NanoGuide

This project aims to develop a sensitive RNA sensor as an early and accessible diagnostic tool for Avian Influenza in both farm animals and humans. In order to accomplish this, Mesoporous Silica Nanoparticles (MSNs) will be synthesized using TEOS and CTAB followed by surface functionalization with APTES. They’ll then be loaded with a fluorophore such as fluorescein by diffusion. Small RNA strands will then be designed and reacted first with EDC in order to conjugate their 5’ terminal phosphate group with the nanoparticle surface. Following this reaction, the RNAs will then be modified at their 3’ ends with Periodate to change their OH groups to aldehydes. These aldehydes can then be crosslinked to a capping molecule that’ll serve as protection for the RNA but also as a molecular cap to the pores. Separately, an E. coli system will be transfected with a plasmid containing: the coding sequence for the Cas13d endonuclease, the sequence for the Guide RNA. The sequence for the gRNA will be designed to be half complementary to a sequence in the cap, and half complementary to a target RNA sequence, in this case to the M gene of Influenza A. The Cas13d-RNA complex will then be isolated and mixed with our nanoparticles. The RNA strand in the cap will then hybridize to the guide RNA and it will be enough to hold the protein complex together near the pores but there won’t be enough complementarity to activate the nuclease activity of the protein. Only when the target RNA is present and hybridizes to the guide RNA, will Cas13d be activated and catalyze a cut in the pore RNA. This cut will open up the pores and allow the fluorescein to be released, we can then analyze our sample through fluorescence microscopy to detect this event. We’ll transfect bacteria with plasmids expressing the RNA of Gene M and then transfect those same cells, as well as some controls, with our sensor. By observing these cells with fluorescence microscopy we’ll be able to assess the functionality of our project. If successful, our sensor can be used as an early detection tool for avian flu infections that requires no expertise to operate in order to help slow the spread of the outbreak, offering a sensor that covers for traditional methods when they fall short. This system won’t just be limited to avian flu. It has the potential to be used as an in-vivo RNA sensor that could aid in research, detection of practically any viral infection, and cancer diagnostics and treatments.