Thermodynamic characterization of tandem mismatches found in naturally occurring RNA

Although all sequence symmetric tandem mismatches and some sequence asymmetric tandem mismatches have been thermodynamically characterized and a model has been proposed to predict the stability of previously unmeasured sequence asymmetric tandem mismatches [Christiansen,M.E. and Znosko,B.M. (2008) Biochemistry, 47, 4329–4336], experimental thermodynamic data for frequently occurring tandem mismatches is lacking. Since experimental data is preferred over a predictive model, the thermodynamic parameters for 25 frequently occurring tandem mismatches were determined. These new experimental values, on average, are 1.0 kcal/mol different from the values predicted for these mismatches using the previous model. The data for the sequence asymmetric tandem mismatches reported here were then combined with the data for 72 sequence asymmetric tandem mismatches that were published previously, and the parameters used to predict the thermodynamics of previously unmeasured sequence asymmetric tandem mismatches were updated. The average absolute difference between the measured values and the values predicted using these updated parameters is 0.5 kcal/mol. This updated model improves the prediction for tandem mismatches that were predicted rather poorly by the previous model. This new experimental data and updated predictive model allow for more accurate calculations of the free energy of RNA duplexes containing tandem mismatches, and, furthermore, should allow for improved prediction of secondary structure from sequence

Thermodynamic characterization of the complete set of sequence symmetric tandem mismatches in RNA and an improved model to predict the free energy contribution of sequence asymmetric tandem mismatches

ABSTRACT: Because of the availability of an abundance of RNA sequence information, the ability to rapidly and accurately predict the secondary structure of RNA from sequence is becoming increasingly important. A common method for predicting RNA secondary structure from sequence is free energy minimization. Therefore, accurate free energy contributions for every RNA secondary structure motif are necessary for accurate secondary structure predictions. Tandem mismatches are prevalent in naturally occurring sequences and are biologically important. A common method for predicting the stability of a sequence asymmetric tandem mismatch relies on the stabilities of the two corresponding sequence symmetric tandem mismatches [Mathews, D. H., Sabina, J., Zuker, M., and Turner, D. H. (1999) J. Mol. Biol. 288, 911-940]. To improve the prediction of sequence asymmetric tandem mismatches, the experimental thermodynamic parameters for the 22 previously unmeasured sequence symmetric tandem mismatches are reported. These new data, however, do not improve prediction of the free energy contributions of sequence asymmetric tandem mismatches. Therefore, a new model, independent of sequence symmetric tandem mismatch free energies, is proposed. This model consists of two penalties to account for destabilizing tandem mismatches, two bonuses to account for stabilizing tandem mismatches, and two penalties to account for A-U and G-U adjacent base pairs. This model improves the prediction of asymmetric tandem mismatch free energy contributions and is likely to improve the prediction of RNA secondary structure from sequence. The three most common base pairs in RNA are the Watson-Crick pairs, G-C and A-U, and the wobble G-U pair. These canonical base pairs are the components of the helical portions of RNA, and they have a regular structure and hydrogen bonding pattern. However, canonical base pairs account for only approximately half of the nucleotides found in RNA (1). The other half are involved in other secondary structure motifs, such as hairpins, bulges, and internal loops. One common RNA secondary structure motif is a tandem mismatch, or 2 × 2 internal loop. Tandem mismatches occur when two adjacent, noncanonical pairs are situated within a helical portion of canonical base pairs. The presence of tandem mismatches has been confirmed in a variety of RNA secondary structures (2-10) ranging from bacteria to trinucleotide repeats in human neurological diseases. Fortunately, due to the pioneering efforts of projects such as the Human Genome Project (11, 12), entire genomes can now be sequenced accurately and efficiently. In recent years, thousands of RNA nucleotide sequences have been made publicly available (13). After a RNA sequence has been determined, the next logical step in better understanding structure and function is to determine an accurate method for predicting the secondary structure of RNA from its primary sequence. The ability to predict secondary structure of RNA from sequence is important for several reasons. The determination of secondary structure can aid in the determination of tertiary structure. Also, because of the direct relationship between structure and function, the ability to predict secondary structure of RNA gives insight into the different functions and roles that RNA may have. In addition, being able to predict the secondary structure of RNA can help with the design of pharmaceuticals by providing an accurate target site for recognition by drugs. The overwhelming importance of being able to predict RNA secondary structure from sequence has led to the development of several computer algorithm

