RNAup calculates the thermodynamics of RNA-RNA interactions by accounting for the cost of making binding sites accessible. Unlike simpler duplex prediction methods, RNAup recognizes that potential binding regions in both RNA molecules are often base-paired within their own secondary structures. Before two RNAs can interact, these intramolecular structures must be disrupted, which costs energy.
The total binding energy RNAup reports combines two components:
The interaction energy (ΔG_interaction) represents the stabilizing base pairing between the two molecules. The opening energy (ΔG_opening) represents the energetic cost of unpairing the binding sites in one or both molecules so they become available for intermolecular pairing. This accessibility-aware approach produces more biophysically realistic predictions than methods that ignore the structural context of binding sites.
Accessibility controls the efficacy of many RNA-mediated processes. MicroRNAs, siRNAs, and bacterial small RNAs all require their target sites to be accessible for efficient binding. If seed sequences are buried within stable secondary structure, the RNA-induced silencing complex (RISC) cannot access them effectively. Studies have shown that target site accessibility correlates directly with siRNA knockdown efficiency and miRNA repression strength.
RNAup predictions are useful for:
RNAup uses partition function calculations over all possible conformations to determine the probability that a binding site remains unpaired in equilibrium. This probability p_u relates to opening energy via:
The algorithm operates in two stages. First, it computes accessibilities for regions of specified length, reporting the free energy required to open each potential binding site. Second, it combines these opening energies with duplex interaction energies to find the optimal interaction.
An efficient O(n³) algorithm computes accessibilities for all intervals within a sequence simultaneously. Earlier implementations could only compute intervals of one fixed length in the same time. This improvement enables scanning for binding sites across a range of interaction lengths without repeated computation.
ProteinIQ provides browser-based access to RNAup, eliminating the need to install the ViennaRNA package locally.
| Input | Description |
|---|---|
RNA Sequence 1 | First RNA sequence in FASTA format or plain sequence. This is typically the longer target sequence (e.g., mRNA). |
RNA Sequence 2 | Second RNA sequence. This is typically the shorter regulatory RNA (e.g., siRNA, miRNA). |
| Setting | Description |
|---|---|
Temperature | Folding temperature in degrees Celsius (0-100, default 37). Affects thermodynamic calculations for both structure prediction and interaction energy. |
Results appear in a spreadsheet with columns for interaction details.
| Column | Description |
|---|---|
Interaction | The pair of sequences analyzed |
Seq 1 Length | Length of the first sequence in nucleotides |
Seq 2 Length | Length of the second sequence in nucleotides |
Structure | Bracket notation showing the interaction structure |
Energy | Total binding energy in kcal/mol (more negative = stronger binding) |
More negative total energy values indicate stronger predicted interactions. When comparing potential binding sites, consider both the total energy and the relative contributions of interaction and opening energy. A site with favorable duplex energy but high opening cost may be less effective than a site with slightly weaker base pairing but greater accessibility.
RNAup assumes that both RNA molecules fold independently before interaction. It does not model cooperative refolding where the interaction itself might stabilize alternative structures. For very long sequences, computation time increases significantly due to the O(n³) complexity.
The method predicts thermodynamic favorability, not kinetic accessibility. A binding site might be thermodynamically accessible but kinetically trapped behind folding barriers. Experimental validation remains essential for confirming predicted interactions.
