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First Energy Constraint Details

Calculate binding energies for all pairs and select pairs with binding energies within 0.5 kcal/mol of WT ASD/SD. This function calls the RNAduplex.exe program of the Vienna Package to be used for our algorithm.

Function: RNAduplexval

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RNAduplex calculates the binding energy (in kcal/mol) of 2 sequences that you input into it. Higher absolute value of binding energies indicates that the two sequences are likely to bind strongly, and vice versa.

Figure S2. Narrowing down based on wild-type ASD (WTASD)/wild-type SD(WTSD) binding energy.

Function: narrow_binding

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Firstly, we find the 16s rRNA sequence using the function get16srRNAseq. We then find the ASD using getASD and find the SD by taking the reverse complement of the 6 bps in the middle of the 4 bp prefix and 2 bp suffix within the wild-type ASD. We calculate the binding energy of the wild-type ASD and wild-type SD sequence by running the WTASD and WTSD through RNAduplex to find their binding energy. Then, for all 4096 ASD-SD pairs, we run RNAduplexval, which gives us the binding energies of those ASD/SD pairs. We then remove all pairs from the library that do not fall within 0.5 kcal/mol binding energy of the wild type. This is because if the ASD/SD binding energy’s absolute value is too high compared to the wild-type, the pair will be too tightly bound and thus inhibiting translation initiation by preventing 30s ribosomal subunit to proceed across the mRNA with elongation. If the binding energy’s absolute value is too little compared to wild-type’s, translation initiation will not occur at all, since the 30s ribosomal subunit will not bind at all to the mRNA. This returns a narrowed library with only ASD/SD pairs within 0.5 kcal/mol above or below wild type binding energies.