Thermodynamic characterization of RNA triloops

ABSTRACT: Relatively few thermodynamic parameters are available for RNA triloops. Therefore, 24 stemloop sequences containing naturally occurring triloops were optically melted, and the thermodynamic parameters ΔH°, ΔS°, ΔG°3 7 , and T M for each stem-loop were determined. These new experimental values, on average, are 0.5 kcal/mol different from the values predicted for these triloops using the model proposed by Mathews et al. [Mathews, D. H., Disney, M. D., Childs, J. L., Schroeder, S. J., Zuker, M., and Turner, D. H. (2004) Proc. Natl. Acad. Sci. U.S. A. 101, 7287-7292]. The data for the 24 triloops reported here were then combined with the data for five triloops that were published previously. A new model was derived to predict the free energy contribution of previously unmeasured triloops. The average absolute difference between the measured values and the values predicted using this proposed model is 0.3 kcal/mol. These new experimental data and updated predictive model allow for more accurate calculations of the free energy of RNA stemloops containing triloops and, furthermore, should allow for improved prediction of secondary structure from sequence. RNA stem-loops containing three nucleotides in the loop, triloops, are common secondary structure motifs found in naturally occurring RNA. For example, bacterial 16S rRNAs strongly favor tetraloops; however, the UUU triloop is the most common replacement (1). In the 16S-like rRNA variable regions, triloops account for 7% of the loops in bacteria and 16% of the loops in eukaryotes (2). Triloops are also found in large subunit rRNAs (3, 4), 5S rRNAs (5), signal recognition particles (6), RNase P RNAs (7), and group I introns (8, 9). More specifically, triloops are found in Brome mosaic virus (þ) strand RNA (10), human rhinovirus isotype 14 (11), iron responsive element RNA (12), and an RNA aptamer for bacteriophage MS2 coat protein (13), to name a few. Although relatively unstable due to the strain in the loop, triloops may be an important structural feature due to the accessibility of the loop nucleotides for recognition by proteins, other nucleic acids, or small molecules. It has been shown that triloops play a role in various biological processes, including virus replication The current model used by secondary structure prediction algorithms to predict the thermodynamic contribution of RNA triloops to stem-loop stability is sequence independent; all triloops contribute 5.4 kcal/mol to stem-loop stability, with the exception of 5 0 CCC3 0 which contributes 6.9 kcal/mol (21). In addition, there are two unstable triloop sequences (5 0 CAACG3 0 and 5 0 GUUAC3 0 ) for which this predictive model is not used; instead, the ΔG°3 7,loop values (6.8 and 6.9 kcal/mol, respectively) for these two triloops are provided in a lookup table (21). An interesting study by the Bevilacqua laboratory (19) used a combinatorial approach and temperature gradient gel electrophoresis to identify stable and unstable RNA triloops. It was discovered that sequence preferences for exceptionally stable triloops included a U-rich loop and C-G as the closing base pair. Although they used 10 mM NaCl during their melting experiments, they suggested that the rules for predicting triloop stability at 1 M NaCl should be modified; however, this has yet to be done. Here, we report the thermodynamic parameters for 24 previously unmeasured RNA triloops in 1 M NaCl and propose a new algorithm for predicting the contribution of triloops to stem-loop stability, which includes two bonuses for stabilizing sequence features. MATERIALS AND METHODS Compiling and Searching a Database for RNA Triloops. The initial aim of this project was to identify the most frequently occurring RNA triloops in nature and to thermodynamically characterize these hairpin triloop sequences. Therefore, a database of 1349 RNA secondary structures containing 123 small subunit rRNAs (22), 223 large subunit rRNAs (3, 4), 309 5S rRNAs (5), 484 tRNAs (23), 91 signal recognition particles (6), 16 RNase P RNAs (7), 100 group I introns (8, 9), and 3 group II introns (24) was compiled. This database was searched for triloops, and the number of occurrences for each type of triloop was tabulated. In this work, G-U pairs are considered to be canonical base pairs. Design of Sequences for Optical Melting Studies. Since most thermodynamic parameters for RNA secondary structure motifs are reported for RNA solutions containing 1 M NaCl, the melting buffer used in this work also contained 1 M NaCl. A major limitation of a thermodynamic analysis of RNA hairpins using this high salt concentration is the possible bimolecular

