5S rRNA, tRNA and miRNA are pseudoknot-free types of RNA. For the 5S rRNA and miRNA sequences chosen in our test set, DotKnot does not introduce any spurious pseudoknots. For two of the tRNA sequences, DotKnot predicts false positive pseudoknots. KnotSeeker, pknotsRG and HotKnots also predict false positive pseudoknots for some of the tRNA sequences. Minimum free energy prediction is known to have low accuracy for tRNAs. This is due to modified bases as well as coaxially stacked helices, which determine the characteristic 3D cloverleaf fold of tRNAs (24, 26) . Coaxial energies are implemented by pknots, which might explain why it does not predict false positive pseudoknots for the tested tRNA sequences.