Design of synthetic RNase-resistant RNA structures

Flaviviruses comprise many human and animal pathogens, such as Dengue, Zika, Yellow Fever, and tick-borne encephalitis viruses. Their single-stranded RNA genome is flanked by heavily structured untranslated regions (UTRs), which do not code for proteins. Inside the UTR at the end of the genome, unique evolutionarily conserved RNA elements, so-called exoribonuclease-resistant RNAs (xrRNAs) protect other parts of the RNA from being broken down by host enzymes known as exoribonuclease. This process, referred to as RNA degradation, results in the production of stable pieces of RNA that accumulate in infected cells, known as subgenomic flavivirus RNAs (sfRNAs), that play a role in how the virus causes disease. We propose to design artificial xrRNAs that can control how quickly exoribonucleases degrade RNA molecules. Combining custom xrRNAs with other RNA elements called aptamers will allow us to create a new class of synthetic biology tools, termed xrRNA riboswitches, which act as molecular gates that enable or disable RNA degradation. These molecular switches will allow us, for example, to control the speed of messenger RNA (mRNA) degradation in biosynthetic networks and living cells. Method-wise, we will use bioinformatics approaches to study the structuredness of xrRNAs, which will provide a better picture of the molecular characteristics required for xrRNA functionality. Based on this, we will design synthetic xrRNAs with varying capacities to protect against exoribonuclease degradation. These xrRNAs will then be experimentally tested in living cells, and in cell-free environments by attaching them to reporter mRNAs, and measuring how long these persist. Additionally, we will combine xrRNAs with aptamers to create new synthetic biology tools that can form protective elements in response to the presence or absence of certain small molecules. We will be able to control how long these RNAs persist by using xrRNA elements that differ in their efficiency to protect against their degradation. In summary, we are developing a new way to control the stability of RNA molecules by re-purposing and engineering viral RNA elements that can control how quickly enzymes break them down. This represents a novel approach in synthetic biology to obtain fine-grained control of the amount of RNA present in specific biological contexts.