Genetically Encoded D-2-HG Fluorescent Indicator for Real-Time Detection

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Current methods for detecting D-2-hydroxyglutarate (D-2-HG), an oncometabolite associated with cancer, are complex, lack sensitivity, and cannot provide real-time quantification or distinguish between D-2-HG and its enantiomer L-2-HG, hindering timely identification of IDH mutations and monitoring of inhibitor therapy effectiveness.

Innovation Solution

Development of a genetically encoded fluorescent sensor, D2HGlo, using a truncated DhdR transcription factor and two fluorescent proteins (ECFP and cpVenus 173) for specific detection and quantification of D-2-HG in biological fluids and living cells, capable of localizing to subcellular compartments.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If liquid chromatography-mass spectroscopy (LC-MS) or gas chromatography-mass spectroscopy (GC-MS) is used to detect D-2-HG, then detection sensitivity is improved, but device complexity and analytical time increase

Engineering Contradiction:

Improvedetection sensitivityVSAvoidtechnical complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The patent replaces complex mechanical chromatography systems with a genetically encoded fluorescent sensor that uses biological components (transcription factor DhdR fused to fluorescent proteins) to detect D-2-HG through fluorescence emission, eliminating the need for LC-MS or GC-MS instrumentation

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The fluorescent sensor performs self-contained detection within living cells or biological samples, where the sensor protein binds D-2-HG and autonomously produces a fluorescent signal without requiring external analytical equipment or complex sample processing

Inventive Principle:

Principle #25Self-service

2Ease of operation

If magnetic resonance spectroscopy (MRS) is used to detect D-2-HG, then non-invasive detection is achieved, but real-time quantification capability is lost

Engineering Contradiction:

Improvenon-invasive detectionVSAvoidreal-time quantification

Core Design Contradiction:

Ease of operationVSLoss of time

Solution Approach 1:

The fluorescent sensor enables continuous real-time monitoring of D-2-HG levels in living cells through sustained fluorescence emission that can be measured immediately upon D-2-HG binding, providing continuous quantification data without the time delays inherent in MRS or other bulk analysis methods

Inventive Principle:

Principle #20Continuity of useful action

3Measurement precision

If existing diagnostic methods such as sequencing or immunohistochemistry are used to identify IDH mutations, then diagnostic accuracy is improved, but analytical time increases to days or weeks

Engineering Contradiction:

Improvediagnostic accuracyVSAvoidanalytical time

Core Design Contradiction:

Measurement precisionVSLoss of time

Solution Approach 1:

The patent detects D-2-HG, the functional metabolite produced by IDH mutations, rather than requiring direct genetic sequencing or protein detection. This preliminary detection of the metabolite's functional effect provides rapid diagnostic information about IDH mutational status without waiting for lengthy sequencing or IHC procedures

Inventive Principle:

Principle #10Preliminary action

4Adaptability or versatility

If D-2-HG detection is performed in complex biological fluids, then clinical relevance is improved, but measurement precision decreases due to interference

Engineering Contradiction:

Improveclinical applicabilityVSAvoiddetection accuracy

Core Design Contradiction:

Adaptability or versatilityVSMeasurement precision

Solution Approach 1:

The patent employs a transcription factor-based sensor with a specific binding pocket that creates a localized high-affinity interaction site for D-2-HG, allowing the sensor to selectively detect D-2-HG even in the presence of complex biological fluids containing other metabolites and molecules that could potentially interfere

Inventive Principle:

Principle #3Local quality

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

D2HGlo provides rapid, accurate, and enantiomer-specific detection of D-2-HG, predicting IDH mutational status with 100% accuracy, and visualizing its intracellular distribution, aiding in monitoring cancer progression and treatment efficacy.

Implementation Method 1

a D-2-HG-sensing domain (derived from the bacterial transcription factor DhdR)

Methodology Applied

Scientific EffectMolecular recognition:

Implementation Method 2

two fluorescent proteins (ECFP and cpVenus 173) for specific detection and quantification of D-2-HG

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Data Source

PatentUS20250314656A1Genetically encoded fluorescent indicator of d-2-hydroxyglutarate

Publication Date: 2025.10.09 PRATT EVAN P S

AI summary

Constructs for detection of d-2-hydroxyglutarate (d-2-HG), and their use in determining the IDH1/2 mutational status of a biological sample obtained from a subject, monitoring a change in D-2-HG levels in a subject, and analysing D-2-HG in a biological sample obtained from a subject, comprising detecting for D-2-HG in the sample or subcellular compartment therein, and methods for the same. Nucleic acid molecules, vectors, cells, and pharmaceutical compositions are also described.