Brent M. Znosko

Thermodynamic contribution and nearest-neighbor parameters of pseudouridine-adenosine base pairs in oligoribonucleotides

Pseudouridine (Ψ) is the most common noncanonical nucleotide present in naturally occurring RNA and serves a variety of roles in the cell, typically appearing where structural stability is crucial to function. Ψ residues are isomerized from native uridine residues by a class of highly conserved enzymes known as pseudouridine synthases. In order to quantify the thermodynamic impact of pseudouridylation on U-A base pairs, 24 oligoribonucleotides, 16 internal and eight terminal Ψ-A oligoribonucleotides, were thermodynamically characterized via optical melting experiments. The thermodynamic parameters derived from two-state fits were used to generate linearly independent parameters for use in secondary structure prediction algorithms using the nearest-neighbor model. On average, internally pseudouridylated duplexes were 1.7 kcal/mol more stable than their U-A counterparts, and terminally pseudouridylated duplexes were 1.0 kcal/mol more stable than their U-A equivalents. Due to the fact that Ψ-A pairs maintain the same Watson-Crick hydrogen bonding capabilities as the parent U-A pair in A-form RNA, the difference in stability due to pseudouridylation was attributed to two possible sources: the novel hydrogen bonding capabilities of the newly relocated imino group as well as the novel stacking interactions afforded by the electronic configuration of the Ψ residue. The newly derived nearest-neighbor parameters for Ψ-A base pairs may be used in conjunction with other nearest-neighbor parameters for accurately predicting the most likely secondary structure of A-form RNA containing Ψ-A base pairs.

A Guanosine-Centric Mechanism for RNA Chaperone Function

Simply Folding RNA chaperones simplify what would otherwise be complex and slow RNA folding events. Grohman et al. (p. 190, published online 7 March) show that the Moloney murine leukemia virus (MuLV) nucleocapsid (NC) protein, which chaperones MuLV RNA dimerization promotes MuLV RNA folding by binding to exposed guanosine bases and destabilizing strong guanosine interactions. With base-pairs being rendered roughly of the same energy, RNA assembly pathways are simplified, promoting proper folding. RNA chaperones promote assembly of complex RNA structures by binding to and “neutralizing” guanosine bases. RNA chaperones are ubiquitous, heterogeneous proteins essential for RNA structural biogenesis and function. We investigated the mechanism of chaperone-mediated RNA folding by following the time-resolved dimerization of the packaging domain of a retroviral RNA at nucleotide resolution. In the absence of the nucleocapsid (NC) chaperone, dimerization proceeded through multiple, slow-folding intermediates. In the presence of NC, dimerization occurred rapidly through a single structural intermediate. The RNA binding domain of heterogeneous nuclear ribonucleoprotein A1 protein, a structurally unrelated chaperone, also accelerated dimerization. Both chaperones interacted primarily with guanosine residues. Replacing guanosine with more weakly pairing inosine yielded an RNA that folded rapidly without a facilitating chaperone. These results show that RNA chaperones can simplify RNA folding landscapes by weakening intramolecular interactions involving guanosine and explain many RNA chaperone activities.

RNA CoSSMos: Characterization of Secondary Structure Motifs—a searchable database of secondary structure motifs in RNA three-dimensional structures

RNA secondary structure is important for designing therapeutics, understanding protein–RNA binding and predicting tertiary structure of RNA. Several databases and downloadable programs exist that specialize in the three-dimensional (3D) structure of RNA, but none focus specifically on secondary structural motifs such as internal, bulge and hairpin loops. The RNA Characterization of Secondary Structure Motifs (RNA CoSSMos) database is a freely accessible and searchable online database and website of 3D characteristics of secondary structure motifs. To create the RNA CoSSMos database, 2156 Protein Data Bank (PDB) files were searched for internal, bulge and hairpin loops, and each loops structural information, including sugar pucker, glycosidic linkage, hydrogen bonding patterns and stacking interactions, was included in the database. False positives were defined, identified and reclassified or omitted from the database to ensure the most accurate results possible. Users can search via general PDB information, experimental parameters, sequence and specific motif and by specific structural parameters in the subquery page after the initial search. Returned results for each search can be viewed individually or a complete set can be downloaded into a spreadsheet to allow for easy comparison. The RNA CoSSMos database is automatically updated weekly and is available at http://cossmos.slu.edu.

