# Development of an improved core technology for efficient genetic code expansion in biomedical research

> **NIH NIH R01** · OREGON STATE UNIVERSITY · 2021 · $359,106

## Abstract

Title: Development of an improved core technology for efficient genetic code expansion in biomedical
research
Principal Investigator: Ryan Mehl
Summary
Genetic code expansion (GCE) allows the introduction of noncanonical amino acids (ncAAs) into proteins.
This provides a powerful toolbox to manipulate and expand biochemical activities, opens new possibilities
to control protein function in cells, and creates new therapeutics against many human pathologies,
including cancer, autoimmune syndromes, and metabolic diseases. First developed in bacterial systems,
biomedical applications of GCE are now rapidly increasing as a consequence of recently expanded abilities
in both mammalian cell culture and model multicellular organisms. Despite these advances and the
enormous potential of the technology, a key challenge to fully implementing GCE – especially for in vivo
applications – is the inefficient synthesis of ncAA-containing proteins in recoded bacterial and mammalian
cells. In particular, a variety of studies in recent years have converged on identifying the key bottleneck in
the technology: creating efficient pairs of orthogonal aminoacyl-tRNA synthetases (o-RS) and transfer
RNAs (o-tRNAs). Indeed, studies indicate that existing o-RS/tRNA pairs are two to five orders of
magnitude less efficient than naturally occurring pairs. Our goal in this proposal is to eliminate this
bottleneck by (1) developing a generally applicable approach for optimizing the function of engineered
o-RS/tRNA pairs, and (2) creating improved “high-efficiency” chassis for current workhorse o-RS/tRNA
pairs. With these advances, in vivo GCE studies to probe cellular functions can be carried out in ways that
generate proteins containing one or more ncAAs at similar concentrations and regulatory control as in
natural cells, and do it consistently without massive amounts of truncation products or excess GCE
components that cause confounding disturbances. No one has yet attempted to systematically attack this
problem. Our approach has two key features as compared to all prior work in the field. First, we will
generate a novel, detailed database correlating in vitro kinetic properties of purified o-RS/tRNA with the
performance of the same o-RS/tRNA pairs in protein synthesis in vivo and in cell-free bacterial and
mammalian extracts. Second, we will develop and apply an iterative process to improve existing o-
RS/tRNA pairs, guided by both computational approaches and experimental determination of allosteric
pathways, in order to optimize the design of libraries covering portions of the protein-tRNA complexes
outside the well-explored primary ncAA binding site. These libraries are then incorporated into directed
evolution approaches to select for catalytically improved pairs. We propose this innovative approach in
response to a specific NIGMS funding opportunity focused on technology development to enable
biomedical research, and it fully embodies the qualities specified by PAR-17-045...

## Key facts

- **NIH application ID:** 10093096
- **Project number:** 5R01GM131168-03
- **Recipient organization:** OREGON STATE UNIVERSITY
- **Principal Investigator:** RYAN A MEHL
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $359,106
- **Award type:** 5
- **Project period:** 2019-02-01 → 2022-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10093096

## Citation

> US National Institutes of Health, RePORTER application 10093096, Development of an improved core technology for efficient genetic code expansion in biomedical research (5R01GM131168-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10093096. Licensed CC0.

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