Project Summary A wide variety of RNA researchers synthesize their RNA using the enzyme T7 RNA polymerase, as this enzyme is robust and can yield large quantities of RNA, at any length scale. However, undesired products typically contaminate the desired RNA, in often unpredictable ways. This is perhaps most impactful currently in the mRNA therapeutics field, where contaminating double stranded RNAs can trigger a potentially lethal innate immune response, but contaminants almost certainly impact other studies as well, from basic research in cell and molecular biology and biochemistry/biophysics, to synthetic biology, to RNA nanotechnology. Gel purifications are tedious, low yield, and imperfect (indeed, the darkest band on the gel may not be the correct product, and even the correct length RNA pool can be heterogeneous!). Longer RNA impurities derive from correct RNA, reducing yields at synthesis, and can be distributed across a wide range of lengths, making gel analysis problematic. Building on new mechanistic understandings, this project will develop systems that limit the conditions that give rise to these impurities, specifically by inhibiting the off-pathway reactions, and will develop simple affinity purification approaches – all with an aim towards achieving monodisperse RNAs of defined length and sequence. Sensitive analytical and functional assays for success will be developed and applied to guide design. Tools will be developed with an eye towards broad adoption by a variety of researchers.