RNAcofold - manual page for RNAcofold 2.6.4


RNAcofold [OPTION]... [FILE]...


RNAcofold 2.6.4

calculate secondary structures of two RNAs with dimerization

The program works much like RNAfold, but allows one to specify two RNA sequences which are then allowed to form a dimer structure. RNA sequences are read from stdin in the usual format, i.e. each line of input corresponds to one sequence, except for lines starting with > which contain the name of the next sequence. To compute the hybrid structure of two molecules, the two sequences must be concatenated using the & character as separator. RNAcofold can compute minimum free energy (mfe) structures, as well as partition function (pf) and base pairing probability matrix (using the -p switch) Since dimer formation is concentration dependent, RNAcofold can be used to compute equilibrium concentrations for all five monomer and (homo/hetero)-dimer species, given input concentrations for the monomers. Output consists of the mfe structure in bracket notation as well as PostScript structure plots and “dot plot” files containing the pair probabilities, see the RNAfold man page for details. In the dot plots a cross marks the chain break between the two concatenated sequences. The program will continue to read new sequences until a line consisting of the single character @ or an end of file condition is encountered.

-h, --help

Print help and exit


Print help, including all details and hidden options, and exit


Print help, including hidden options, and exit

-V, --version

Print version and exit

-v, --verbose

Be verbose.


I/O Options:

Command line options for input and output (pre-)processing


Change the default output format.


The following output formats are currently supported:

ViennaRNA format (V), Delimiter-separated format (D) also known as CSV



Change the delimiting character for Delimiter-separated output format, such as CSV.


Delimiter-separated output defaults to comma separated values (CSV), i.e. all data in one data set is delimited by a comma character. This option allows one to change the delimiting character to something else. Note, to switch to tab-separated data, use $'\t' as delimiting character.


Do not print header for Delimiter-separated output, such as CSV.


-j, --jobs[=number]

Split batch input into jobs and start processing in parallel using multiple threads. A value of 0 indicates to use as many parallel threads as computation cores are available.


Default processing of input data is performed in a serial fashion, i.e. one sequence pair at a time. Using this switch, a user can instead start the computation for many sequence pairs in the input in parallel. RNAcofold will create as many parallel computation slots as specified and assigns input sequences of the input file(s) to the available slots. Note, that this increases memory consumption since input alignments have to be kept in memory until an empty compute slot is available and each running job requires its own dynamic programming matrices.


Do not try to keep output in order with input while parallel processing is in place.


When parallel input processing (--jobs flag) is enabled, the order in which input is processed depends on the host machines job scheduler. Therefore, any output to stdout or files generated by this program will most likely not follow the order of the corresponding input data set. The default of RNAcofold is to use a specialized data structure to still keep the results output in order with the input data. However, this comes with a trade-off in terms of memory consumption, since all output must be kept in memory for as long as no chunks of consecutive, ordered output are available. By setting this flag, RNAcofold will not buffer individual results but print them as soon as they have been computated.


Do not automatically substitute nucleotide “T” with “U”.



Automatically generate an ID for each sequence. (default=off)

The default mode of RNAcofold is to automatically determine an ID from the input sequence data if the input file format allows to do that. Sequence IDs are usually given in the FASTA header of input sequences. If this flag is active, RNAcofold ignores any IDs retrieved from the input and automatically generates an ID for each sequence. This ID consists of a prefix and an increasing number. This flag can also be used to add a FASTA header to the output even if the input has none.


Prefix for automatically generated IDs (as used in output file names).


If this parameter is set, each sequence will be prefixed with the provided string. Hence, the output files will obey the following naming scheme: “prefix_xxxx_ss.ps” (secondary structure plot), “prefix_xxxx_dp.ps” (dot-plot), “prefix_xxxx_dp2.ps” (stack probabilities), etc. where xxxx is the sequence number. Note: Setting this parameter implies --auto-id.


Change the delimiter between prefix and increasing number for automatically generated IDs (as used in output file names).


This parameter can be used to change the default delimiter “_” between the prefix string and the increasing number for automatically generated ID.


Specify the number of digits of the counter in automatically generated alignment IDs.


When alignments IDs are automatically generated, they receive an increasing number, starting with 1. This number will always be left-padded by leading zeros, such that the number takes up a certain width. Using this parameter, the width can be specified to the users need. We allow numbers in the range [1:18]. This option implies --auto-id.


Specify the first number in automatically generated IDs.


When sequence IDs are automatically generated, they receive an increasing number, usually starting with 1. Using this parameter, the first number can be specified to the users requirements. Note: negative numbers are not allowed. Note: Setting this parameter implies to ignore any IDs retrieved from the input data, i.e. it activates the --auto-id flag.


