# Genetic code expansion for the construction of beyond rule-of-5 compliant macrocyclic peptide libraries

> **NIH NIH R01** · VIRGINIA COMMONWEALTH UNIVERSITY · 2022 · $108,798

## Abstract

Project Summary
Many intracellular targets involve intracellular protein-protein interactions that are “undruggable” because the
binding surfaces are too large and featureless to be blocked by a standard rule-of-5 compliant small molecule.
Recently, there have been attempts to catalog molecules that are orally bioavailable but lie beyond the rule of
five (bRo5) to access these targets. Macrocyclic peptides can inhabit this bRo5 space, and a key advantage to
using peptides as bRo5 molecules is that there are many mature techniques for finding peptide binders from
vast libraries. Arguably, the most powerful of these techniques is mRNA display, which allows creation of
peptide libraries containing over 10 trillion variants, 6-7 orders of magnitude larger than a standard peptide
library prepared on beads. The extreme diversity of these libraries has enabled many successes in inhibitor
development. Yet these successes are disconnected from real drug discovery, because the peptides
uncovered are much too large to be bRo5 compliant. Libraries that are short in sequence and bRo5 compliant
can be created by mRNA display, but these libraries lack the diversity needed to uncover potent inhibitors
because standard mRNA display is limited by the genetic code to ~20 variants at each position. In this
proposal two strategies to enhance this positional diversity will be pursued. The first involves breaking the
degeneracy of the standard genetic code through isolation of fully modified tRNA isoacceptors. Based on
codon reading rules it is predicted that this will allow the addition of 10 non-canonical amino acids (ncAAs) to
the code. The second involves insertion of an unnatural base pair (UBP) to the code. The addition of a single
UBP into the genetic code at a single codon position opens 32 new empty codons that can be exploited for the
introduction of novel ncAAs to the code. tRNAs that read each of these codons will be prepared and the codon
reading preferences will be validated. Putting the two strategies together should allow expansion of the
genetic code to the use of 40 monomers at each position. With carefully chosen building blocks, this will allow
for the creation of bRo5 compliant libraries containing billions of variants for use in drug discovery.

## Key facts

- **NIH application ID:** 10652818
- **Project number:** 3R01GM143396-02S1
- **Recipient organization:** VIRGINIA COMMONWEALTH UNIVERSITY
- **Principal Investigator:** Matthew C Hartman
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $108,798
- **Award type:** 3
- **Project period:** 2021-08-01 → 2025-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10652818

## Citation

> US National Institutes of Health, RePORTER application 10652818, Genetic code expansion for the construction of beyond rule-of-5 compliant macrocyclic peptide libraries (3R01GM143396-02S1). Retrieved via AI Analytics 2026-06-12 from https://api.ai-analytics.org/grant/nih/10652818. Licensed CC0.

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