Most widely used tools for RNA Secondary Structure Prediction

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RNA secondary structure prediction tools

In this article, some software/tools are mentioned that are most widely used for RNA secondary structure prediction.

1. mfold

mfold is the most widely used tool for RNA secondary structure prediction based on thermodynamic methods [1]. The mfold software is freely accessible and can be downloaded from here. mfold is currently available for Unix, Linux, and Mac OS. A webserver for mfold can be accessed here. However, it has been replaced by UNAfold that is much easier to install and run. UNAfold provides additional computing parameters and can be downloaded from here.

2. RNAfold

It predicts the secondary structure of RNAs with a limit to 75oo nucleotides for partition function prediction and 10,000 nucleotides for minimum free energy predictions. The webserver of RNAfold can be freely accessed at The minimum free energy structure prediction is based on a loop-based energy model and a dynamic programming algorithm. The energy minimized RNA structure is predicted according to the given energy parameter set and temperature [2].

3. RNAstructure

RNAstructure predicts RNA and DNA secondary structures based on different algorithms. It also provides additional features including biomolecular structure prediction, base-pairing probabilities, and equilibrium binding affinities prediction [3]. The latest version of RNAstructure Version6.2 is available to download here after filling out a registration form. It comes with a graphical user interface and command-line interface for Windows, Linux, and Mac OSs.


SPOT-RNA uses an ensemble of deep neural networks and transfer learning for RNA secondary structure prediction [4]. It is completely based on machine learning. A limitation of SPOT-RNA is that it is trained by short RNAs (<500 nucleotides). But the reported prediction accuracy is high on the tested data [4]. SPOT-RNA web server can be accessed here. The source code can be downloaded from GitHub.

5. RNAsoft

RNAsoft is a collection of RNA structure prediction algorithms and design [5]. It offers several online webservers for secondary structure prediction and RNA design. These web servers offer to predict RNA secondary structure using different algorithms, lowest minimum free energy, and pseudoknots [5]. It can be easily accessed here.


  1. Zuker, M. (2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic acids research31(13), 3406-3415.
  2. Lorenz, R., Bernhart, S. H., Zu Siederdissen, C. H., Tafer, H., Flamm, C., Stadler, P. F., & Hofacker, I. L. (2011). ViennaRNA Package 2.0. Algorithms for molecular biology6(1), 26.
  3. Reuter, J. S., & Mathews, D. H. (2010). RNAstructure: software for RNA secondary structure prediction and analysis. BMC bioinformatics11(1), 1-9.
  4. Singh, J., Hanson, J., Paliwal, K., & Zhou, Y. (2019). RNA secondary structure prediction using an ensemble of two-dimensional deep neural networks and transfer learning. Nature communications10(1), 1-13.
  5. Andronescu, M., Aguirre-Hernandez, R., Condon, A., & Hoos, H. H. (2003). RNAsoft: a suite of RNA secondary structure prediction and design software tools. Nucleic acids research31(13), 3416-3422.
Tariq is founder of Bioinformatics Review and a professional Software Developer at IQL Technologies. His areas of expertise include algorithm design, phylogenetics, MicroArray, Plant Systematics, and genome data analysis. If you have questions, reach out to him via his homepage.

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