# Structure, Function and Applications of Inteins

> **NIH NIH R01** · PRINCETON UNIVERSITY · 2024 · $584,219

## Abstract

SUMMARY: Chemistry-driven strategies that allow proteins to be manipulated in ways not
permitted by standard genetics are now indispensable tools in both basic and applied
aspects of biomedical science. Of the various platform technologies in common use, those
that rely on manipulation of intein-mediated protein splicing have proven especially
powerful owing to broad compatibility with both in vitro and in vivo applications. Work
performed during the current funding period has led to the discovery, characterization and
eventual engineering of several inteins, especially those split into two mutually-dependent
fragments that support protein trans-splicing upon complexation. We have employed
these ‘protein ligases’ to solve a number of biochemical problems, primarily in the
chromatin biology area. In the next phase of this program, we will exploit recent advances
in our understanding of protein trans-splicing, to develop split intein tools designed to
address a series of problems also in the area of epigenetics. Accordingly, a concerted
protein ‘transposition’ reaction will be developed that allows replacement of internal
regions of intact target proteins with synthetic cassettes containing chemical probes such
as crosslinkers and photosensitizers (Aim 1). This cut-and paste system will be used to
define and manipulate epigenetic complexes involved in higher-order chromatin structure.
We will also develop various controllable versions of split inteins for use in both basic
science and synthetic biology applications. In one line of enquiry (Aim 2), we will use these
tools to rapidly generate oncofusion proteins such AML-ETO, thereby allowing
characterization of the early stages of cell transformation. Lastly, in Aim 3, we will develop
protein ‘actuators’ that use conditional protein splicing to convert a biochemical input of
choice into a user-defined biochemical output. These ‘actuators’ will form the basis of
epigenetic circuits that are dependent on Boolean logic operations and that allow higher-
order chromatin complexes to be monitored in cells. While our focus is in the epigenetic
area, we imagine that the technologies developed in the context of this work will expand
the type of protein systems, and hence biological problems, accessible to interrogation
through chemical biology strategies.

## Key facts

- **NIH application ID:** 10915573
- **Project number:** 5R01GM086868-27
- **Recipient organization:** PRINCETON UNIVERSITY
- **Principal Investigator:** Tom Muir
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $584,219
- **Award type:** 5
- **Project period:** 1999-09-01 → 2026-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10915573

## Citation

> US National Institutes of Health, RePORTER application 10915573, Structure, Function and Applications of Inteins (5R01GM086868-27). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10915573. Licensed CC0.

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