method to deduce RNA secondary structure on the basis of data from microarrays of 2'-O-methyl RNA 9-mers immobilized in agarose film on glass slides is tested with a 249 nucleotide RNA from the 3' end of the R2 retrotransposon from Bombyx mori. Various algorithms incorporating binding data and free-energy minimization calculations were compared for interpreting the data to provide possible secondary structures. Two different methods give structures with 100 and 87% of the base pairs determined by sequence comparison. In contrast, structures predicted by free-energy minimization alone by Mfold and RNAstructure contain 52 and 72% of the known base pairs, respectively. This combination of high throughput microarray techniques with algorithms using free-energy calculations has potential to allow for fast determination of RNA secondary structure. It should also facilitate the design of antisense and siRNA oligonucleotides.

A previous NMR structure of the duplex 5'GGU GGA GGCU/PCCG AAG CCG5' revealed an unusually stable RNA internal loop with three consecutive sheared GA pairs. Here, we report NMR studies of two duplexes, 5'GGU GGA GGCU/PCCA AAG CCG5' (replacing the UG pair with a UA closing pair) and 5'GGU GAA GGCU/PCCG AAG CCG5' (replacing the middle GA pair with an AA pair). An unusually stable loop with three consecutive sheared GA pairs forms in the duplex 5'GGU GGA GGCU/PCCA AAG CCG5'. The structure contrasts with that reported for this loop in the crystal structure of the large ribosomal subunit of Deinococcus radiodurans [Harms, J., Schluenzen, F., Zarivach, R., Bashan, A., Gat, S., Agmon, I., Bartels, H., Franceschi, F., and Yonath, A. (2001) Cell 107, 679-688]. The middle AA pair in the duplex 5'GGU GAA GGCU/PCCG AAG CCG5' rapidly exchanges orientations, resulting in alternative base stacking and pseudosymmetry with exclusively sheared pairs. The U GAA G/G AAG C internal loop is 2.1 kcal/mol less stable than the U GGA G/G AAG C internal loop at 37 degrees C. Structural, energetic, and dynamic consequences upon functional group substitutions within related 3 x 3 and 3 x 6 internal loops are also reported.

Internal loops in RNA are important for folding and function. Consecutive noncanonical pairs can form in internal loops having at least two nucleotides on each side. Thermodynamic and structural insights into such internal loops should improve approximations for their stabilities and predictions of secondary and three-dimensional structures. Most natural internal loops are purine rich. A series of oligoribonucleotides that form purine-rich internal loops of 5-10 nucleotides, including kink-turn loops, were studied by UV melting, exchangeable proton and phosphorus NMR. Three consecutive GA pairs with the motif 5' Y GGA/3' R AAG or GGA R 3'/AAG Y 5' (i.e., 5' GGA 3'/3' AAG 5' closed on at least one side with a CG, UA, or UG pair with Y representing C or U and R representing A or G) stabilize internal loops having 6-10 nucleotides. Certain motifs with two consecutive GA pairs are also stabilizing. In internal loops with three or more nucleotides on each side, the motif 5' U G/3' G A has stability similar to 5' C G/3' G A. A revised model for predicting stabilities of internal loops with 6-10 nucleotides is derived by multiple linear regression. Loops with 2 x 3 nucleotides are predicted well by a previous thermodynamic model.

Determining RNA secondary structure is important for understanding structure-function relationships and identifying potential drug targets. This paper reports the use of microarrays with heptamer 2'-O-methyl oligoribonucleotides to probe the secondary structure of an RNA and thereby improve the prediction of that secondary structure. When experimental constraints from hybridization results are added to a free-energy minimization algorithm, the prediction of the secondary structure of Escherichia coli 5S rRNA improves from 27 to 92% of the known canonical base pairs. Optimization of buffer conditions for hybridization and application of 2'-O-methyl-2-thiouridine to enhance binding and improve discrimination between AU and GU pairs are also described. The results suggest that probing RNA with oligonucleotide microarrays can facilitate determination of secondary structure.

