RNAsubopt

Enumerate suboptimal RNA secondary structures.

Input

RNA Sequences

6 credits

Output

Configure inputs to begin

Set options on the left, then click “Submit job”.

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 structures limit 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.

Related tools

RNAdos

RNAdos calculates density-of-states summaries for RNA sequences, reporting representative structures and state counts across energy bands.

RNAeval

RNAeval calculates the free energy of an RNA secondary structure for a given sequence. Evaluates if a proposed structure is thermodynamically favorable.

RNAfold

RNAfold predicts RNA secondary structure using minimum free energy (MFE) algorithms and optionally returns partition-function ensemble metrics when explicitly enabled.

RNALfold

RNALfold reports locally stable RNA secondary structures within a sliding window and returns their start and end positions on the input sequence.

RNAplfold

RNAplfold computes local base pair probabilities using a sliding window approach. Useful for analyzing accessibility and identifying binding sites in long RNA sequences.

ViennaRNA

ViennaRNA supports a curated set of scientifically faithful ViennaRNA 2.7.2 workflows for RNA folding, density-of-states analysis, interaction prediction, local accessibility, plotting, inverse folding, and structure analysis.

RNAcofold

RNAcofold predicts the joint secondary structure of two interacting RNA molecules and optionally reports partition-function and concentration-dependent equilibrium metrics.

RNAdistance

RNAdistance compares RNA secondary structures using the selected native ViennaRNA distance representation and comparison mode.

RNAduplex

RNAduplex computes the hybridization structure between two RNA sequences. Predicts the optimal duplex formation and binding energy.

RNAplex

RNAplex predicts fast query-target RNA interactions, reporting parsed hit coordinates, structures, and energies.

What is RNAsubopt?

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

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

Practical constraints:

Sequences beyond ~100 nucleotides become computationally intractable for exhaustive enumeration
The Maximum structures limit 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.