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OligoWalk is used to predict the accessible regions in an RNA target to oligonucleotide hybridization. It calculates thermodynamic features of sense-antisense hybidization and predicts the free energy changes of oligonucleotides binding to a target RNA. The secondary structures of the oligomer and target mRNA are considered in the OligoWalk algorithm. It can be used to design efficient siRNA targeting a given mRNA sequence. 

OligoWalk Schematic

USAGE: OligoWalk <sequence file> <report file> [options]

Required parameters:

<sequence file> The name of a sequence file (SEQ, FASTA) or structure file (CT, DBN) containing the input sequence.
<report file>

The name of an output file to which report results will be written.

Options that do not require added values:

-d, --dna Specify that the oligomers are DNA. (The target sequence is still assumed to be RNA). This causes DNA:RNA hybridization energies to be used.
(In the absense of this flag, the default behavior is to assume both the oligomers and the target sequence are RNA)
-h, --help Display the usage details message.
-v, --version Display the RNAstructure package version information.
--html Write the report in HTML format instead of plain text.
--no-header The default behavior is to include a summary of the parameters and options used in the calulation directly in the output report file. This option DISABLES that behavior. (I.e. the header information will not be included in the report file if this flag is present.)
-score Score the siRNA prefilter.
-w, --webserver Use special output for running OligoWalk as a webserver. This implies HTML=true and it sends the header (which summarizes the parameters and options used in the calulation) to STDOUT instead of including them in the report. It also turns off progress updates.
-write Write sav files to save time in test mode.

Options that require added values:

-c, -co, --conc

Specify the molar concentration of oligos. e.g. "1.5E-6", "1.5uM", or "0.0000015" (1.5 micromolar).

This may be used in conjunction with the '--unit' flag. See '--unit for more examples.
Unit Abbreviations: mM=10-3, uM=10-6, nM=10-9, pM=10-12

-u, --unit

Specifies a power-of-ten factor to multiply the concentration by.
This parameter is not required, because it is entirely possible to specify the concentration directly in scientific notation (e.g. "--conc 4.2E-3"  or  "--conc 4.2mM". However there may be some circumstances when it is convenient to separate the concentration from the units, e.g. "--conc 4.2 --unit -3"  or  "--conc 4.2 --unit mM"
This might be useful, for example, when the program is run as a backend to a graphical or web interface and the user has selected the units separately from the concentration (from a drop-down list etc).


  • 2.5 millimolar can be specified in any of the following ways:
    • --conc 2.5mM
    • --conc 2.5E-3
    • --conc 2.5  --unit -3
    • --conc 2.5  --unit mM
  • 5x10-5 M can be specified in any of the following ways:
    • --conc 5E-5
    • --conc 50uM
    • --conc 5    --unit -5
    • --conc 50   --unit uM
    • --conc 0.05 --unit mM

Unit Abbreviations: mM=10-3, uM=10-6, nM=10-9, pM=10-12

-l, --length Length of oligomers for hybridization.
-dist Limit the maximum distance between nucleotides that can pair.
-st, --start Truncate the target sequence to end at this nucleotide position. This can improve runtime since only a subset of the original sequence will be folded and scanned. Use this in conjunction with the --end parameter.
-en, --end Truncate the target sequence (see the --start parameter).
-fi, --filter Whether to use the siRNA prefilter to prefill functional siRNA.
0=No Prefilter (default); 1=Use Prefilter
-fold Only fold a fragment with the specified size (plus the length of the oligomer), which is centered on the binding region.
When FOLDSIZE > 1, MODE (-m) and SUBOPTIMAL (-s) must both be 2.
-ss, --from Start position of scanning on folded region of target.
-se, --to End position of scanning on folded region of target.
-m, --mode

Determines how target structure is used:

1=Break Local Structure.
2=Refold target RNA after oligo binding.
3=No target structure considered.

-s, --suboptimal

Determines suboptimal structure options:

0=Do not consider suboptimal structures.
1=Use AllSub to generate suboptimal structures.
2=Use Partition Function to generate all suboptimal structures.
3=Use a heuristic method for both oligo-free and oligo-bound RNA.
4=Use stochastic sampling to generate 1000 structures.

-sh, --shape Specify a SHAPE data file to be used to generate restraints. These restraints specifically use SHAPE pseudoenergy restraints.
Default is no SHAPE data file specified.
-test Perform self-tests. The parameter should be a list of space-separated test numbers, e.g.: -test '1 2 5'


  1. J. Lu and D.H. Mathews. (2008) "Efficient siRNA selection using hybridization thermodynamics"
    Nucleic Acids Research, 36:640-647.
  2. Reynolds, A., Leake, D., Boese, Q., Scaringe, S., Marshall, W.S. and Khvorova, A. (2004) Rational siRNA design for RNA interference. Nat Biotechnol., 22, 326-330.
  3. Lu, Z.J., Turner, D.H. and Mathews, D.H. (2006) A set of nearest neighbor parameters for predicting the enthalpy change of RNA secondary structure formation. Nucleic Acids Res., 34, 4912-4924.
  4. D.H. Mathews, M.D. Disney, J.L. Childs, S.J. Schroeder, M. Zuker, and D.H. Turner. "Incorporating Chemical Modification Constraints into a Dynamic Programming Algorithm for Prediction of RNA Secondary Structure"
    Proceedings of the National Academy of Sciences USA, 101, 7287-7292, (2004).
  5. D.H. Mathews, J. Sabina, M. Zuker, and D. H. Turner "Expanded Sequence Dependence of Thermodynamic Parameters: Improves Prediction of RNA Secondary Structure" Journal of Molecular Biology, 288, 911-940, (1999).
  6. N. Sugimoto, S. Nakano, A. Katoh, H. Nakamura, T. Ohmichi, M. Yoneyama, and M. Sasaki
    "Thermodynamic Parameters to Predict the Stability of RNA/DNA Hybrid Duplexes"
    Biochemistry, 34, 11211-11216, (1995).