Some experimental results for the thermodynamics of RNA folding cannot be explained by simple pairwise hydrogen-bonding models. Such effects include the stabilities of isoguanosine-isocytidine (iG-iC) base pairs and of various 2 x 2 nucleotide internal loops. Presumably, these results can be explained by base stacking effects, which can be partitioned into Coulombic and overlap effects. We review experimental measurements that provide benchmarks for testing the approximations and theories used for modeling nucleic acids. Quantitative agreement between experiment and theory will indicate understanding of the interactions determining RNA stability and structure.

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

The J4/5 loop of group I introns has tertiary interactions with the P1 helix that position the P1 substrate for the self-splicing reaction. The J4/5 loop of Candida albicans and Candida dubliniensis, 5'GAAGG3'/3'UAAUU5', potentially contains two A.A pairs flanked by one G.U pair on one side and two G.U pairs on the other side. Results from optical melting, nuclear magnetic resonance spectroscopy, and functional group substitution experiments with a mimic of the C. albicans and C. dubliniensis J4/5 loop are consistent with the adenosines forming tandem sheared A.A pairs with a cross-strand stack and only the G.U pair not adjacent to an A.A pair forming a static wobble G.U pair. The two G.U pairs adjacent to the tandem A.A pairs are likely in a dynamic equilibrium between multiple conformations. Although Co(NH(3))(6)(3+) stabilizes the loop by several kilocalories per mole at 37 degrees C, addition of Mg(2+) or Co(NH(3))(6)(3+) has no effect on the structure of the loop. The tandem G.U pairs provide a pocket of negative charge for Co(NH(3))(6)(3+) to bind. The results contribute to understanding the structure and dynamics of purine-rich internal loops and potential G.U pairs adjacent to internal loops.

Internal loops in RNA are important for folding and function. The 3 x 3 nucleotide internal loops are the smallest size symmetric loops with a potential noncanonical base pair (middle pair) flanked on both sides by a noncanonical base pair (loop-terminal pair). Thermodynamic and structural insights acquired for 3 x 3 loops should improve approximations for stabilities of 3 x 3 and larger internal loops. Most natural 3 x 3 internal loops are purine rich, which is also true of other internal loops. A series of oligoribonucleotides containing different 3 x 3 internal loops were studied by UV melting and imino proton NMR. Both loop-terminal and middle pairs contribute to the thermodynamic stabilities of 3 x 3 loops. Extra stabilization of -1.2 kcal/mol was found for a GA middle pair when flanked by at least one non-pyrimidine-pyrimidine loop-terminal pair. A penalty of approximately 1 kcal/mol was found for loops with a single loop-terminal GA pair that has a U 3' to the G of the GA pair. A revised model for predicting stabilities of 3 x 3 loops is derived by multiple linear regression.

The J4/5 loop of the group I intron in the mouse-derived fungal pathogen Pneumocystis carinii is the docking site for the first step of the RNA-catalyzed self-splicing reaction and thus is a model of a potential drug target. This purine-rich asymmetric internal loop, 5'GGAAG/3'UAGU, is also thermodynamically more stable than other internal loops with two GU closing pairs and three nucleotides opposite two nucleotides. The results from optical melting, nuclear magnetic resonance spectroscopy, and functional group substitution experiments suggest that the GU closing pairs form and that sheared GA pairs form in the internal loop. The NMR spectra show evidence of conformational dynamics, and several GA pairings are possible. Thus, this dynamic loop presents several possible structures for potential binding of drugs that target group I self-splicing introns. The results also contribute to understanding the structural and dynamic basis for the function and thermodynamic stability of this loop.

