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What is RNAsubopt?
RNAsubopt generates all RNA secondary structures within a specified energy range above the minimum free energy (MFE). Unlike RNAfold, which returns only the single most stable structure, RNAsubopt exhaustively enumerates alternative conformations that are thermodynamically plausible.
This exhaustive approach matters because RNA molecules do not exist in a single conformation. At physiological temperatures, an RNA population samples many structures from its Boltzmann ensemble, and functionally important conformations may not be the MFE structure. RNAsubopt reveals this structural diversity.
The algorithm was first described by Wuchty et al. (1999) and implemented in the ViennaRNA Package. It uses dynamic programming to systematically trace back through the energy landscape, collecting all structures whose free energy falls within the specified delta of the optimal.
When to use RNAsubopt
RNAsubopt is particularly useful for:
- Riboswitch analysis: Riboswitches function by adopting alternative structures in response to ligand binding. Enumerating suboptimal structures reveals these functional conformations.
- Kinetic accessibility studies: Structures close in energy to the MFE may be kinetically more accessible than the global minimum.
- Functional site accessibility: Determining whether regulatory sites like Shine-Dalgarno sequences are exposed in the ensemble.
- Folding kinetics: Combined with the barriers program, suboptimal structures enable analysis of RNA folding pathways.
How to use RNAsubopt online
Paste one or more RNA sequences, set an energy range above the MFE, and RNAsubopt returns every secondary structure within that window. Results appear as a sortable table of dot-bracket structures with their free energies, ready for download.
Input
| Field | Description |
|---|---|
RNA Sequences | One or more RNA sequences in FASTA format or as plain text. Accepts .fasta, .fa, or .txt files up to 10 MB. |
Settings
Suboptimal enumeration
| Setting | Description |
|---|---|
Energy range | Delta above MFE in kcal/mol (1-20, default 5). Structures within this range are returned. Larger values yield exponentially more structures. |
Maximum structures | Cap on returned structures (10-1000, default 100). Prevents runaway enumeration on long sequences. |
Prediction options
| Setting | Description |
|---|---|
Temperature | Folding temperature in Celsius (0-100, default 37). Affects energy calculations and which structures fall within the range. |
Disallow lonely pairs | When enabled, excludes structures containing isolated base pairs (helices of length 1). Reduces output volume and may improve biological relevance. |
Dangling ends | Treatment of unpaired nucleotides adjacent to helices. Double dangles (default) is recommended for most applications. |
Output
Results are returned as a table with one row per enumerated structure:
| Column | Description |
|---|---|
Sequence ID | Identifier from FASTA header or auto-generated. |
Structure # | Index of this structure (1 = MFE, higher = less stable). |
Length | Sequence length in nucleotides. |
Secondary Structure | Dot-bracket notation showing base pairs. |
Energy | Free energy in kcal/mol. |
Is MFE | Whether this is the minimum free energy structure. |
Limitations
The number of suboptimal structures grows exponentially with both sequence length and energy range. For a 50-nucleotide sequence, an energy range of 5 kcal/mol might yield hundreds of structures, while the same range on a 100-nucleotide sequence could produce millions.
Practical constraints:
- Sequences beyond ~100 nucleotides become computationally intractable for exhaustive enumeration
- The
Maximum structureslimit prevents memory exhaustion but means results may be incomplete - Very large energy ranges (>10 kcal/mol) are rarely useful and dramatically increase computation time
For longer sequences, consider RNAplfold for local structure analysis, or use RNAsubopt with the Disallow lonely pairs option enabled to reduce output volume.