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RNAstructure Installation and Overview
Software Package Components

Single Sequence Methods

Feature Text Interface Program JAVA GUI Menu Item Class Library and Function Name
Free energy minimization and generation of all suboptimal structures
(References: Duan et al. 2006, Wuchty et al. 1999)
AllSub Generate All Suboptimal RNA Structures RNA::GenerateAllSuboptimalStructures
Predict canonical and non-canonical pairs. CycleFold N/A N/A
Design a sequence to fold with low ensemble defect to a target structure.
(Reference: Bellaousov et al. 2018)
Design N/A Design
Drawing secondary structure diagrams draw Draw RNA::DetermineDrawingCoordinates
Ensemble defect calculator.
(Reference: Bellaousov et al. 2018)
EDcalculator N/A N/A
Efn2 (Energy function 2)
(Reference: Mathews, Sabina et al. 1999)
efn2 Efn2 RNA RNA::CalculateFreeEnergy
Free energy minimization structure prediction
(Reference: Mathews et al. 2004)
Fold Fold RNA Single Strand RNA::FoldSingleStrand
Maximum expected accuracy structure prediction
(Reference: Lu et al. 2009)
MaxExpect MaxExpect: Predict RNA MEA Structure RNA::MaximumExpectedAccuracy
(Reference: Hart et al. 2008)
Estimate base pairing probabilities and probabilities of structures.
Partition function
(Reference: Mathews 2004)
partition Partition Function RNA RNA::PartitionFunction
Partition function, implemented in parallel for CUDA GPUs partition-cuda N/A N/A
Prediction of structures with pairs above specified pairing probability threshold
(Reference: Mathews 2004)
ProbablePair Output Probable Structures RNA::PredictProbablePairs
Prediction of secondary structures including pseudoknots
(Reference: Bellaousov and Mathews 2010)
ProbKnot ProbKnot: Predict RNA MEA Structure Including Pseudoknots RNA::ProbKnot
Calculation of loop probabilities
(Reference: Sloma and Mathews 2016)
ProbScan N/A ProbScan
Remove Pseudoknots
(Reference: Smit et al. 2008)
RemovePseudoknots Break Pseudoknots RNA::BreakPseudoknot
Predict multiple folding conformations, guided by SHAPE data.
(Reference: Spasic et al. 2018)
Rsample N/A Rsample
Predict structures that may contains pseudoknots, restrained by SHAPE mapping data. ShapeKnots N/A N/A
Stochastic sampling of structures
(Reference: Ding and Lawrence, 2003)
stochastic Stochastic RNA Sampling RNA::Stochastic

Multiple Sequence Methods (Bimolecular or Common Folding)

Feature Text Interface Program JAVA GUI Menu Item Class Library and Function Name
Predict bimolecular base pairs while accounting for preexisting structure.DiChiacchio et al 2016. AccessFold N/A HybridRNA::AccessFold
Bimolecular structure prediction with intramolecular pairs bifold Fold RNA Bimolecular HybridRNA::FoldBimolecular
Bimolecular partition function (no intramolecular pairs) bipartition Partition Function RNA Bimolecular HybridRNA::PartitionFunctionBimolecular
Predict conserved pairs, both canonical and non-canonical. CycleFold N/A N/A
Bimolecular structure prediction without intramolecular pairs DuplexFold N/A HybridRNA::FoldDuplex
Predict a secondary structure common to two homologs.
(References: Harmanci et al. 2007, Uzilov et al 2006, Mathews and Turner 2002)
dynalign RNA Dynalign Dynalign_object::Dynalign
Predict a secondary structure common to two homologs, allowing structure domain inserts.
Dynalign II
dynalign_ii N/A

Dynalign_object::Dynalign (Built by defining DYNALIGN_II at precompile time)