The contribution of amino groups to the thermodynamics, structure, and dynamics of tandem A.A mismatches is investigated by substitution of purine (P) for adenine (A) within the RNA duplex, 5'(rGGCAAGCCU)(2), to give 5'(rGGCPAGCCU)(2), 5'(rGGCAPGCCU)(2), and 5'(rGGCPPGCCU)(2). The 5'(rGGCAAGCCU)(2) duplex has sheared A(anti).A(anti) (A.A trans Hoogsteen/Sugar-edge) pairs in which the A5 amino group is involved in hydrogen bonds but the A4 amino group is not [Znosko, B. M., Burkard, M. E., Schroeder, S. J., Krugh, T. R., and Turner, D. H. (2002) Biochemistry 41, 14969-14977]. In comparison to 5'(rGGCAAGCCU)(2), replacing the amino group of A4 with a hydrogen stabilizes the duplex by 1.3 kcal/mol, replacement of the A5 amino group destabilizes the duplex by 0.6 kcal/mol, and replacement of both A4 and A5 amino groups destabilizes the duplex by 0.8 kcal/mol. In NMR structures, the P.A noncanonical pairs of the 5'(rGGCPAGCCU)(2) duplex have a sheared anti-anti structure (P.A trans Hoogsteen/Sugar-edge) with P4.A5 interstrand hydrogen bonding and A5 bases that interstrand stack, similar to the structure of 5'(rGGCAAGCCU)(2). In contrast, the A.P pairs of the 5'(rGGCAPGCCU)(2) duplex have a face-to-face conformation (A.P trans Watson-Crick/Watson-Crick) with intrastrand stacking resembling typical A-form geometry. Although the P5 bases in 5'(rGGCPPGCCU)(2) are involved in an interstrand stack, the loop region is largely undefined. The results illustrate that both hydrogen-bonded and non-hydrogen-bonded amino groups play important roles in determining the thermodynamic, structural, and dynamic characteristics of purine rich internal loops.

The 5'(rGGCAAGCCU)(2) duplex contains tandem A.A pairs. The three-dimensional structure of the 5'(rGGCAAGCCU)(2) duplex was modeled by molecular dynamics and energy minimization with NMR-derived distance and dihedral angle restraints. Although the 5'(rCAAG)(2) loop is thermodynamically destabilizing by 1.1 kcal/mol, the tandem A.A pairs adopt a predominant conformation: a sheared anti-anti (A.A trans Hoogsteen/Sugar-edge) alignment similar to that observed in the crystal structure of the P4-P6 domain of the Tetrahymena thermophila intron [Cate, J. H., Gooding, A. R., Podell, E., Zhou, K., Golden, B. L., Kundrot, C. E., Cech, T. R., and Doudna, J. A. (1996) Science 273, 1678-1685]. The NMR-derived structure of the 5'(rGGCAAGCCU)(2) duplex exhibits cross-strand hydrogen bonds from N3 of A4 to an amino hydrogen of A5 and from the 2' oxygen of the A4 sugar to the other amino hydrogen of A5. An intrastrand hydrogen bond is formed from the 2' OH hydrogen of A4 to O5' of A5. The cross-strand A5 bases are stacked. The Watson-Crick G-C regions are essentially A-form. The sheared anti-anti (A.A trans Hoogsteen/Sugar-edge) alignment provides potential contact sites for tertiary interactions and, therefore, is a possible target site for therapeutics. Thus, thermodynamically destabilizing internal loops can be preorganized for tertiary interactions or ligand binding.

A G.A pair at position -5 in the P1 helix of the Candida albicans ribozyme contributes to tertiary binding of the 5' exon substrate [Disney, M. D., Haidaris, C. G., and Turner, D. H. (2001) Biochemistry 40, 6507-6519]. Here, the G in the G.A pair is replaced with inosine (I) in both semisynthetic ribozymes and oligonucleotide mimics of the internal guide sequence. Comparisons of oligonucleotide binding affinity for these and other sequences indicate that the G.A pair is in an imino conformation where the exocyclic amine of G contributes approximately 1.4 kcal/mol to tertiary interactions that help dock the ribozyme's P1 helix. Furthermore, replacement of the G.A pair with a G-C pair produces less favorable interactions with the 2'-hydroxyl group at the -3 position and a less favorable K(M) for pG in a ribozyme-catalyzed transesterification reaction. These results are also consistent with the G.A pair promoting docking of the P1 helix into the catalytic core. Evidently, tertiary interactions with the exocyclic amino group of a G in a single G.A pair can increase the equilibrium constant for tertiary folding of RNA by roughly 10-fold at 37 degrees C. Results with a G.U or G.G pair replacing the G.A pair at the -5 position suggest similar tertiary interactions with these pairs.

