Saturday, April 20, 2024
4/20/2024
PROJECT SUMMARY CMV disease is the largest cause of post-transplant patient mortality. After a transplant, patients have to undergo extensive screening and prophylactic therapy in order to prevent reactivation of the virus. In many cases, even if these precautions are taken, the virus will reactivate after the patients finish antiviral therapy. This is due to the virus establishing latency in cells of the myeloid lineage. Because the antivirals target and inhibit virus replication, CMV is very difficult to target in its latent stage due to low levels of replication. This proposal aims to develop molecular tools that have the potential to be used in clinic to prevent CMV-related mortality, by combining the fields of protein and DNA engineering. In the first aim, a CMV detection platform will be developed that combines the techniques of rolling circle amplification and hybridization chain reaction to then turn on a luminescent protein biosensor that can be detected with a cell phone camera. This tool will allow identification of active CMV infection with high sensitivity by performing the one-pot reaction at room temperature with no special equipment for detection other than a cell phone. Patients will be able to self-screen at home using this low-cost alternative to PCR that does not require trained personnel or expensive equipment. In the second aim, DNA self-assembly technique of toehold-dependent strand displacement will be utilized to trigger a toxic protein switch in response to CMV-specific RNA input. The components will be introduced into a population of cells that may be latently infected with CMV, and the switch will turn on in response to a latency-associated CMV RNA that is expressed in target cells. This will cause specific death of CMV-infected cells. The potential application of this switch is its use in bone marrow transplants—the switch may be introduced in the cells of donor bone marrow, and any infected cells can be killed before transplantation into the recipient. Development of DNA/RNA-activated protein switches have been limited to natural cas proteins so far. There is a strong desire for the discovery of more cas proteins to extend the toolbox of CRISPR. The techniques developed in this proposal will circumvents that need by “CRISPR-izing” proteins of choice to perform any desired function based on a nucleic acid input. In aim 1 of this proposal, an RNA-activated luminescent protein switch will be constructed, and in aim 2, an RNA-activated toxic protein. These findings will give rise to a new class of proteins to perform functions unattainable by cas proteins in nature.
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