Chemogenetic Tools for Controlling Protein Phase Separation

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Solution Overview

Problem

Existing technologies lack effective chemogenetic tools to manipulate protein liquid-liquid phase separation (LLPS) in living cells, which is crucial for understanding biomolecular condensates' formation and function in normal cell physiology and disease states.

Innovation Solution

Compositions comprising a first amino acid sequence with a zinc finger domain and a second amino acid sequence with a cereblon (CRBN) domain are used to induce or dissociate phase separation, allowing for the isolation and manipulation of proteins and nucleic acids, and can be encapsulated in particles for in vivo use.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If optogenetic tools are used to induce protein droplet formation, then protein phase separation can be controlled in living cells, but the complexity of the system increases and requires specialized equipment

Engineering Contradiction:

Improvecontrol over protein phase separationVSAvoidsystem complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent replaces optogenetic tools (which require optical equipment and light delivery systems) with chemogenetic tools that use small molecule inducers. This substitution eliminates the need for complex optical instrumentation while maintaining controlled induction of protein phase separation through chemical means, thereby reducing device complexity while preserving reliability of control

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The patent introduces small molecule inducers as intermediaries that bind to protein domains (such as FKBP-FRB pairs or other chemogenetic modules) to trigger phase separation. This intermediary approach allows indirect control of protein condensation through chemical signaling, simplifying the overall system by replacing direct optical activation with a chemical mediator that can be administered systemically

Inventive Principle:

Principle #24Intermediary (Mediator)

2Quantity of substance

If protein phase separation is induced to form condensates, then protein-protein interactions can be visualized and isolated, but the resolution and characterization of low concentration interactions becomes difficult

Engineering Contradiction:

Improveconcentration of protein complexesVSAvoidresolution of protein-protein interactions

Core Design Contradiction:

Quantity of substanceVSMeasurement precision

Solution Approach 1:

The patent applies local quality by creating distinct phase-separated condensates with specific compositional properties. By engineering proteins with specific phase-separation domains (such as IDRs or multivalent interaction modules), the system creates localized regions with enhanced concentration of specific protein interactions, allowing high-resolution characterization of particular protein-protein interactions while maintaining the ability to study low-abundance interactors within the condensed phase

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The patent segments the cellular environment into distinct phase-separated compartments that can be independently studied. By creating discrete condensates through controlled phase separation, the system isolates specific protein interaction networks from the bulk cytoplasm, enabling focused analysis of particular protein complexes even at low concentrations through their enrichment within discrete condensate structures

Inventive Principle:

Principle #1Segmentation

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

These compositions enable precise control over protein phase separation, facilitating the isolation and characterization of proteins, visualization of protein-protein interactions, and potential therapeutic applications such as mutating endogenous DNA sequences.

Implementation Method 1

a system of amino acid sequences that, in the presence of an inducer, phase separate from a fluid into a solid or semi-solid granule, free of lipid membrane

Methodology Applied

Scientific EffectLiquid-liquid phase separation: Phase Change

Data Source

PatentUS20250314661A1Chemigenetic tools and methods of controlling and assessing protein phase separation

Publication Date: 2025.10.09 RGT UNIV OF CALIFORNIA

AI summary

The present disclosure is directed to compositions useful in, for example, screening for active compounds, inducing phase separation in a solution, and isolating a protein from a solution. Also disclosed are vaccines and compositions useful therein, which can be used to mutate an endogenous DNA sequence in a subject. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.