Basic Research to Diagnostics and Surveillance in Lower Resource Environments Foundation for Applied Molecular Evolution Steven A. Benner ABSTRACT We will deliver to the NIAID and CDC communities, through basic research, a scientific understanding of pairing, mispairing, and enzymology of natural DNA and RNA (collectively xNA) that goes deeper than the axiom that "A pairs with T, and G pairs with C". The experiments are designed to learn: (a) Why robust multiplexed PCR (mPCR) for clinical use seems impossible with more than 20-30 targets. (b) Why conventional expedients (including careful primer and probe design, internal nesting, and external tagging) fail to robustly support multiplexing beyond ~30 targets. (c) Why those failures are not reproducible from sample to sample. (d) Why conventional multiplexes targeting n targets often collapse when an n+1th target is added. This prevents, when a new pathogen emerges (as for 2019-nCoV), a diagnostics maker from simply adding a new target to an existing mPCR kit, thereby meeting the emergency need. (e) Why manufacturing specs become increasingly more demanding as the level of multiplexing increases. These problems restrain 21st century diagnostics to two 20th century design and regulatory paradigms. (i) A "guess-then-test" paradigm for singleplexed molecular diagnosis, which requires physician to guess which pathogen might be associated with patient malaise, prescribe a ~$150 singleplexed test based on that guess, and re-prescribe further tests until a guess proves correct. (ii) The "inflexible-multiplexed-panel" paradigm. Here, assays are bundled into a multiplex appropriate for a specific sample and symptom set; failure (d) prevents that multiplex from changing for emerging diseases. By developing the science of both natural and unnatural DNA (including artificially expanded genetic information systems, AEGIS, and self avoiding molecular recognition systems, SAMRS), this project will deliver to researchers, manufacturers, and the FDA science to meet the 21st century NIAID mission. We will: Task 1. Complete thermodynamic and enzyme rules to place SAMRS optimally in primers that target both DNA and RNA. Rules will be metricked by comparing predictions made with these rules to experiments. Task 2. Metric, by deep sequencing, mPCR failures (a) through (e). Task 3. Metric how AEGIS and SAMRS mitigate or eliminate failures (a) through (e). Task 4. Identify failure modes that arise with RNA targets specifically. Since RNA has folding options not available to DNA, these modes may be especially resistant to nucleic acid innovations. Task 5. Build a body of statistical knowledge for AEGIS-SAMRS mPCR, especially with respect to "add-ons", quantitative amplification, and manufacturing tolerances. This will help move away from "guess-then- test" and "inflexible-multiplexed-panel" paradigms, lowing cost, supporting FDA regulatory processes, and better managing pandemics. 1