Structural characterization of naturally occurring RNA single mismatches

RNA is known to be involved in several cellular processes; however, it is only active when it is folded into its correct 3D conformation. The folding, bending and twisting of an RNA molecule is dependent upon the multitude of canonical and non-canonical secondary structure motifs. These motifs contribute to the structural complexity of RNA but also serve important integral biological functions, such as serving as recognition and binding sites for other biomolecules or small ligands. One of the most prevalent types of RNA secondary structure motifs are single mismatches, which occur when two canonical pairs are separated by a single non-canonical pair. To determine sequence–structure relationships and to identify structural patterns, we have systematically located, annotated and compared all available occurrences of the 30 most frequently occurring single mismatch-nearest neighbor sequence combinations found in experimentally determined 3D structures of RNA-containing molecules deposited into the Protein Data Bank. Hydrogen bonding, stacking and interaction of nucleotide edges for the mismatched and nearest neighbor base pairs are described and compared, allowing for the identification of several structural patterns. Such a database and comparison will allow researchers to gain insight into the structural features of unstudied sequences and to quickly look-up studied sequences

Effect of Sodium Ions on RNA Duplex Stability

The standard sodium concentration for RNA optical melting experiments is 1.021 M. Algorithms that predict <i>T</i>_m, Δ<i>G</i>°₃₇, and secondary structure from sequence generally rely on parameters derived from optical melting experiments performed in 1.021 M sodium. Physiological monovalent cation concentrations are much lower than 1.021 M. In fact, many molecular biology techniques require buffers containing monovalent cation concentrations other than 1.021 M. Predictions based on the 1.021 M Na⁺ parameters may not be accurate when the monovalent cation concentration is not 1.021 M. Here, we report thermodynamic data from optical melting experiments for a set of 18 RNA duplexes, each melted over a wide range of sodium ion concentrations (71, 121, 221, and 621 mM). Using these data and previously published data for the same sequences melted in 1.021 M Na⁺, we report <i>T</i>_m and Δ<i>G</i>°₃₇ correction factors to scale the standard 1.021 M Na⁺ RNA parameters to other sodium ion concentrations. The recommended <i>T</i>_m correction factor predicts the melting temperature within 0.7 °C, and the recommended Δ<i>G</i>°₃₇ correction factor predicts the free energy within 0.14 kcal/mol. These correction factors can be incorporated into prediction algorithms that predict RNA secondary structure from sequence and provide <i>T</i>_m and Δ<i>G</i>°₃₇ values for RNA duplexes

Thermodynamic characterization of naturally occurring RNA tetraloops

Although tetraloops are one of the most frequently occurring secondary structure motifs in RNA, less than one-third of the 30 most frequently occurring RNA tetraloops have been thermodynamically characterized. Therefore, 24 stem–loop sequences containing common tetraloops were optically melted, and the thermodynamic parameters ΔH°, ΔS°, ΔG°37, and TM for each stem–loop were determined. These new experimental values, on average, are 0.7 kcal/mol different from the values predicted for these tetraloops using the model proposed by Vecenie CJ, Morrow CV, Zyra A, Serra MJ. 2006. Biochemistry 45: 1400–1407. The data for the 24 tetraloops reported here were then combined with the data for 28 tetraloops that were published previously. A new model, independent of terminal mismatch data, was derived to predict the free energy contribution of previously unmeasured tetraloops. The average absolute difference between the measured values and the values predicted using this proposed model is 0.4 kcal/mol. This new experimental data and updated predictive model allow for more accurate calculations of the free energy of RNA stem–loops containing tetraloops and, furthermore, should allow for improved prediction of secondary structure from sequence. It was also shown that tetraloops within the sequence 5′-GCCNNNNGGC-3′ are, on average, 0.6 kcal/mol more stable than the same tetraloop within the sequence 5′-GGCNNNNGCC-3′. More systemic studies are required to determine the full extent of non-nearest-neighbor effects on tetraloop stability