Advancing viral RNA structure prediction: measuring the thermodynamics of pyrimidine-rich internal loops

Abstract

Accurate thermodynamic parameters improve RNA structure predictions and thus accelerate understanding of RNA function and the identification of RNA drug binding sites. Many viral RNA structures, such as internal ribosome entry sites, have internal loops and bulges that are potential drug target sites. Current models used to predict internal loops are biased toward small, symmetric purine loops, and thus poorly predict asymmetric, pyrimidine-rich loops with >6 nucleotides (nt) that occur frequently in viral RNA. This article presents new thermodynamic data for 40 pyrimidine loops, many of which can form UU or protonated CC base pairs. Uracil and protonated cytosine base pairs stabilize asymmetric internal loops. Accurate prediction rules are presented that account for all thermodynamic measurements of RNA asymmetric internal loops. New loop initiation terms for loops with >6 nt are presented that do not follow previous assumptions that increasing asymmetry destabilizes loops. Since the last 2004 update, 126 new loops with asymmetry or sizes greater than 2 × 2 have been measured. These new measurements significantly deepen and diversify the thermodynamic database for RNA. These results will help better predict internal loops that are larger, pyrimidine-rich, and occur within viral structures such as internal ribosome entry sites.

Thermodynamic examination of pH and magnesium effect on U6 RNA internal loop

“…This can be attributed to protonation dependent stabilization of the helix that leads to a single-nucleotide-like bulge loop. The literature value for the 1 × 2 internal loop penalty in 1 M NaCl for ΔG o 37,loop is 2.2 ± 0.1 kcal/mol (Schroeder et al 1996;Phan et al 2017). The individual ΔG o 37,loop measured values in Badhwar et al varied between 2.47 and 5.29 kcal/mol for the 1 × 2 bulge loop identical to the U6 wild-type construct when placed in different helical stems (Badhwar et al 2007), indicating that non-nearest neighbor effects play a significant role in RNA stability.…”

“…In the presence of magnesium ions, the penalty for breaking the helix decreases by as much as 1.25 kcal/ mol over 1 M KCl. The C68U construct was uniquely destabilizing (ΔG o 37,loop = 5.25 kcal/mol), likely indicating that the G•U base pair at the end of the helix causes significant changes to the loop, perhaps leading to a potential 2 × 3 internal loop, for which ΔG o 37,loop of 3.5 kcal/ mol is utilized in prediction models (Phan et al 2017). The presence of magnesium ions or lowering the pH did not decrease the penalty.…”

Challenges and approaches to predicting RNA with multiple functional structures

“…Free energy minimization can provide a secondary structure model for a single or multiple RNA sequences. The thermodynamic database that forms the foundation for free energy minimization is still being updated with improved thermodynamic parameters for noncanonical RNA loop motifs (Phan et al 2017;Zuber et al 2017). One approximation of the Zuker-Steigler algorithm is that substructures with suboptimal energies will not be combined, which very efficiently reduces the search for the minimum energy structure (Zuker and Stiegler 1981;Mathews 2006).…”

Quantitative high-throughput tests of ubiquitous RNA secondary structure prediction algorithms via RNA/protein binding

“…In contrast, only a small fraction (<10%) of the 1024 1 possible tetraloops have been measured (11,14), with several displaying exceptionally low or high stabilities that span a range of 5 kcal/mol at 37 ˚C. The coverage becomes even lower for features such as asymmetric internal loops, despite their common occurrence in biological RNAs: e.g., fewer than 0.1% of possible 3x5 internal loops sequences have been measured (7 of >10 4 ) (15).…”