# Quadruplet Decoding for Multiplexed Non-Canonical Amino Acid Incorporation

> **NIH NIH F31** · MASSACHUSETTS INSTITUTE OF TECHNOLOGY · 2020 · $43,414

## Abstract

Project Summary/Abstract
 Protein therapeutics have played an ever-increasing role in drug discovery during the past 20 years,
leading to some 380 FDA-approved drugs on the market today. All these biologics use only the tiny fraction of
chemical space accessible to the standard 20 amino acids. The ability to incorporate Non-Canonical Amino
Acids (NCAAs) into protein therapeutics is a promising strategy that would greatly improve the chemical
sophistication of biologics, increase their bioavailability through cyclization or PEGylation, and facilitate
chemical conjugation to create immunoconjugates. NCAA incorporation can be accomplished both in vitro and
in vivo, but the cost of in vitro systems renders them unsuitable for production of therapeutics at scale, and
cellular production is crippled by the lack of free triplet codons. I propose to harness powerful new directed
evolution techniques to develop a method capable of efficiently incorporating numerous NCAAs into proteins in
vivo, thereby paving the way toward low-cost, chemically-diverse protein therapeutics.
 Techniques in use today are limited to incorporation of no more than two NCAAs into the same protein
chain. Here, I aim to improve upon this technical capability. If successful, this approach will pave the way
toward production of genetically-encoded materials entirely composed of NCAAs, a technical capability that
would have immediate applications for therapeutics as well as material science more broadly. I focus on
frameshift suppression, an extensible technique for NCAA incorporation that offers 256 codons, rather than just
two. I propose to develop a reporter for frameshift suppression, allowing me to quantify suppression efficiency
more robustly than was previously possible. Next, I propose to evolve two independent molecular targets that
limit frameshift suppression efficiency: suppressor tRNAs and ribosomal rRNA, in favor of improved efficiency.
Existing work with traditional engineering and small libraries has seen modest success already toward the goal
of improved efficiency, suggesting that my approach, which leverages a powerful continuous evolution
technique, will be successful. Together, this project investigates an extensible approach to the important
capability of NCAA incorporation, and aims to develop technology capable of alleviating technical difficulties
that presently limit the utility of this approach.

## Key facts

- **NIH application ID:** 9863748
- **Project number:** 5F31AI145181-02
- **Recipient organization:** MASSACHUSETTS INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** Erika Alden DeBenedictis
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $43,414
- **Award type:** 5
- **Project period:** 2019-01-25 → 2020-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9863748

## Citation

> US National Institutes of Health, RePORTER application 9863748, Quadruplet Decoding for Multiplexed Non-Canonical Amino Acid Incorporation (5F31AI145181-02). Retrieved via AI Analytics 2026-06-11 from https://api.ai-analytics.org/grant/nih/9863748. Licensed CC0.

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