Revisiting the base pair maximization approach for RNA secondary structure prediction with SQUARNA

Non-coding RNAs play diverse roles in a wide range of cellular processes, with their spatial structure being pivotal to their function. RNA secondary structure is a key determinant of its overall fold. Given the scarcity of experimentally determined RNA 3D structures, understanding secondary structure is vital for discerning RNA function. Currently, there is no universally effective solution for de novo RNA secondary structure prediction. Existing methods are becoming increasingly complex without marked improvements in accuracy and often overlook critical features such as pseudoknots and alternative folds. Here, we introduce SQUARNA, a new approach to de novo RNA secondary structure prediction that is suitable for both individual RNA analysis and large-scale structural searches. SQUARNA revisits the concept of base pair maximization and develops it into a stem maximization idea coupled with the widely used free energy minimization (MFE) framework. SQUARNA can predict alternative structures and handle pseudoknots of arbitrary complexity. Benchmarking shows that SQUARNA outperforms existing methods, including deep learning models, in both single-sequence and alignment-based RNA secondary structure prediction. SQUARNA seamlessly integrates sequence and alignment information with experimental data, such as residue reactivities obtained by chemical probing, as well as other structural restraints, including automated searches for Rfam database templates, G-quadruplex patterns, and protein-binding motifs. SQUARNA is available as a standalone tool at https://github.com/febos/SQUARNA and as a web server at https://larnal.imol.institute.