Difference between revisions of "Template:CLSB-UK Model NUPACK"

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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.
 
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.
  
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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 maximizes the toehold switch output. This discussed in more depth along with the other important parameters for toehold switch design on the [[Team:CLSB-UK/Design|design page]].
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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 maximizes the toehold switch output. This is discussed in more depth along with the other important parameters for toehold switch design on the [[Team:CLSB-UK/Design|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.
 
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.
  
 
{{CLSB-UK Model NUPACK Diagrams}}
 
{{CLSB-UK Model NUPACK Diagrams}}

Revision as of 02:17, 2 November 2017

NUPACK model


NUPACK is a software tool that can model the secondary structures of RNA systems.[1] We used NUPACK to optimize the design of our toehold switches.

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 maximizes the toehold switch output. This is 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.

View structures of toehold switch for miRNA:


RBSStart codonTriggerBindingSiteFree energy of secondary structure: -26.40 kcal/molMFE structure at 37.0°CACGUTriggerBindingSitemiRNAStart codonRBSFree energy of secondary structure: -43.98 kcal/molMFE structure at 37.0°CACGU
Start codonRBSTriggerBindingSiteFree energy of secondary structure: -18.80 kcal/molMFE structure at 37.0°CACGUTriggerBindingSitemiRNAStartcodonRBSFree energy of secondary structure: -46.98 kcal/molMFE structure at 37.0°CACGU
anti-miRNAmiRNAFree energy of secondary structure: -18.57 kcal/molMFE structure at 35.0°CACGU Free energy of secondary structure: -15.27 kcal/molMFE structure at 35.0°CACGUTrigger Binding SiteStart CodonRBS Free energy of secondary structure: -68.61 kcal/molMFE structure at 35.0°CACGUStart CodonTrigger Binding Siteanti-miRNAmiRNARBS
anti-miRNAmiRNAFree energy of secondary structure: -21.70 kcal/molMFE structure at 35.0°CACGU RBSStart codonTriggerBindingSiteFree energy of secondary structure: -14.08 kcal/molMFE structure at 35.0°CACGU anti-miRNAmiRNATriggersitebindingRBSStartcodonFree energy of secondary structure: -72.09 kcal/molMFE structure at 35.0°CACGU

Base diagrams created in NUPACK, highlighted regions of interest by us.

  1. ↑ Zadeh, J. N., Steenberg, C. D., Bois, J. S., Wolfe, B. R., Pierce, M. B., Khan, A. R., ... & Pierce, N. A. (2011). NUPACK: analysis and design of nucleic acid systems. Journal of computational chemistry, 32(1), 170-173.