RNA questions span secondary structure, ensembles, accessibility, intermolecular pairing, sequence design, and three-dimensional complexes. Choose the scale and context before selecting a model.
Match the method to the question
| Question | Tools to investigate | Key assumption |
|---|---|---|
| Base pairing and secondary structure | RNAfold, RNAsubopt | Thermodynamic model and alternative folds |
| Local accessibility | RNAplfold | Window and pairing assumptions |
| Two-RNA interactions | RNAcofold, RNAup | Joint folding versus accessibility-aware interaction energy |
| Sequence design for a target structure | RNAinverse, RhoDesign | Secondary-structure versus 3D inverse-folding target |
| RNA with protein or ligand partners | Appropriate co-folding methods | Complete molecular context and supported chemistry |
Prepare the system
Preserve RNA identity, orientation, construct boundaries, modifications, and interacting partners. Do not substitute DNA or remove molecular context merely to satisfy a generic sequence field. Check temperature and other exposed physical assumptions in thermodynamic workflows.
Interpret alternatives
A minimum-free-energy structure is one model outcome, not the only solution conformation. Inspect pairing probabilities, accessibility, and alternate states where available. Interaction energies from different methods may include different contributions and should not be compared as a single calibrated affinity scale.
Design and validate
For inverse folding, test whether proposed sequences support the intended fold using an appropriate prediction method and relevant alternative states. Retain constraints and candidate diversity, then evaluate expression, stability, and function experimentally.
See RNA design solutions for the tool catalog and input preparation for representation checks.