What is RNAcofold?
RNAcofold computes the secondary structure of two RNA sequences that interact to form a dimer. Part of the ViennaRNA Package, it extends the single-sequence folding capabilities of RNAfold to predict how two RNA molecules base-pair with each other while also forming internal secondary structures.
The algorithm concatenates both sequences and applies thermodynamic folding calculations, treating the junction between sequences as an exterior loop. This approach identifies the minimum free energy (MFE) structure of the RNA-RNA complex, including both intramolecular base pairs within each sequence and intermolecular base pairs between them.
Applications
RNAcofold is used to study:
- sRNA-mRNA regulation: Small regulatory RNAs that bind and regulate messenger RNA targets
- Ribozyme-substrate interactions: Self-cleaving RNA complexes and their target binding
- Antisense oligonucleotide design: Predicting how synthetic sequences will bind RNA targets
- RNA dimerization: Studying homo- and heterodimer formation in viral genomes
How does RNAcofold work?
RNAcofold joins two RNA sequences with a virtual cut point, then applies dynamic programming to find the structure with lowest free energy. The cut point is treated specially: the loop containing it behaves like an exterior loop rather than an internal loop, allowing both sequences to remain distinct entities while forming intermolecular base pairs.
Energy calculations use nearest-neighbor thermodynamic parameters (Turner model), summing contributions from base pair stacking, loop entropy, and dangling ends. The partition function mode extends this to compute the full ensemble of structures, yielding base-pair probabilities and equilibrium constants.
Because dimer formation depends on molecular concentrations, RNAcofold can also compute equilibrium concentrations for all five possible species: two monomers, two homodimers, and one heterodimer. Given initial concentrations, the program uses the relationship K_AB = Z_AB / (Z_A × Z_B) to determine how molecules distribute at equilibrium.
Limitations
RNAcofold uses the Zuker algorithm, which prohibits pseudoknots. Some biologically important interaction motifs cannot be predicted:
- Kissing hairpins: Loop-loop interactions where two hairpin loops base-pair
- Pseudoknotted duplexes: Interactions that would cross when drawn on a 2D diagram
For these cases, RNAup provides an alternative that can model certain pseudoknotted configurations, though it considers only a single interaction site rather than multiple binding regions.
How to use RNAcofold online
ProteinIQ provides browser-based access to RNAcofold without command-line setup or local installation.
Inputs
| Input | Description |
|---|---|
RNA Sequence 1 | First RNA sequence in FASTA format or plain text (A, C, G, U characters) |
RNA Sequence 2 | Second RNA sequence in FASTA format or plain text |
Settings
Prediction options
| Setting | Description |
|---|---|
Temperature | Folding temperature in degrees Celsius (0-100, default 37). Affects base-pair stability and free energy calculations. |
Disallow lonely pairs | When enabled, helices must contain at least two consecutive base pairs. Single isolated pairs are forbidden. |
Energy parameters
| Setting | Description |
|---|---|
Dangling ends | Controls treatment of unpaired nucleotides adjacent to helices. Ignore dangling ends excludes their energy contribution. Only unpaired on either side counts dangles conservatively. Double dangles (default) includes both 5' and 3' contributions. Allow coaxial stacking enables stacking between adjacent helices. |
Results
Results include the dimer structure in dot-bracket notation and the minimum free energy.
| Column | Description |
|---|---|
Interaction | Identifier for the RNA pair |
Seq 1 Length | Length of the first RNA sequence in nucleotides |
Seq 2 Length | Length of the second RNA sequence in nucleotides |
Dimer Structure | Dot-bracket notation showing paired (parentheses) and unpaired (dots) positions, with & marking the sequence boundary |
MFE (kcal/mol) | Minimum free energy of the dimer structure. More negative values indicate stronger, more stable interactions. |
Interpreting MFE values
| MFE Range | Interpretation |
|---|---|
| < -20 kcal/mol | Very stable complex, strong interaction |
| -10 to -20 kcal/mol | Moderate stability, typical for regulatory interactions |
| -5 to -10 kcal/mol | Weak interaction, may be transient |
| > -5 kcal/mol | Unstable, unlikely to form under physiological conditions |
Related tools
- RNAfold: Single-sequence RNA secondary structure prediction
- RNAduplex: Simplified duplex hybridization without internal structure
- RNAplex: Fast identification of multiple potential binding sites
- RNAup: Interaction prediction accounting for accessibility costs
- RNAalifold: Consensus structure from aligned RNA sequences
- ViennaRNA: Access all 14 ViennaRNA methods through a unified interface