Model
To begin computational research, Huntingtin mutants and their percentage of occurrence were located on various genetic databases, mainly NCBI. Our goal was to achieve toehold strand displacement of mutated HTT with a corrected strand. Sequences were run through protein-folding softwares to select viable candidates for the project. By aligning proteins, hairpin loops could be identified and targeted. Candidates had to be created for chaperone and promoter sequences, with approximately 40 CAG repeats within the sequence. The computational team was split up to test for feasible sequences. When attempting to order strands, the sequence was not practical, so it was revised.
Our goal was to attempt a toehold strand displacement of mutated HTT with a corrected strand. We used mFold software packages to model RNA sequence folds in order to find a tractable hairpin within the 5’ UTR. However, using mFold did not provide enough information on any full sequences the size of HTT. We switched to the Vienna package as it provided a much better model of the data. Model folding calculations/visualizations allowed prediction of the position of a usable a hairpin loop for strand displacement.
After running our sequence through Vienna, it was apparent that the mRNA molecule was still too large. One software prediction was not enough. Many online sites with protein folding capabilities proved difficult to use or were not being maintained. Genstrip and RNAI designer were two programs provided multiples sequences for us to target. UGENE was used to view and align these sequences, allowing us to target the optimal hairpin loop and figure out exactly where to begin targeting.
