# Programmable Synthetic Organelles Built from Disordered Proteins for Cellular Engineering

> **NIH NIH R01** · UNIVERSITY OF PENNSYLVANIA · 2021 · $414,791

## Abstract

Abstract
The engineering of cellular decision-making – such as entry into the cell cycle or stem cell differentiation – is
critical to our ability to regenerate functional cells and tissues. The development of these technologies is limited
by the extent to which one can manipulate the endogenous cellular machinery. Membraneless organelles are
naturally-occurring assemblies of intrinsically disordered proteins (IDPs) that form protein-rich, fluid-like phases
in cells. With insightful engineering, these organelles can provide a means for directing cell physiology through
the compartmentalization and release of regulatory molecules. Recently, we developed a novel platform for the
assembly of membraneless organelles endowed with a myriad of novel properties and control motifs. Our
methods are based on engineering intrinsically-disordered arginine-glycine rich RGG domains that are sufficient
to drives phase separation. The premise of our application is the development of numerous, state-of-the-art IDP
materials for assembly of new, designer membraneless organelles. We have developed methods to dynamically
modulate the multivalency of RGG sequences and introduced protein-interaction motifs to allow sequestration
of designated cargo. We propose to develop strategies for controlling the rapid release and sequestration of
cargo proteins in response to optical and thermal stimuli. Further, we propose the develop optical and enzymatic
controllers for organelle assembly and disassembly. Importantly, we have already demonstrated the expression
of the sequences is sufficient to generate membraneless organelle in yeast and mammalian cell lines. Here we
propose a novel, innovative paradigm in synthetic biology for controlling cellular responses through the
manipulation of membraneless organelles. These organelles will be designed to sequester factors that control
cell cycle commitment or cell fate decisions on cue, in response to light or biochemical stimulus. The ultimate
goal of the proposed work is to control the dynamic progression through the cell cycle and cell fate decision-
making in hematopoietic stem cells for regenerative medicine. In aim 1, we will expand the toolbox of self-
assembling IDPs by engineering optical and thermal switches within disordered protein materials for dynamic
gating of organelle assembly, composition and release. In aim 2, we will use membranless organelles for cell
decision-making, rapidly sequestering and releasing target proteins to dynamically control cell proliferation and
stem cell fate. Harnessing the power of compartmentalization, our designer organelles promise a tunable
biochemical niche and a generalizable strategy for precisely engineering cell systems capable of responding to
specific stimuli with predictable outcomes.

## Key facts

- **NIH application ID:** 10220971
- **Project number:** 5R01EB028320-03
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** Matthew Charlton Good
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $414,791
- **Award type:** 5
- **Project period:** 2019-09-15 → 2023-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10220971

## Citation

> US National Institutes of Health, RePORTER application 10220971, Programmable Synthetic Organelles Built from Disordered Proteins for Cellular Engineering (5R01EB028320-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10220971. Licensed CC0.

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