RNAup

What is RNAup?

RNAup predicts RNA-RNA interactions while accounting for the cost of making binding sites accessible. This is the central idea that separates RNAup from simpler interaction tools: before two RNA molecules can hybridize, each must unfold any internal structure that blocks the interaction site. RNAup calculates this "opening energy" for both strands and adds it to the intermolecular binding energy, producing a total energy that reflects the true thermodynamic cost of the interaction.

The total energy reported by RNAup is the sum of three terms:

• Interaction energy: the free energy gained from forming intermolecular base pairs between the two strands
• Opening energy (target): the cost of unfolding the target RNA's internal structure at the binding site
• Opening energy (query): the cost of unfolding the query RNA's internal structure at the binding site

This decomposition makes RNAup particularly valuable for studying interactions where one or both molecules have stable internal folds. A binding site that looks favorable based on intermolecular pairing alone may turn out to be thermodynamically unfavorable once the opening cost is included.

RNAup vs. other interaction tools

Tool	Considers accessibility	Speed	Best for
RNAduplex	No	Very fast	Initial screening, pre-filtering large sets
RNAup	Yes (full calculation)	Moderate	Accurate binding energy with opening costs
RNAplex	Yes (precomputed profiles)	Fast	Scanning long targets for interaction sites
RNAcofold	Implicit (joint folding)	Moderate	Full dimer structure including intra- and intermolecular pairs

RNAduplex is a good first pass, but it systematically overestimates binding stability for structured RNAs because it ignores opening costs. RNAup corrects this by computing accessibility from the full ensemble of each strand's internal structures.

How to use RNAup online

RNAup on ProteinIQ takes two RNA sequences (a target and a query), computes accessibility-aware interaction energies using ViennaRNA's RNAup program, and returns interaction coordinates, energy decompositions, and downloadable output files. No local ViennaRNA installation is needed.

Inputs

Input	Description
Target RNA	The RNA sequence to scan for binding sites. FASTA or plain nucleotide format.
Query RNA	The RNA sequence that binds to the target. FASTA or plain nucleotide format.

Supported file formats: .fasta, .fa, .txt

Settings

Interaction options

Setting	Type	Default	Description
Interaction mode	select	binding-site	Controls the RNAup workflow. The binding-site mode reports optimal interaction sites; the default mode uses RNAup's standard output format.
Unpaired region length	slider (1-50)	1	Maximum length of the unpaired region considered for accessibility (-u flag). Larger values allow RNAup to evaluate longer stretches of unpaired nucleotides as potential binding sites.
Window size	slider (1-200)	25	Size of the sliding window used for accessibility computation (-w flag). Should be at least as large as the unpaired region length.
Report energy contributions	switch	off	Request contribution-oriented output from RNAup where supported.
Include both strands	switch	off	Report interaction results for both orientations (5'>3' and 3'>5').
Extend 5'	text	default	Optional extension of the interaction region in the 5' direction.
Extend 3'	text	default	Optional extension of the interaction region in the 3' direction.

Thermodynamic parameters

Setting	Type	Default	Description
Temperature	slider (0-100)	37	Folding temperature in degrees Celsius.
Salt concentration	text	default	Monovalent salt concentration for the ViennaRNA energy model.
Initial salt correction	text	default	Initial salt correction parameter.
Parameter file content	textarea	none	Inline ViennaRNA parameter file content passed via --paramFile.

Energy model

Setting	Type	Default	Description
Dangling ends	select	2 (double dangles)	How unpaired nucleotides adjacent to helices contribute to the energy. Options: 0 (ignore), 1 (one side), 2 (both sides), 3 (coaxial stacking).
Disallow lonely pairs	switch	off	Prohibit helices of length 1 (isolated base pairs).

Leaving optional text fields blank preserves the native ViennaRNA CLI defaults.

Output

Results table

Column	Description
Interaction	Identifier for the interaction pair
Query ID	Identifier of the query sequence
Target ID	Identifier of the target sequence
Query Start / Query End	Nucleotide positions of the interaction on the query strand
Target Start / Target End	Nucleotide positions of the interaction on the target strand
Interaction Energy (kcal/mol)	Free energy from intermolecular base pairing alone
Opening Energy (kcal/mol)	Combined cost of unfolding internal structure on both strands at the interaction site
Total Energy (kcal/mol)	Sum of interaction energy and opening energy. More negative values indicate more favorable interactions.

Files

The Files tab holds rnaup.out, the native output RNAup writes for each sequence pair, one pair after another in input order.