Change the delimiting character used in sanitized filenames.


This parameter can be used to change the delimiting character used while sanitizing filenames, i.e. replacing invalid characters. Note, that the default delimiter ALWAYS is the first character of the “ID delimiter” as supplied through the --id-delim option. If the delimiter is a whitespace character or empty, invalid characters will be simply removed rather than substituted. Currently, we regard the following characters as illegal for use in filenames: backslash \, slash /, question mark ?, percent sign %, asterisk *, colon :, pipe symbol |, double quote ", triangular brackets < and >.


Use full FASTA header to create filenames. (default=off)

This parameter can be used to deactivate the default behavior of limiting output filenames to the first word of the sequence ID. Consider the following example: An input with FASTA header >NM_0001 Homo Sapiens some gene usually produces output files with the prefix “NM_0001” without the additional data available in the FASTA header, e.g. “NM_0001_ss.ps” for secondary structure plots. With this flag set, no truncation of the output filenames is done, i.e. output filenames receive the full FASTA header data as prefixes. Note, however, that invalid characters (such as whitespace) will be substituted by a delimiting character or simply removed, (see also the parameter option --filename-delim).


Select additional algorithms which should be included in the calculations. The Minimum free energy (MFE) and a structure representative are calculated in any case.

-p, --partfunc[=INT]

Calculate the partition function and base pairing probability matrix in addition to the mfe structure. Default is calculation of mfe structure only.


In addition to the MFE structure we print a coarse representation of the pair probabilities in form of a pseudo bracket notation, followed by the ensemble free energy, as well as the centroid structure derived from the pair probabilities together with its free energy and distance to the ensemble. Finally it prints the frequency of the mfe structure, and the structural diversity (mean distance between the structures in the ensemble). See the description of pf_fold() and mean_bp_dist() and centroid() in the RNAlib documentation for details. Note that unless you also specify -d2 or -d0, the partition function and mfe calculations will use a slightly different energy model. See the discussion of dangling end options below.

An additionally passed value to this option changes the behavior of partition function calculation:

In order to calculate the partition function but not the pair probabilities

use the -p0 option and save about

50% in runtime. This prints the ensemble free energy dG=-kT ln(Z).

-a, --all_pf[=INT]

Compute the partition function and free energies not only of the hetero-dimer consisting of the two input sequences (the AB dimer), but also of the homo-dimers AA and BB as well as A and B monomers.


The output will contain the free energies for each of these species, as well as 5 dot plots containing the conditional pair probabilities, called “ABname5.ps”, “AAname5.ps” and so on. For later use, these dot plot files also contain the free energy of the ensemble as a comment. Using -a automatically switches on the -p option. Base pair probability computations may be turned off altogether by providing 0 as an argument to this parameter. In that case, no dot plot files will be generated.


Set the scaling of the Boltzmann factors. (default=”1.”)

The argument provided with this option is used to scale the thermodynamic temperature in the Boltzmann factors independently from the temperature of the individual loop energy contributions. The Boltzmann factors then become exp(- dG/(kT*betaScale)) where k is the Boltzmann constant, dG the free energy contribution of the state and T the absolute temperature.

-S, --pfScale=DOUBLE

In the calculation of the pf use scale*mfe as an estimate for the ensemble free energy (used to avoid overflows).


The default is 1.07, useful values are 1.0 to 1.2. Occasionally needed for long sequences.

-c, --concentrations

In addition to everything listed under the -a option, read in initial monomer concentrations and compute the expected equilibrium concentrations of the 5 possible species (AB, AA, BB, A, B).


Start concentrations are read from stdin (unless the -f option is used) in [mol/l], equilibrium concentrations are given realtive to the sum of the two inputs. An arbitrary number of initial concentrations can be specified (one pair of concentrations per line).

-f, --concfile=filename

Specify a file with initial concentrations for the two sequences.

The table consits of arbitrary many lines with just two numbers (the concentration of sequence A and B). This option will automatically toggle the -c (and thus -a and -p) options (see above).


Compute the centroid structure. (default=off)

Additionally to the MFE structure, compute the centroid representative of the structure ensemble. Here, we apply the base pair distance as distance measure, and report the structure that minimizes its Boltzmann weighted base pair distance to the rest of the ensemble. Computing the centroid structure requires equilibrium base pair probabilities. Therefore, this option implies the -p switch. For historical reasons, the centroid structure output is deactivated by default.


Compute MEA (maximum expected accuracy) structure.