Algorithms for predicting RNA secondary structure require approximations for the free energies of multibranch loops, also called junctions. The stabilities of 62 RNA duplexes with three- and four-way multibranch loops were determined by optical melting. To account for the observed sequence dependence, a revised loop free-energy approximation is proposed that accounts for the strain in three-way junctions with fewer than two unpaired nucleotides, penalizes asymmetry in the distribution of unpaired nucleotides, and gives a bonus for four-way loops relative to three-way loops. Parameters for this equation were determined by linear regression.

The final step of tRNA splicing in Saccharomyces cerevisiae requires 2'-phosphotransferase (Tpt1) to transfer the 2'-phosphate from ligated tRNA to NAD, producing mature tRNA and ADP ribose-1' '-2' '-cyclic phosphate. To address how Tpt1 protein recognizes substrate RNAs, we measured the steady-state kinetic parameters of Tpt1 protein with 2'-phosphorylated ligated tRNA and a variety of related substrates. Tpt1 protein has a high apparent affinity for ligated tRNA (K(m,RNA), 0.35 nM) and a low turnover rate (k(cat), 0.3 min(-1)). Tpt1 protein recognizes both tRNA and the internal 2'-phosphate of RNAs. Steady-state kinetic analysis reveals that as RNAs lose structure and length, K(m,RNA) and k(cat) both increase commensurately. For a 2'-phosphorylated octadecamer derived from the anticodon stem-loop of ligated tRNA, K(m,RNA) and k(cat) are 5- and 8-fold higher, respectively, than for ligated tRNA, whereas for a simple substrate like pApA(p)pA, K(m,RNA) and k(cat) are 430- and 150-fold higher, respectively. Tpt1 is not detectably active on a trimer with a terminal 5'- or 3'-phosphate and is very inefficient at removal of a terminal 2'-phosphate unless there is an adjacent 3'-phosphate or phosphodiester. The K(m,NAD) for Tpt1 is substrate dependent: K(m,NAD) is 10 microM with ligated tRNA, 200 microM with pApA(p)pA, and 600 microM with pApApA(p). Preliminary analysis of KptA, a functional Tpt1 protein homologue from Escherichia coli, reveals that KptA protein is strikingly similar to yeast Tpt1 in its kinetic parameters, although E. coli is not known to have a 2'-phosphorylated RNA substrate.

UV melting experiments show that C5-(1-propynyl)ation of seven pyrimidines to give a fully propynylated oligodeoxynucleotide (PrODN) heptamer increases the thermodynamic stability of six Watson-Crick paired DNA:RNA duplexes by 8.2 kcal/mol, on average, at 37 degrees C. About 2.5 kcal/mol of this enhancement is due to long-range cooperativity between the propynylated pyrimidines, Y(p)'s. On average, penalties for dU(p):rG, dC(p):rA, dU(p):rC, and dC(p):rC mismatches are enhanced by 2.9 kcal/mol in PrODN:RNA duplexes over those in unmodified duplexes. This results in penalties as large as 10 kcal/mol for a single mismatch. Removing a single propyne two base pairs away from a mismatch in a PrODN:RNA duplex eliminates the enhancement in specificity. Evidently, enhanced specificity is directly linked to long-range cooperativity between Y(p)'s. In most cases, the enhanced specificity is larger for internal than for terminal mismatches. PrODN:RNA duplexes are destabilized by full phosphorothioate backbone substitution to give S-PrODN:RNA duplexes. The S-PrODN:RNA duplexes retain enhanced mismatch penalties, however. These results provide insight for utilizing long-range cooperativity and enhanced specificity to improve nucleic acid based probe and drug design.

