Overview

The MSA Tool is a secondary analytical module developed as part of our modelling and software framework. While our main software (see Software and Software Technicalities pages) focuses on designing and simulating functional RNA switches, this complementary tool was created to explore the evolutionary and structural conservation of naturally occurring RNA elements.

We specifically designed the MSA Tool during our attempt to use an SCR element as our Functional RNA Element (FRE) (see Model and Software pages). Because the SCR's structure was not well characterized, we needed a way to test whether it showed conserved structural motifs that could indicate a functional secondary structure.

T When studying protein function, researchers often rely on structure prediction tools such as AlphaFold^[2] or conservation analysis pipelines that use evolutionary information to identify key residues. However, for RNA, there is no equivalent integrated tool that allows researchers to visualise structural conservation in an evolutionary context.
This tool provided the framework to perform such an analysis — allowing us to visualize structural conservation directly on the RNA secondary structure. Ultimately, experiments designed using the insights from this tool confirmed that no important structure was conserved, helping us move forward with other FRE candidates.

Access Points

How It Works

The MSA Tool takes a Multiple Sequence Alignment (MSA) file and analyzes both sequence conservation and predicted RNA secondary structures, integrating evolutionary information with RNA folding predictions.

Supported formats:

• FASTA (.fasta, .fa) and Clustal (.aln) — up to 200 MB per file
• You can drag and drop your file, or upload it manually

A folder named "examples" is available in the tool's web interface — it includes a sample FASTA file you can try directly on the platform.

The first sequence in the alignment is considered the reference sequence, and its predicted structure is used as a baseline for comparison and visualization.

Output Overview

Once the file is processed, three result tabs are generated:

1. General Information

This section provides the predicted structure and folding energy of the reference sequence, alongside a conservation map showing how conserved each base is across the alignment.

Example structure:

.((.(((.....))).)).....(((((.(((.....((((........))))....)))))))). 
          Energy: -15.00 kcal/mol

The conservation map displays a score between 0 (variable) and 1 (fully conserved) for each nucleotide, visualized on the RNAFold-predicted structure. High conservation across base-paired regions indicates possible structural or functional importance.

2. Clustering Analysis

This tab summarizes the structural diversity observed across the alignment — showing how many unique structures exist and how they cluster together.

Example:

Total Structures: 20
Unique Structures: 4
Cluster 0 — 9 structures
Cluster 1 — 9 structures
Cluster 2 — 1 structure
Cluster 3 — 1 structure

A low number of unique clusters, compared with the number of sequences, indicates a highly conserved overall fold, while higher diversity suggests that the sequence may not rely on a stable conserved structure for its function.

3. Visualise All Structures

This section allows you to explore each predicted structure individually — including sequence, folding energy, and base-pairing pattern — for a deeper look into how mutations may influence the RNA's folding.

Significance

The MSA Tool was designed to shed light on the potential structural role of naturally occurring RNA elements, offering an evidence-based approach rather than working "in the dark."

By integrating evolutionary conservation, structure prediction, and clustering analysis, it helps determine:

In our case, it ultimately demonstrated that the SCR element lacked significant structural conservation, leading us to conclude that it was unlikely to function as a reliable FRE. This insight was crucial in guiding the next steps of our design–build–test–learn cycle.