The expected accuracy is computed from the pair probabilities: each base pair (i,j) receives a score 2*gamma*p_ij and the score of an unpaired base is given by the probability of not forming a pair. The parameter gamma tunes the importance of correctly predicted pairs versus unpaired bases. Thus, for small values of gamma the MEA structure will contain only pairs with very high probability. Using --MEA implies -p for computing the pair probabilities.


Set the threshold/cutoff for base pair probabilities included in the postscript output.


By setting the threshold the base pair probabilities that are included in the output can be varied. By default only those exceeding 1e-5 in probability will be shown as squares in the dot plot. Changing the threshold to any other value allows for increase or decrease of data.

-g, --gquad

Incoorporate G-Quadruplex formation into the structure prediction algorithm.


Structure Constraints:

Command line options to interact with the structure constraints feature of this program


Set the maximum base pair span.


-C, --constraint[=filename]

Calculate structures subject to constraints. (default=””)

The program reads first the sequence, then a string containing constraints on the structure encoded with the symbols:

. (no constraint for this base)

| (the corresponding base has to be paired

x (the base is unpaired)

< (base i is paired with a base j>i)

> (base i is paired with a base j<i)

and matching brackets ( ) (base i pairs base j)

With the exception of |, constraints will disallow all pairs conflicting with the constraint. This is usually sufficient to enforce the constraint, but occasionally a base may stay unpaired in spite of constraints. PF folding ignores constraints of type |.


Use constraints for multiple sequences. (default=off)

Usually, constraints provided from input file only apply to a single input sequence. Therefore, RNAcofold will stop its computation and quit after the first input sequence was processed. Using this switch, RNAcofold processes multiple input sequences and applies the same provided constraints to each of them.


Remove non-canonical base pairs from the structure constraint.



Enforce base pairs given by round brackets ( ) in structure constraint.



Use SHAPE reactivity data to guide structure predictions.


Select SHAPE reactivity data incorporation strategy.


The following methods can be used to convert SHAPE reactivities into pseudo energy contributions.

D: Convert by using the linear equation according to Deigan et al 2009.

Derived pseudo energy terms will be applied for every nucleotide involved in a stacked pair. This method is recognized by a capital D in the provided parameter, i.e.: --shapeMethod=”D” is the default setting. The slope m and the intercept b can be set to a non-default value if necessary, otherwise m=1.8 and b=-0.6. To alter these parameters, e.g. m=1.9 and b=-0.7, use a parameter string like this: --shapeMethod=”Dm1.9b-0.7”. You may also provide only one of the two parameters like: --shapeMethod=”Dm1.9” or --shapeMethod=”Db-0.7”.

Z: Convert SHAPE reactivities to pseudo energies according to Zarringhalam

et al 2012. SHAPE reactivities will be converted to pairing probabilities by using linear mapping. Aberration from the observed pairing probabilities will be penalized during the folding recursion. The magnitude of the penalties can affected by adjusting the factor beta (e.g. --shapeMethod=”Zb0.8”).

W: Apply a given vector of perturbation energies to unpaired nucleotides

according to Washietl et al 2012. Perturbation vectors can be calculated by using RNApvmin.


Select method for SHAPE reactivity conversion.


This parameter is useful when dealing with the SHAPE incorporation according to Zarringhalam et al. The following methods can be used to convert SHAPE reactivities into the probability for a certain nucleotide to be unpaired.

M: Use linear mapping according to Zarringhalam et al. C: Use a cutoff-approach to divide into paired and unpaired nucleotides (e.g. “C0.25”) S: Skip the normalizing step since the input data already represents probabilities for being unpaired rather than raw reactivity values L: Use a linear model to convert the reactivity into a probability for being unpaired (e.g. “Ls0.68i0.2” to use a slope of 0.68 and an intercept of 0.2) O: Use a linear model to convert the log of the reactivity into a probability for being unpaired (e.g. “Os1.6i-2.29” to use a slope of 1.6 and an intercept of -2.29)


Read additional commands from file

Commands include hard and soft constraints, but also structure motifs in hairpin and interior loops that need to be treeted differently. Furthermore, commands can be set for unstructured and structured domains.

Energy Parameters:

Energy parameter sets can be adapted or loaded from user-provided input files

-T, --temp=DOUBLE

Rescale energy parameters to a temperature of temp C. Default is 37C.


-P, --paramFile=paramfile

Read energy parameters from paramfile, instead of using the default parameter set.

Different sets of energy parameters for RNA and DNA should accompany your distribution. See the RNAlib documentation for details on the file format. The placeholder file name DNA can be used to load DNA parameters without the need to actually specify any input file.

-4, --noTetra

Do not include special tabulated stabilizing energies for tri-, tetra- and hexaloop hairpins.