Thermodynamic Characterization of RNA Triloops

Relatively few thermodynamic parameters are available for RNA triloops. Therefore, 24 stem-loop sequences containing naturally occurring triloops were optically melted, and the thermodynamic parameters ΔH°, ΔS°, ΔG°(37), 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 stem-loops containing triloops and, furthermore, should allow for improved prediction of secondary structure from sequence.

Thermodynamic Characterization of Naturally Occurring RNA Single Mismatches with G-U Nearest Neighbors

Due to their prevalence and roles in biological systems, single mismatches adjacent to G-U pairs are important RNA structural elements. Since there are only limited experimental values for the stability of single mismatches adjacent to G-U pairs, current algorithms using free energy minimization to predict RNA secondary structure from sequence assign predicted thermodynamic values to these types of single mismatches. Here, thermodynamic data are reported for frequently occurring single mismatches adjacent to at least one G-U pair. This experimental data can be used in place of predicted thermodynamic values in algorithms that predict secondary structure from sequence using free energy minimization. When predicting the thermodynamic contributions of previously unmeasured single mismatches, most algorithms apply the same thermodynamic penalty for an A-U pair adjacent to a single mismatch and a G-U pair adjacent to a single mismatch. A recent study, however, suggests that the penalty for a G-U pair adjacent to a tandem mismatch should be 1.2 +/- 0.1 kcal/mol, and the penalty for an A-U pair adjacent to a tandem mismatch should be 0.5 +/- 0.2 kcal/mol [Christiansen, M. E. and Znosko, B. M. (2008) Biochemistry 47, 4329-4336]. Therefore, the data reported here are combined with the existing thermodynamic dataset of single mismatches, and nearest neighbor parameters are derived for an A-U pair adjacent to a single mismatch (1.1 +/- 0.1 kcal/mol) and a G-U pair adjacent to a single mismatch (1.4 +/- 0.1 kcal/mol).

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

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.

Effects of Non-Nearest Neighbors on the Thermodynamic Stability of RNA GNRA Hairpin Tetraloops

Currently, several models for predicting the secondary structure of RNA exist, one of which is free energy minimization using the Nearest Neighbor Model. This model predicts the lowest-free energy secondary structure from a primary sequence by summing the free energy contributions of the Watson-Crick nearest neighbor base pair combinations and any noncanonical secondary structure motif. The Nearest Neighbor Model also assumes that the free energy of the secondary structure motif is dependent solely on the identities of the nucleotides within the motif and the motifs nearest neighbors. To test the current assumption of the Nearest Neighbor Model that the non-nearest neighbors do not affect the stability of the motif, we optically melted different stem-loop oligonucleotides to experimentally determine their thermodynamic parameters. In each of these oligonucleotides, the hairpin loop sequence and the adjacent base pairs were held constant, while the first or second non-nearest neighbors were varied. The experimental results show that the thermodynamic contributions of the hairpin loop were dependent upon the identity of the first non-nearest neighbor, while the second non-nearest neighbor had a less obvious effect. These results were then used to create an updated model for predicting the thermodynamic contributions of a hairpin loop to the overall stability of the stem-loop structure.

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.

Computational model for predicting experimental RNA and DNA nearest-neighbor free energy rankings.

Hydrogen-bonding, intrastrand base-stacking, and interstrand base-stacking energies were calculated for RNA and DNA dimers at the MP2(full)/6-311G** level of theory. Standard A-form RNA and B-form DNA geometries from average fiber diffraction data were employed for all base monomer and dimer geometries, and all dimer binding energies were obtained via single-point calculations. The effects of water solvation were considered using the PCM model. The resulting dimer binding energies were used to calculate the 10 unique RNA and 10 unique DNA computational nearest-neighbor energies, and the ranking of these computational nearest neighbor energies are in excellent agreement with the ranking of the experimental nearest-neighbor free energies. These results dispel the notion that average fiber diffraction geometries are insufficient for calculating RNA and DNA stacking energies.

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.