Project Summary/Abstract The synthesis of proteins takes place by ribosomal translation of mRNA molecules. Despite its central importance in biology, there are only a handful of techniques to analyze ribosome::mRNA complexes in vivo, and none of these are able to identify sites of ribosome binding on intact and individual mRNA molecules. A facile technique to simultaneously sequence mRNA molecules and visualize sites of ribosome::mRNA interactions would greatly expand the ability to study protein synthesis, with impacts from model organism biology through human clinical applications. This application’s broad, long-term objective is to develop a technology to simultaneously identify all sites of ribosome binding on a single mRNA molecule, sequence the mRNA molecule, and do so across all mRNA molecules in a sample. To achieve the objective, we will combine the relatively old technology of chemical footprinting to mark sites of ribosome::mRNA interaction and the nascent technology of single molecule mRNA sequencing on the Oxford nanopore device to identify modification sites. Specifically, we will: (1) Identify appropriate chemical footprinting reagents and evaluate nanopore performance in modification detection and sequence identification on RNAs treated with the reagents; (2) Chemically footprint ribosome::mRNA complexes, and compare information obtained to the existing state of the art (ribosome footprint profiling); (3) Develop computational algorithms to identify footprints via statistical inference, using both simulated and real-world data. Results from these aims will provide critical data on the feasibility of ribosome footprinting with whole transcript single molecule sequencing. The resulting protocol will equip researchers and clinicians to visualize and quantify facets of protein synthesis in their experimental system of choice for diagnostic purposes, high-throughput phenotyping, and/or mechanistic analysis of protein synthesis.