Exploring the accuracy of ab initio prediction methods for viral pseudoknotted RNA structures

The prediction of tertiary RNA structures is significant to the field of medicine (e.g. mRNA vaccines, genome editing), and the exploration of viral transcripts. Though many RNA folding software exist, few studies have condensed their locus of attention solely to viral pseudoknotted RNA. These regulatory pseudoknots play a role in genome replication, gene expression, and protein synthesis. This study explores five RNA folding engines that compute either the minimum free energy (MFE) or the maximum expected accuracy (MEA). These folding engines were tested against 26 experimentally derived short pseudoknotted sequences (20-150nt) using metrics that are commonly applied to software prediction accuracy (e.g. F₁scoring, PPV). This paper reports higher accuracy RNA prediction engines, such as pKiss, when compared to previous iterations of the software, and when compared to older folding engines. They show that MEA folding software does not always outperform MFE folding software in prediction accuracy when assessed with metrics such as percent error, sensitivity, PPV, and F₁scoring when applied to viral pseudoknotted RNA. Moreover, the results suggest that thermodynamic model parameters will not ensure accuracy if auxiliary parameters such as Mg²⁺binding, dangling end options, and H-type penalties are not applied. The observations reported in this paper highlight the quality between differentab initioprediction methods while enforcing the idea that a better understanding of intracellular thermodynamics is necessary for a more efficacious screening of RNAs.