Contribution

This guide serves as an intuitive instruction set for new users to design their own riboswitch sequences. Avoiding advanced options, there are 5 critical aspects to the process:

The Riboswitch Calculator’s Design Mode requires multiple inputs to have enough information to run. Let’s walk through each one:

Title
Name your riboswitch something relevant to what it intends to bind to and regulate.

Target Translation Rate without Ligand and Target Translation Rate with Excess Ligand
The default value is 10 for without ligand and 200,000 with excess ligand. If you are looking to design an ON switch (where the presence of the ligand of interest turns expression ON), then leave this as it is. If you are looking to design an OFF switch, (where the presence of the ligand of interest turns expression OFF), then swap these values.

Select a previously characterized aptamer domain
Ignore this part, and move on to the Aptamer Sequence.

Aptamer Sequence
You will need to paste your aptamer sequence into this section. More information on selecting your aptamers can be found in the next section.
NOTE: riboswitches are mRNA sequences, so you will need to use an RNA aptamer!

Aptamer Structure
Aptamer structure must be in ().x notation, which will be explained more in section 3. Determine your Aptamer Structures. Most RNA aptamers form hairpin structures, and you will need to know which nucleotides bind to each other to form that structure.

Promoter Name and Promoter Sequence
Paste in your promoter name and sequence of choice.

Protein CDS Name and Protein CDS Sequence
Your protein CDS is the nucleotide sequence for the gene you want your riboswitch to regulate. You will often find that your protein of interest will be in the form of an amino acid sequence. You can use De Novo DNA’s CDS Calculator to codon optimize your amino acid sequence and produce a nucleotide acid sequence that you can plug into the Riboswitch Calculator.

Again, remember that riboswitches are mRNA sequences, so they will only work with RNA aptamers! Aptagen’s Apta-Index is a library that documents many of these aptamers. You can filter your search for RNA aptamers under the “Aptamer Chemistry” option. Note that riboswitches will work better with shorter aptamer sequences, since sequences that are too long may structurally inhibit the ribosome binding site, even when not intended. RNA aptamers under 70-nucleotides long will work best. Also, make sure that the aptamer has a high binding affinity. The smaller the number, the higher the affinity. Values for binding affinity in the 0-100 nanomolar range are good, and picomolar is best.
Aptagen is not the only source for RNA aptamers. RNA aptamers can be found in literature as they are discovered with the SELEX process, but be wary as they may not always be well tested.

Inputting aptamer structure is easier than it looks. Let’s take a look at the aptamer structure for basic fibroblast growth factor (bFGF).

The structure of this apatmer would be denoted as (((((((((xxx((xxxx))xx))xxx))))))). It is read from 5’ to 3’. The parentheses indicate nucleotides that are bound, and each “x” represents an unbound nucleotide. Each “(“ corresponds to the respective “)”, representing a nucleotide and the nucleotide bound to it. For example, the first “(“ represents the “G” on the 5’ end, and the last “)” represents the “C” on the 3’ end that is bound to it.
Occasionally, you may stumble upon an RNA aptamer sequence without a diagram of the structure. Using a website like RNAfold can help predict a 2D structure that you can use for your Riboswitch Calculator predictions.

Aptagen only provides the aptamers’ the binding affinity as its equilibrium dissociation constant, KD. To run the calculator, you will need the ligand-aptamer binding free energy, which can be calculated using KD. This can easily be calculated with the equation ΔG = RT*ln(KD). After finding the binding free energy, you are now ready to run the algorithm!

Paste in all the inputs in their appropriate places, the submit the job to the queue. Make sure to refresh your results to see when the job finishes (for us, it typically took between 10-20 minutes. After this, your results will be ready! The Riboswitch Calculator will produce many predicted sequences with varying activation rations, ON/OFF rates, and binding free energies. Test multiple, unique designs to select the most optimal riboswitch.