Many internal loops that form tertiary contacts in natural RNAs have GU closing pairs; examples include the tetraloop receptor and P1 helix docking site in group I introns. Thus, thermodynamic parameters of internal loops with GU closing pairs can contribute to the prediction of both secondary and tertiary structure. Oligoribonucleotide duplexes containing small internal loops with GU closing pairs were studied by optical melting, one-dimensional imino proton NMR, and one-dimensional phosphorus NMR. The thermodynamic stabilities of asymmetric internal loops with GU closing pairs relative to those of loops with GC closing pairs may be explained by hydrogen bonds. In contrast, the free energy increments for symmetric internal loops of two noncanonical pairs with GU closing pairs relative to loops with GC closing pairs show much more sequence dependence. Imino proton and phosphorus NMR spectra suggest that some GA pairs adjacent to GU closing pairs may form an overall thermodynamically stable but non-A-form conformation.

RNA multibranch loops (junctions) are loops from which three or more helices exit. They are nearly ubiquitous in RNA secondary structures determined by comparative sequence analysis. In this study, systems in which two strands combine to form three-way junctions were used to measure the stabilities of RNA multibranch loops by UV optical melting and isothermal titration calorimetry (ITC). These data were used to calculate the free energy increment for initiation of a three-way junction on the basis of a nearest neighbor model for secondary structure stability. Imino proton NMR spectra were also measured for two systems and are consistent with the hypothesized helical structures. Incorporation of the experimental data into the mfold and RNA structure computer programs has contributed to an improvement in prediction of RNA secondary structure from sequence.

Candida albicans is one of many infectious pathogens that are evolving resistance to current treatments. RNAs provide a large class of targets for new therapeutics for fighting these organisms. One strategy for targeting RNAs uses short oligonucleotides that exhibit binding enhancement by tertiary interactions in addition to Watson-Crick pairing. A potential RNA target in C. albicans is the self-splicing group I intron in the LSU rRNA precursor. The recognition elements that align the 5' exon splice site for a ribozyme derived from this precursor are complex [Disney, M. D., Haidaris, C. G., and Turner, D. H. (2001) Biochemistry 40, 6507-6519]. These recognition elements have been used to guide design of hexanucleotide mimics of the 5' exon that have backbones modified for nuclease stability. These hexanucleotides bind as much as 100000-fold more tightly to a ribozyme derived from the intron than to a hexanucleotide mimic of the intron's internal guide sequence, r(GGAGGC). Several of these oligonucleotides inhibit precursor self-splicing via a suicide inhibition mechanism. The most promising suicide inhibitor is the ribophosphoramidate rn(GCCUC)rU, which forms more trans-spliced than cis-spliced product at oligonucleotide concentrations of >100 nM at 1 mM Mg(2+). The results indicate that short oligonucleotides modified for nuclease stability can target catalytic RNAs when the elements of tertiary interactions are complex.

A group I intron precursor and ribozyme were cloned from the large subunit rRNA of the human pathogen Candida albicans. Both the precursor and ribozyme are functional as determined from in vitro assays. Comparisons of dissociation constants for oligonucleotide binding to the ribozyme and to a hexanucleotide mimic of its internal guide sequence lead to a model for recognition of the 5' exon substrate by this intron. In particular, tertiary contacts with the P1 helix that help align the splice site include three 2'-hydroxyl groups, a G.U pair that occurs at the intron's splice junction, and a G.A pair. The free energy contribution that each interaction contributes to tertiary binding is determined. When the G.A pair is replaced with a G-C pair, tertiary interactions to 5' exon mimic 2'-hydroxyl groups are significantly weakened. When the G.A pair is replaced with a G.U pair, tertiary interactions are retained and binding is 10-fold tighter. These results expand our knowledge of substrate recognition by group I introns, and also provide a basis for rational design of oligonucleotide-based therapeutics for targeting group I introns by binding enhancement by tertiary interactions and suicide inhibition strategies.

