Revision as of 23:36, 28 October 2017

NUPACK modelling

NUPACK is a software tool that can analyse the secondary structures of RNA systems. To design our toehold switches we first modelled them in NUPACK.

We ran our simulations at 37°C, with 100 picomoles of miRNA, 20 nanomoles each of DNA and anti-miRNA. We chose this temperature as it is the optimum for E. coli derived enzymes.

Using NUPACK, we checked our switches had no base pairing in the ribosome binding site in both the off and on state and minimal secondary structure around the start codon in the on state. We also modelled the bound state, ensuring the structure around the RBS and start codon was correct in both the off and on state.

Having demonstrated that our design would work, we minimized the |∆GRBS-Linker| via site directed mutagenesis (varying the sequence at specific sites). Making the |∆GRBS-Linker| as close to zero as possible maximises the toehold switch output. This discussed in more depth along with the other important parameters for toehold switch design on the design page.

However, NUPACK struggled to predict the specificity of the second series of switches as they had multi-step reactions. Additionally, we found that NUPACK was unable to match experimental data for molecular beacon specificity in our new form of riboregulators. , Despite this, NUPACK was suitable for accurately designing our first series of switches due to their simpler and more predictable nature.

TODO: Pictures and diagrams