Predict a common secondary structure to multiple homologs.
(Reference: Xu and Mathews, 2011)
multilign RNA Multilign Multilign_object::ProgressiveMultilign
Assess the duplex formation thermodynamics for a set of oligonucleotides.
Reference: Mateeva et al. 2003)
oligoscreen OligoScreen Oligowalk_object::OligoScreen
Find high affinity oligonucleotides to a structured RNA target.
(References: Lu and Mathews, 2008, Lu and Mathews, 2007, Mathews, Burkard et al. 1999)
OligoWalk OligoWalk Oligowalk_object::OligoWalk
(References: Harmanci et al. 2009, Harmanci et al. 2008)
Predict the common secondary structure and multiple sequence alignment for multiple sequence homologs.
References: Harmanci et al. 2011, Tan et al. 2017)
TurboFold RNA TurboFold TurboFold::fold
Predict a secondary structure by homology modeling when the structure and alignment is available for sequence homologs.
TurboHomology N/A TurboHomology


Feature Text Interface Program JAVA GUI Menu Item Class Library and Function Name
Circular structure comparison CircleCompare N/A N/A
CT file to dot bracket file conversion ct2dot N/A N/A
Dot bracket file to CT file conversion dot2ct N/A N/A
Create a dot plot from a Dynalign save file DynalignDotPlot Dot Plot Dynalign N/A
Create a dot plot from a folding energy save file EnergyPlot Dot Plot N/A
Ensemble energy calculation EnsembleEnergy N/A N/A
Create a dot plot from a partition function save file ProbabilityPlot Dot Plot Partition Function N/A
Refold from a previously folded sequence. refold Refold From Save File RNA::ReFoldSingleStrand
Scoring comparison of two structures
(Reference: Mathews, Sabina et al. 1999)
scorer N/A N/A
Graphical Secondary Structure Editing and Drawing Standalone Program: StructureEditor
Free energy calculation library C++ Source Code (class library) RNAstructure/efn_lib
Written by Max Ward (PhD Candidate, University of Western Australia)


  1. Bellaousov, S., Kayedkhordeh, M., Peterson, R. J. & Mathews, D. H. (2018).
    Accelerated RNA Secondary Structure Design Using Pre-Selected Sequences for Helices and Loops
    . 24: 1555-1567.
  2. Spasic, A., Assmann, S. M., Bevilacqua, P. C., & Mathews, D. H. (2018).
    Modeling RNA Secondary Structure Folding Ensembles Using SHAPE Mapping Data
    Nucleic Acids Research
    . 46: 314-323.
  3. Tan, Z., Fu, Y., Sharma, G., & Mathews, D. H. (2017).
    TurboFold II: RNA structural alignment and secondary structure prediction informed by multiple homologs.

