The <scp>RNA</scp> ‐Puzzles Assessments of <scp>RNA</scp> ‐Only Targets in <scp>CASP16</scp>

RNA-Puzzles was launched in 2011 as a collaborative effort dedicated to advancing and improving RNA 3D structure prediction. The automatic evaluation protocols for comparisons between prediction and experiment developed within RNA-Puzzles are applied to the 2024 CASP16 competition. The scores evaluate stereochemical parameters, Watson-Crick pairs, non-Watson-Crick pairs, and base stacking in addition to standard global parameters such as RMSD, TM-score, GDT, or lDDT. Several targets were particularly difficult owing to their size or multimerization. As noted in previous evaluations, although predictions that perform well on secondary structure may also achieve acceptable overall folds, they are insufficient to guarantee chemical precision or to correctly identify residues involved in non-Watson-Crick interactions. Both are essential for obtaining a valid three-dimensional architecture and for understanding the biological function of RNAs.