1. Plug-n-Play with RNA Isaacs & Collins Deepti & John S

2. Motivation What is a plug-and-play system? Why do we want these? –Easy to use –Increase functionality quickly and reliably –…Implement this into biosystems using mRNA switches…

3. mRNA Switches RNA is key to many mediating gene expression at the translational level In Eukaryotes… –Micro RNA Cleavage & Degradation Complementary strand binding In Prokaryotes…

4. Prokaryotic translational regulation Riboregulators –non-coding regulatory RNA which binds to coding mRNA preventing gene expression Early transcription termination Inhibition of translational initiation Enhancement of mRNA degradation mRNA riboregulator 5’ 3’

5. Prokaryotic translational regulation Ribozymes –Non-coding regulatory RNA which cleaves the mRNA when a specific metabolite attaches to a ligand motif (binding site) Ligand motif can be changed easily to suit system Ligand can be essentially any small molecule mRNA ribozyme CLEAVAGE! The ribozyme acts as a RNAase enzyme to cleave the mRNA and thus…prevent translation! Metabolite binds to aptamer

6. Bayer & Smolke’s Hybrid System A combination of both riboregulator and ribozyme action! –Ligand-Aptamer based control from ribozyme –Antisense domain complementary to target mRNA adapted from riboregulator Two types of hybrid ‘antiswitches’ –On Antiswitch… –Off Antiswitch…

9. Features of the ‘Antiswitch’ Sensitive to small amounts of metabolite –Is this good or bad? Noise? Speed? Ability to alter the antisense sequence of riboregulator to change the strength of the stem-loop structure (self-binding action to prevent activation) –Number of point mutations will dictate strength

10. Plug-n-Play Features Ability to change ligand and ligand motifs –Essentially any small molecules (dyes, proteins, ions, etc.) Ability to change the specificity of target mRNA complementary strand –Point mutations to determine target sequence

11. Extension to the antiswitch Ability to combine more than one antiswitch system in a cell simultaneously –More than one ligand can be implemented so that e.g. tetracycline can activate one antiswitch whilst theophylline can activate another antiswitch –Possibility of using two on one antiswitch to create a 2-input logic gate…

12. Further (random!)Thoughts Cancer control in eukaryotic cell –microRNA will bind to mRNA which code for proteins with regulatory functions Speed up switching functionality –Target inhibition at the translational level instead of transcriptional level Self-Regulation – feedback loops –Use of the protein products as metabolites which bind to aptamer on riboregulator to inhibit/promote translation of protein product itself (negative or positive feedback loop)