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.

Pneumocystis carinii is a mammalian pathogen that infects and kills immunocompromised hosts such as cancer and AIDS patients. The LSU rRNA precursor of P. carinii contains a conserved group I intron that is an attractive drug target because humans do not contain group I introns. The oligonucleotide r(AUGACU), whose sequence mimics the 3'-end of the 5'-exon, binds to a ribozyme derived from the intron with a K(d) of 5.2 nM, which is 61000-fold tighter than expected from base-pairing alone [Testa, S. M., Haidaris, G. C., Gigliotti, F., and Turner, D. H. (1997) Biochemistry 36, 9379-9385]. Thus, oligonucleotide binding is enhanced by tertiary interactions. To localize interactions that give rise to this tertiary stability, binding to the ribozyme has been measured as a function of oligonucleotide length and sequence. The results indicate that 4.3 kcal/mol of tertiary stability is due to a G.U pair that forms at the intron's splice junction. Eliminating nucleotides at the 5'-end of r(AUGACU) does not affect intron binding more than expected from differences in base-pairing until r((___)ACU), which binds much more tightly than expected. Adding a C at the 5'- or 3'-end that can potentially form a C-G pair with the target has little effect on binding affinity. Truncated oligonucleotides were tested for their ability to inhibit intron self-splicing via a suicide inhibition mechanism. The tetramer, r((__)GACU), retains similar binding affinity and reactivity as the hexamer, r(AUGACU). Thus oligonucleotides as short as tetramers might serve as therapeutics that can use a suicide inhibition mechanism to inhibit self-splicing. Results with a phosphoramidate tetramer and thiophosphoramidate hexamer indicate that oligonucleotides with backbones stable to nuclease digestion retain favorable binding and reactivity properties.

Nucleotides in RNA that are not Watson-Crick-paired form unique structures for recognition or catalysis, but determinants of these structures and their stabilities are poorly understood. A single noncanonical pair of two guanosines (G) is more stable than other noncanonical pairs and can potentially form pairing structures with two hydrogen bonds in four different ways. Here, the energetics and structure of single GG pairs are investigated in several sequence contexts by optical melting and NMR. The data for r(5'GCAGGCGUGC3')(2), in which G4 and G7 are paired, are consistent with a model in which G4 and G7 alternate syn glycosidic conformations in a two-hydrogen-bond pair. The two distinct structures are derived from nuclear Overhauser effect spectroscopic distance restraints coupled with simulated annealing using the AMBER 95 force field. In each structure, the imino and amino protons of the anti G are hydrogen bonded to the O6 and N7 acceptors of the syn G, respectively. An additional hydrogen-bond connects the syn G amino group to the 5' nonbridging pro-R(p) phosphate oxygen. The GG pair fits well into a Watson-Crick helix. In r(5'GCAGGCGUGC3')(2), the G4(anti), G7(syn) structure is preferred over G4(syn), G7(anti). For single GG pairs in other contexts, exchange processes make interpretation of spectra more difficult but the pairs are also G(syn), G(anti). Thermodynamic data for a variety of duplexes containing pairs of G, inosine, and 7-deazaguanosine flanked by GC pairs are consistent with the structural and energetic interpretations for r(5'GCAGGCGUGC3')(2), suggesting similar GG conformations.