    Nucleic Acids Research
    . 45: 11570-11581.
  4. Sloma, M.F., & Mathews, D.H. (2016).
    Exact calculation of loop formation probability identifies folding motifs in RNA secondary structures.
    . 22: 1808-1818.
  5. DiChiacchio, L., Sloma, M. F., & Mathews, D. H. (2016).
    AccessFold: Predicting RNA-RNA Interactions with Consideration for Competing Self-Structure.
    Bioinformatics. 32: 1033-1039.
  6. Harmanci, A.O., Sharma, G. and Mathews, D.H. (2011).
    TurboFold: Iterative probabilistic estimation of secondary structures for multiple RNA sequences.
    BMC Bioinformatics. 12:108.
  7. Xu, Z., and Mathews, D.H. (2011).
    Multilign: An algorithm to predict secondary structures conserved in multiple RNA sequences.
    Bioinformatics, 27:626-632.
  8. Bellaousov, S., and Mathews, D. H. (2010.
    ProbKnot: fast prediction of RNA secondary structure including pseudoknots.
    RNA. 16:1870-1880
  9. Harmanci, A.O., Sharma, G. and Mathews, D.H. (2009).
    Stochastic sampling of the RNA structural alignment space.
    Nucleic Acids Res., 37:4063-4075.
  10. Lu, Z.J., Gloor, J.W. and Mathews, D.H. (2009).
    Improved RNA Secondary Structure Prediction by Maximizing Expected Pair Accuracy.
    RNA, 15:1805-1813.
  11. Harmanci, A.O., Sharma, G. and Mathews, D.H. (2008).
    PARTS: Probabilistic Alignment for RNA joinT Secondary structure prediction.
    Nucleic Acids Res., 36:2406-2417.
  12. Hart, J.M., Kennedy, S.D., Mathews, D.H. and Turner, D.H. (2008).
    NMR-assisted prediction of RNA secondary structure: identification of a probable pseudoknot in the coding region of an R2 retrotransposon.
    J. Am. Chem. Soc., 130:10233-10239.
  13. Lu, Z.J. and Mathews, D.H. (2008).
    Fundamental Differences in the Equilibrium Considerations for siRNA and Antisense Oligodeoxynucleotide Design.
    Nucleic Acids Res., 36:3738-3745.
  14. Smit, S., Rother, K., Heringa, J. and Knight, R. (2008).
    From knotted to nested RNA structures: a variety of computational methods for pseudoknot removal.
    RNA, 14:410-416.
  15. Harmanci, A.O., Sharma, G. and Mathews, D.H. (2007).
    Efficient Pairwise RNA Structure Prediction Using Probabilistic Alignment Constraints in Dynalign.
    BMC Bioinformatics, 8:130.
  16. Lu, Z.J. and Mathews, D.H. (2007).
    Efficient siRNA Selection Using Hybridization Thermodynamics.
    Nucleic Acids Res., 36:640-647.
  17. Duan, S., Mathews, D.H. and Turner, D.H. (2006).
    Interpreting oligonucleotide microarray data to determine RNA secondary structure: application to the 3' end of Bombyx mori R2 RNA.
    Biochemistry, 45:9819-9832.
  18. Uzilov, A.V., Keegan, J.M. and Mathews, D.H. (2006).
    Detection of non-coding RNAs on the basis of predicted secondary structure formation free energy change.
    BMC Bioinformatics, 7:173.
  19. Mathews, D.H. (2004).
    Using an RNA secondary structure partition function to determine confidence in base pairs predicted by free energy minimization.
    RNA, 10:1178-1190.
  20. Mathews, D.H., Disney, M.D., Childs, J.L., Schroeder, S.J., Zuker, M. and Turner, D.H. (2004).
    Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure.
    Proc. Natl. Acad. Sci. USA, 101:7287-7292.
  21. Ding, Y. and Lawrence, C.E. (2003).
    A statistical sampling algorithm for RNA secondary structure prediction.
    Nucleic Acids Res., 31:7280-7301.
  22. Matveeva, O.V., Mathews, D.H., Tsodikov, A.D., Shabalina, S.A., Gesteland, R.F., Atkins, J.F. and Freier, S.M. (2003).
    Thermodynamic criteria for high hit rate antisense oligonucleotide design.
    Nucleic Acids Res., 31:4989-4994.
  23. Mathews, D.H. and Turner, D.H. (2002).
    Dynalign: An algorithm for finding the secondary structure common to two RNA sequences.
    J. Mol. Biol., 317:191-203.
  24. Mathews, D.H., Burkard, M.E., Freier, S.M., Wyatt, J.R. and Turner, D.H. (1999).
    Predicting oligonucleotide affinity to nucleic acid targets.
    RNA, 5:1458-1469.
  25. Mathews, D.H., Sabina, J., Zuker, M. and Turner, D.H. (1999).
    Expanded sequence dependence of thermodynamic parameters provides improved prediction of RNA secondary structure.
    J. Mol. Biol., 288:911-940.
  26. Wuchty, S., Fontana, W., Hofacker, I.L. and Schuster, P. (1999).
    Complete suboptimal folding of RNA and the stability of secondary structures.
    Biopolymers, 49:145-165.