# Engineering Proteins with Noncanonical Amino Acids

> **NIH NIH R35** · RICE UNIVERSITY · 2024 · $381,190

## Abstract

PROJECT SUMMARY/ABSTRACT
With few exceptions, all organisms encode encodes 20 amino acid building blocks used in the synthesis of
proteins. The overall goal of the PI's research program is to develop interdisciplinary tools to reprogram the
genetic code to precisely probe and manipulate biological systems. Our ability to incorporate noncanonical amino
acids (ncAAs) with diverse physical, chemical, and biological properties into proteins of interest is central to the
reprogramming of genetic codes. The overall goal of this proposal is to develop cells able to biosynthesize and
utilize ncAAs and explore the utility of these unnatural organisms in the biosynthesis of bioactive drugs and in
vivo sensors for posttranslational modification enzymes. To achieve this goal, the first research goal is to identify
more ncAA biosynthetic pathways and use them for the generation of prokaryotic and eukaryotic organisms with
additional amino acids. The additional amino acid can be biosynthesized without exogenous addition, followed
by the genetic incorporation of it into proteins using a bioorthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA
pair in a site-specific manner. The biosynthesis pathway of ncAAs will be obtained via metabolic engineering or
using bioinformatic strategies. The resulting organisms with 21st amino acids will be used to prepare bioactive
compounds with novel activities as well as the development of in vivo sensors for posttranslational modification
enzymes. To biosynthesize bioactive compounds with ncAAs, we will first introduce the bioorthogonal aaRS for
aminoacylation of tRNAs with the ncAA, and then engineer synthetases of natural products towards the tRNA
charged with the ncAA. The resulting natural product derivatives will exhibit enhanced biological activities or
unprecedented biophysiological properties. Next, we will explore the utility of these unnatural organisms with
additional protein building blocks for monitoring the activities of posttranslational modification enzymes in vivo.
The eukaryotic cells able to biosynthesize and utilize amino acids with posttranslational modification handles will
be used to produce caged luciferase or fluorescent proteins. The resulting engineered cells will allow for the
evaluation of posttranslational modification enzymes' activities in real-time, thus providing a strategy to guide the
treatment of epigenetic inhibitors in vivo. Our efforts in this project will yield a collection of organisms with a 21st
amino acid, and will result in new platforms for the evolution of ncAA-based therapeutic molecules or novel living
sensors.

## Key facts

- **NIH application ID:** 10765446
- **Project number:** 2R35GM133706-06
- **Recipient organization:** RICE UNIVERSITY
- **Principal Investigator:** Han Xiao
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $381,190
- **Award type:** 2
- **Project period:** 2019-09-01 → 2029-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10765446

## Citation

> US National Institutes of Health, RePORTER application 10765446, Engineering Proteins with Noncanonical Amino Acids (2R35GM133706-06). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10765446. Licensed CC0.

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