Optical melting experiments were used to determine the thermodynamic parameters for oligoribonucleotides containing small asymmetric internal loops. The results show a broad range of thermodynamic stabilities, which depend on loop size, asymmetry, sequence, closing base pairs, and length of helix stems. Imino proton NMR experiments provide evidence for possible hydrogen bonding in GA and UU mismatches in some asymmetric loops. The stabilizing effects of GA, GG, and UU mismatches on the thermodynamic stability of internal loops vary depending on the size and asymmetry of the loop. The dependence of loop stability on Watson-Crick closing base pairs may be explained by an account of hydrogen bonds. Models are presented for approximating the free energy increments of 2 x 3 and 1 x 3 internal loops.

G.U pairs occur frequently and have many important biological functions. The stability of symmetric tandem G.U motifs depends both on the adjacent Watson-Crick base pairs, e.g., 5'G > 5'C, and the sequence of the G.U pairs, i.e., 5'-UG-3' > 5'-GU-3', where an underline represents a nucleotide in a G.U pair [Wu, M., McDowell, J. A., and Turner, D. H. (1995) Biochemistry 34, 3204-3211]. In particular, at 37 degrees C, the motif 5'-CGUG-3' is less stable by approximately 3 kcal/mol compared with other symmetric tandem G.U motifs with G-C as adjacent pairs: 5'-GGUC-3', 5'-GUGC-3', and 5'-CUGG-3'. The solution structures of r(GAGUGCUC)(2) and r(GGCGUGCC)(2) duplexes have been determined by NMR and restrained simulated annealing. The global geometry of both duplexes is close to A-form, with some distortions localized in the tandem G.U pair region. The striking discovery is that in r(GGCGUGCC)(2) each G.U pair apparently has only one hydrogen bond instead of the two expected for a canonical wobble pair. In the one-hydrogen-bond model, the distance between GO6 and UH3 is too far to form a hydrogen bond. In addition, the temperature dependence of the imino proton resonances is also consistent with the different number of hydrogen bonds in the G.U pair. To test the NMR models, U or G in various G.U pairs were individually replaced by N3-methyluridine or isoguanosine, respectively, thus eliminating the possibility of hydrogen bonding between GO6 and UH3. The results of thermal melting studies on duplexes with these substitutions support the NMR models.

Pneumocystis carinii is a mammalian pathogen that contains a self-splicing group I intron in its large subunit rRNA precursor. We report the binding of methylphosphonate/DNA chimeras and neutral methylphosphonate oligonucleotides to a ribozyme that is a truncated form of the intron. At 15 mM Mg(2+), the nuclease-resistant all-methylphosphonate hexamer, d(AmTmGmAmCm)rU, with a sequence that mimics the 3' end of the precursor's 5' exon, binds with a dissociation constant of 272 nM. The hexamer's dissociation constant for binding by base-pairing alone to the ribozyme's binding site sequence is 8.3 mM. Thus there is a 30 000-fold binding enhancement by tertiary interactions (BETI), which is close to the 60 000-fold enhancement previously observed with the all-ribo hexamer, r(AUGACU). Evidently, backbone charge and 2' hydroxyl groups are not required for BETI. At 3-15 mM Mg(2+), the all-methylphosphonate and DNA oligonucleotides trans-splice to a truncated form of the rRNA precursor, but do not compete with cis-splicing when pG is present. These results suggest that uncharged or partially charged backbones may be used to design therapeutics to target RNAs through binding enhancement by tertiary interactions and suicide inhibition strategies.

