The past decade has brought an escalating growth of important new commercial applications for RNA. RNA molecules have emerged as the preeminent regulators of gene expression. They have a unique ability to edit genomes and destabilize gene products. New diagnostic applications for RNA are continually being discovered. Last but not least, modified mRNA nanoparticle formulations are enabling the development of new vaccines and cellular therapies with a speed and efficacy unrivaled by other processes. This newfound success has driven increased interest and efforts in many areas of RNA research. Researchers are constantly discovering important new roles for RNA in the regulation of cellular processes. It has also opened the door to many new commercial opportunities for startups and large pharmaceutical and diagnostic companies alike. Accordingly, massive effort is now directed to production scale-up and large-scale manufacturing of RNA molecules. As production increases, there is a far greater need for care and control of manufacturing processes. This is especially true for RNA since it is quite vulnerable to degradation by contaminating enzymes in the surrounding environment. As production scales and costs increase, more attention must be paid to strict process control and quality management. For example, manufacturing of RNA for regulated therapeutic and diagnostic applications must be performed in a certified RNAse-free environment. Current commercial methods to verify the absence of RNAse require expensive equipment and hours to perform. The cost and time required to perform RNAse testing hinders the productivity of manufacturers and researchers alike. A new rapid tool to test for unwanted RNA contamination would great assist researchers and manufacturers who must frequently test their environments and supply chain materials. In Phase I, we will create a novel prototype lateral flow test for the sensitive detection of RNAse. Our work will produce a test kit product with several important advantages over currently available tools: Convenience: Our test will be portable and not require bulky, expensive lab equipment. Sensitivity: Our test will be at least 5-times more sensitive than current methods. Time: Users will see their results in less than 15 minutes rather than hours. These combined advantages of our proposed product will create a highly effective tool to further support the continuing innovation in RNA therapeutics and research. Specific Aim #1: Optimize the incubation buffer and RNA oligonucleotide sequence to improve RNAse assay sensitivity. Specific Aim #2: Produce a more sensitive fluorescent version of the lateral flow RNAse detection assay. Specific Aim #3: Enable testing of solid surfaces using swab wipes.
Public Health Relevance Statement: Narrative RNA-based vaccines are a leading tool in the fight against COVID-19 and other diseases. mRNA molecules used to make these vaccines are extremely vulnerable to ribonuclease contaminants during the manufacturing process. We will develop a rapid and portable test to detect ribonucleases in RNA laboratories and manufacturing facilities. Terms:
