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.