Data S1 Solid-phase fractionation data. (XLS) Data S2 Liquid-phase fractionation data. (XLS) Predicting pseudoknotted structures across two RNA sequences Motivation: Laboratory RNA structure determination is demanding and costly and thus, computational structure prediction is an important task. Single sequence methods for RNA secondary structure prediction are limited by the accuracy of the underlying folding model, if a structure is supported by a family of evolutionarily related sequences, one can be more confident that the prediction is accurate. RNA pseudoknots are functional elements, which have highly conserved structures. However, few comparative structure prediction methods can handle pseudoknots due to the computational complexity. Results: A comparative pseudoknot prediction method called DotKnot-PW is introduced based on structural comparison of secondary structure elements and H-type pseudoknot candidates. DotKnot-PW outperforms other methods from the literature on a hand-curated test set of RNA structures with experimental support. Availability: DotKnot-PW and the RNA structure test set are available at the web site http://dotknot.csse.uwa.edu.au/pw. Contact: janaspe@csse.uwa.edu.au Supplementary information: Supplementary data are available at Bioinformatics online. Macromolecules such as DNA, RNA and proteins have the ability to form diverse tertiary structures, which enable functionality and thus, life. For many decades, proteins were deemed the global players in the cell until RNA entered the spotlight. For example, RNA structures have been found to be catalytically active, which was assumed to be the privilege of proteins. Furthermore, small RNAs are known to regulate gene expression and RNA viruses employ a plethora of structure elements to invade the host cell.