Chemogenetic Intracellular pH Modulation With Subcellular Specificity

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Solution Overview

Problem

Current methods for manipulating intracellular pH and hydrogen sulfide levels lack the precision and specificity needed to study their role in cellular physiology and pathophysiology, particularly in subcellular locales, due to off-target effects and limited spatio-temporal resolution.

Innovation Solution

A chemogenetic approach using the Salmonella typhimurium-derived enzyme stDCyD, which converts β-Chloro D-alanine to hydrochloric acid or D-cysteine to hydrogen sulfide, allowing precise manipulation of pH and H2S levels with high spatio-temporal resolution, using genetically encoded biosensors for real-time detection.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If conventional methods (micropipettes, genetic manipulation of proton pumps, optogenetic approaches, small chemical inhibitors) are used to manipulate intracellular pH, then pH levels can be altered, but off-target effects occur and spatio-temporal resolution is limited

Engineering Contradiction:

Improvespecificity of pH manipulationVSAvoidoff-target effects

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The invention divides the cell into specific subcellular compartments (mitochondria, nucleus, cytosol, ER, Golgi, caveolae) and expresses targeted chemogenetic tools in each compartment. This spatial segmentation allows pH manipulation to occur only in the desired location without affecting other cellular regions, thereby eliminating off-target effects while maintaining high specificity.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention uses chemogenetic enzymes (such as Rhodobacter sphaeroides bacteriorhodopsin variants) as intermediary molecules that convert extracellular substrates into intracellular pH changes. These enzymes act as specific mediators that respond only to their designated substrates, providing precise temporal and spatial control over pH manipulation without the non-specific effects of conventional chemical inhibitors.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Manufacturing precision

If optogenetic approaches are used to manipulate intracellular pH, then high spatio-temporal resolution is achieved, but the tools cannot cover acidic pH ranges and require expensive facilities

Engineering Contradiction:

Improvespatio-temporal resolution of pH manipulationVSAvoidpH range coverage

Core Design Contradiction:

Manufacturing precisionVSAdaptability or versatility

Solution Approach 1:

The invention employs multiple chemogenetic enzyme variants with different kinetic properties and pH optima. By selecting and expressing appropriate enzyme variants, the system can manipulate pH across a broad range from acidic to alkaline conditions. This parameter diversification allows coverage of the full physiological pH spectrum, including acidic ranges that optogenetic tools cannot access.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The chemogenetic approach uses a universal enzymatic mechanism (substrate conversion) that can be adapted to produce both acidification and alkalization effects. The same basic platform (chemogenetic enzyme expression) can be tuned to achieve different pH outcomes by selecting appropriate enzyme variants and substrates, providing universal applicability across different pH ranges and cell types without requiring separate specialized tools.

Inventive Principle:

Principle #6Universality (Multi-functionality)

3Area of stationary object

If current chemogenetic tools are used, then controlled manipulation in deep tissues is possible, but tools for acidic pH range manipulation are lacking

Engineering Contradiction:

Improvedepth of tissue penetrationVSAvoidacidic pH manipulation capability

Core Design Contradiction:

Area of stationary objectVSAdaptability or versatility

Solution Approach 1:

The invention uses genetically encoded chemogenetic enzymes that can be delivered to deep tissues through viral or plasmid transfection. Once expressed, these enzymes replicate the pH manipulation function locally within the tissue, enabling controlled acidification without requiring external device penetration. The genetic copy of the enzyme persists in the tissue, providing sustained capability for acidic pH manipulation at the desired depth.

Inventive Principle:

Principle #26Copying

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

Enables precise and reversible manipulation of intracellular pH and H2S levels in specific cellular compartments, providing a tool for studying their roles in various pathologies and therapeutic strategies with minimal off-target effects.

Implementation Method 1

the biochemical conversion of β-Chloro D-alanine to hydrochloric acid or D-cysteine to hydrogen sulfide, respectively, when the substrates are provided to the enzyme

Methodology Applied

Scientific EffectEnzymatic conversion: Enzyme

Implementation Method 2

the biochemical conversion of β-Chloro D-alanine to hydrochloric acid or D-cysteine to hydrogen sulfide, respectively, when the substrates are provided to the enzyme

Methodology Applied

Scientific EffectEnzymatic conversion: Enzyme

Implementation Method 3

using genetically encoded biosensors for real-time detection

Methodology Applied

Scientific EffectBiosensing:

Data Source

PatentUS20260023087A1A Novel Chemogenetic Tool Permitting Modulation of Intracellular pH

Publication Date: 2026.01.22 T C ISTANBUL MEDIPOL UNIVERSITESI

AI summary

The invention relates to a new chemogenetic tool manipulating intracellular pH levels and hydrogen sulfide levels in living cells and tissues with high spatio-temporal resolution.