Antisense compounds are designed to optimize selective hybridization of an exogenous oligonucleotide to a cellular target. Typically, Watson-Crick base pairing between the antisense compound and target provides the key recognition element. Uridine (U), however, not only stably base pairs with adenosine (A) but also with guanosine (G), thus reducing specificity. Studies of duplex formation by oligonucleotides with either an internal or a terminal 2- or 4-thiouridine (s(2)U or s(4)U) show that s(2)U can increase the stability of base pairing with A more than with G, while s(4)U can increase the stability of base pairing with G more than with A. The latter may be useful when binding can be enhanced by tertiary interactions with a s(4)U-G pair. To test the effects of s(2)U and s(4)U substitutions on tertiary interactions, binding to a group I intron ribozyme from mouse-derived Pneumocystis carinii was measured for the hexamers, r(AUGACU), r(AUGACs(2)U), and r(AUGACs(4)U), which mimic the 3' end of the 5' exon. The results suggest that at least one of the carbonyl groups of the 3' terminal U of r(AUGACU) is involved in tertiary interactions with the catalytic core of the ribozyme and/or thio groups change the orientation of a terminal U-G base pair. Thus thio substitutions may affect tertiary interactions. Studies of trans-splicing of 5' exon mimics to a truncated rRNA precursor, however, indicate that thio substitutions have negligible effects on overall reactivity. Therefore, modified bases can enhance the specificity of base pairing while retaining other activities and, thus, increase the specificity of antisense compounds targeting cellular RNA.

The thermodynamic properties and structures of single mismatches in short RNA duplexes were studied in optical melting and imino proton NMR experiments. The free energy increments at 37 degrees C measured for non-GU single mismatches range from -2.6 to 1.7 kcal/mol. These increments depend on the identity of the mismatch, adjacent base pairs, and the position in the helix. UU and AA mismatches are more stable close to a helix end, but GG mismatch stability is essentially unaffected by the position in the helix. Approximations are suggested for predicting stabilities of single mismatches in short RNA duplexes.

Improved thermodynamic parameters for prediction of RNA duplex formation are derived from optical melting studies of 90 oligoribonucleotide duplexes containing only Watson-Crick base pairs. To test end or base composition effects, new sets of duplexes are included that have identical nearest neighbors, but different base compositions and therefore different ends. Duplexes with terminal GC pairs are more stable than duplexes with the same nearest neighbors but terminal AU pairs. Penalizing terminal AU base pairs by 0.45 kcal/mol relative to terminal GC base pairs significantly improves predictions of DeltaG degrees37 from a nearest-neighbor model. A physical model is suggested in which the differential treatment of AU and GC ends accounts for the dependence of the total number of Watson-Crick hydrogen bonds on the base composition of a duplex. On average, the new parameters predict DeltaG degrees37, DeltaH degrees, DeltaS degrees, and TM within 3.2%, 6.0%, 6.8%, and 1.3 degreesC, respectively. These predictions are within the limit of the model, based on experimental results for duplexes predicted to have identical thermodynamic parameters.

Pneumocystis carinii is the most common lethal opportunistic pathogen infecting Acquired Immune Deficiency Syndrome (AIDS) patients, and more effective therapeutics for it are needed. P. carinii, but not humans, contain RNA self-splicing group I introns, so these functionally important RNAs are potential anti-fungal targets. In vitro, d(ATGACT), which mimics the 3' end of the 5' exon of a conserved ribosomal RNA group I intron from mouse-derived Pneumocystis carinii binds to a ribozyme that is a truncated form of this intron. The binding is about 30,000 times tighter than expected for simple base-pairing because binding is enhanced by tertiary interactions. Here we report the effects of modifying the phosphodiester backbone of d(ATGACT) with phosphorothioate and of d(ATGAC)rU with N3'-->P5' phosphoramidate linkages. The enhancement of binding by tertiary interactions is not substantially decreased, and in some cases is increased when single Rp and Sp phosphorothioate substitutions are made, although overall binding is weaker by up to 6-fold. A mixture of 5' exon mimic isomers that each contain five phosphorothioate linkages binds to the ribozyme at least 14-fold less tightly than the corresponding phosphodiester mimic. In contrast, the 5' exon mimic with five internal N3'-->P5' phosphoramidate linkages binds 4-fold more tightly than d(ATGAC)rU. This increased binding is largely due to more favorable base-pairing, but tertiary interactions still enhance binding by more than 2, 000-fold. These results indicate that chemically modified, nuclease stable 5' exon mimics can act as antisense agents with binding enhanced by tertiary interactions (BETI). This strategy permits design of short antisense agents with high specificity.