hybrid-ss — fold an RNA or DNA sequence


hybrid-ss [OPTION]... FILE


hybrid-ss computes a partition function for the folding of the sequence in FILE. If the suffix option is used, the folding is computed using the energy rules with the suffix extension; otherwise, it is computed at each temperature from tMin to tMax degrees Celsius, in increments of tInc degrees. All output files are named with a prefix which consists of the file name with the extension .seq removed. The partition function and associated free energy at each temperature are written to prefix.dG. The probabilities of each possible base pair occurring are written to prefix.temperature.plot and the probabilities of each base and each pair of adjacent bases being single-stranded are written to prefix.temperature.ext. A summary of the options the program was invoked with is written to


set nucleic acid type to RNA or DNA. Default is RNA.
-t, --tmin=REAL
set minimum temperature to REAL °C. Default is 0.
-i, --tinc=REAL
set temperature increment to REAL C°. Default is 1.
-T, --tmax=REAL
set maximum temperature to REAL °C. Default is 100.
-N, --sodium=REAL
set Sodium ion concentration to REAL molar. Default is 1.
-M, --magnesium=REAL
set Magnesium ion concentration to REAL molar. Default is 0.
-p, --polymer
use salt corrections for polymers instead of oligomers (the default).
-s, --suffix=STRING
use energy rules with the suffix STRING. Overrides --tmin, --tinc, --tmax, --sodium, --magnesium and --polymer.
-o, --output=STRING
name output files with the prefix STRING.
-f, --force=i,j[,k]
force all basepairs in the helix from i,j to i+k-1,j-k+1. If j is 0, force bases i to i+k-1 to be double-stranded; if i is 0, force bases j to j-k+1 to be double-stranded. k defaults to 1 if not specified.
-r, --prohibit=i,j[,k]
prohibit all basepairs in the helix from i,j to i+k-1,j-k+1. If j is 0, prohibit bases i to i+k-1 from pairing at all; if i is 0, prohibit bases j to j-k+1 from pairing at all. k defaults to 1 if not specified.
-R, --prohibit-range=i,j,k,l
prohibit any base from i to j from pairing with any base from k to l.
-E, --energy-only
skip computation of probabilities and output only prefix.dG and This mode uses less time and memory.
-I, --noisolate
prohibit all isolated basepairs. Isolated basepairs are helices of length 1; that is, they do not stack on another basepair on either side. (See also the --prefilter and --nopostfilter options below.)
-k, --tracebacks=NUMBER
compute NUMBER stochastic tracebacks and write them to prefix.temperature.ct. Stochastic tracebacks are computed according to the Boltzmann probability distribution so that the probability of a structure is its Boltzmann factor divided by the partition function.
-m, --maxbp=NUMBER
bases farther apart than NUMBER cannot form. Default is no limit.
-c, --constraints[=FILE]
read a list of constraints from FILE. Constraints must be in the form "F i j k", "P i j k" or "R i-j k-l". These are equivalent to specifying "--force=i,j,k", "--prohibit=i,j,k" or "--prohibit-range=i,j,k,l", respectively. If FILE is not specified, it defaults to prefix.aux.
-b, --basepairs=FILE
read a list of allowable helices from FILE. Each helix consists of three whitespace-delimited numbers, which specify the starting basepair and the length of the helix. When this option is used, all basepairs except those in FILE are prohibited from forming.
treat sequences as circular rather than linear.


allow basepairs to form between any two nucleotides. When --allpairs is not specified, only Watson-Crick and wobble basepairs are allowed.
-d, --debug
set the maximum size of bulge/interior loops to NUMBER. Default is 30.
set the maximum asymmetry of bulge/interior loops to NUMBER. Default is 30.
remove single-base stacking from consideration.
make the penalty for multibranch loops constant rather than affine.
set the prefilter to filter out all basepairs except those in groups of value2 adjacent basepairs of which value1 can form. value2 is the same as value1 if unspecified. Default is 2 of 2. (See also the --noisolate option above.)
disable the postfilter. The postfilter, which is enabled by default, removes from consideration all structures that consist of only one basepair.
scale the partition function by the factor REAL per base.


an alternate location from which to read the energy rules. The default energy rules can be overridden with files in the current directory or in the directory pointed to by UNAFOLDDAT. hybrid-ss looks for each file first in the current directory, then in the directory specified by UNAFOLDDAT and last in /usr/local/share/unafold (or wherever the energy rules were installed).


Markham, N. R. and Zuker, M. (2008) UNAFold: software for nucleic acid folding and hybridization. In Keith, J. M., editor, Bioinformatics, Volume II. Structure, Functions and Applications, number 453 in Methods in Molecular Biology, chapter 1, pages 3-31. Humana Press, Totowa, NJ. ISBN 978-1-60327-428-9.

Other references which may be useful may be found at


hybrid-ss-min(1), hybrid(1)


Nick Markham <> and Michael Zuker <>


Copyright (c) 2006-2021, Rensselaer Polytechnic Institute.


Both commercial and non-commercial use of UNAFold require a license from RPI; see