Mostly for testing.


Set salt concentration in molar (M). Default is 1.021M.


Provide salt correction for duplex initialization (in kcal/mol).

-m, --modifications[=STRING]

Allow for modified bases within the RNA sequence string.


Treat modified bases within the RNA sequence differently, i.e. use corresponding energy corrections and/or pairing partner rules if available. For that, the modified bases in the input sequence must be marked by their corresponding one-letter code. If no additional arguments are supplied, all available corrections are performed. Otherwise, the user may limit the modifications to a particular subset of modifications, resp. one-letter codes, e.g. -mP6 will only correct for pseudouridine and m6A bases.

Currently supported one-letter codes and energy corrections are:

7: 7-deaza-adenonsine (7DA)

I: Inosine

6: N6-methyladenosine (m6A)

P: Pseudouridine

9: Purine (a.k.a. nebularine)

D: Dihydrouridine


Use additional modified base data from JSON file.

Model Details:

Tweak the energy model and pairing rules additionally using the following parameters

-d, --dangles=INT

How to treat “dangling end” energies for bases adjacent to helices in free ends and multi-loops.


With -d1 only unpaired bases can participate in at most one dangling end. With -d2 this check is ignored, dangling energies will be added for the bases adjacent to a helix on both sides in any case; this is the default for mfe and partition function folding (-p). The option -d0 ignores dangling ends altogether (mostly for debugging). With -d3 mfe folding will allow coaxial stacking of adjacent helices in multi-loops. At the moment the implementation will not allow coaxial stacking of the two interior pairs in a loop of degree 3 and works only for mfe folding.

Note that with -d1 and -d3 only the MFE computations will be using this setting while partition function uses -d2 setting, i.e. dangling ends will be treated differently.


Produce structures without lonely pairs (helices of length 1).


For partition function folding this only disallows pairs that can only occur isolated. Other pairs may still occasionally occur as helices of length 1.


Do not allow GU pairs.



Do not allow GU pairs at the end of helices.



Allow other pairs in addition to the usual AU,GC,and GU pairs.

Its argument is a comma separated list of additionally allowed pairs. If the first character is a “-” then AB will imply that AB and BA are allowed pairs, e.g. --nsp=”-GA” will allow GA and AG pairs. Nonstandard pairs are given 0 stacking energy.

-e, --energyModel=INT

Set energy model.

Rarely used option to fold sequences from the artificial ABCD… alphabet, where A pairs B, C-D etc. Use the energy parameters for GC (-e 1) or AU (-e 2) pairs.


Set the helical rise of the helix in units of Angstrom.


Use with caution! This value will be re-set automatically to 3.4 in case DNA parameters are loaded via -P DNA and no further value is provided.


Set the average backbone length for looped regions in units of Angstrom.


Use with caution! This value will be re-set automatically to 6.76 in case DNA parameters are loaded via -P DNA and no further value is provided.


Command line options for changing the default behavior of structure layout and pairing probability plots


Do not produce postscript drawing of the mfe structure.



If you use this program in your work you might want to cite:

R. Lorenz, S.H. Bernhart, C. Hoener zu Siederdissen, H. Tafer, C. Flamm, P.F. Stadler and I.L. Hofacker (2011), “ViennaRNA Package 2.0”, Algorithms for Molecular Biology: 6:26

I.L. Hofacker, W. Fontana, P.F. Stadler, S. Bonhoeffer, M. Tacker, P. Schuster (1994), “Fast Folding and Comparison of RNA Secondary Structures”, Monatshefte f. Chemie: 125, pp 167-188

R. Lorenz, I.L. Hofacker, P.F. Stadler (2016), “RNA folding with hard and soft constraints”, Algorithms for Molecular Biology 11:1 pp 1-13

S.H.Bernhart, Ch. Flamm, P.F. Stadler, I.L. Hofacker, (2006), “Partition Function and Base Pairing Probabilities of RNA Heterodimers”, Algorithms Mol. Biol.

The energy parameters are taken from:

D.H. Mathews, M.D. Disney, D. Matthew, J.L. Childs, S.J. Schroeder, J. Susan, M. Zuker, D.H. Turner (2004), “Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure”, Proc. Natl. Acad. Sci. USA: 101, pp 7287-7292

D.H Turner, D.H. Mathews (2009), “NNDB: The nearest neighbor parameter database for predicting stability of nucleic acid secondary structure”, Nucleic Acids Research: 38, pp 280-282


Ivo L Hofacker, Peter F Stadler, Stephan Bernhart, Ronny Lorenz


If in doubt our program is right, nature is at fault. Comments should be sent to rna@tbi.univie.ac.at.