DHFR Biosensor NADPH Detection via RET Domain Insertion

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Solution Overview

Problem

There is no suitable binding protein (BP) identified for the development of a biosensor to detect reduced nicotinamide adenine dinucleotide phosphate (NADPH), a crucial cofactor involved in various cellular processes, limiting the ability to quantify NADPH levels effectively in clinical samples.

Innovation Solution

A resonance energy transfer (RET)-based sensor is developed using dihydrofolate reductase (DHFR) with a heterologous protein domain inserted at specific regions, enhancing NADPH-dependent affinity, allowing for the detection of NADPH through fluorescence or bioluminescence resonance energy transfer (FRET or BRET) mechanisms.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If dihydrofolate reductase (DHFR) is used as the binding protein for NADPH detection, then the sensor can detect NADPH through RET mechanisms, but the binding affinity and specificity are insufficient without modification

Engineering Contradiction:

ImproveNADPH detection reliabilityVSAvoidNADPH binding affinity

Core Design Contradiction:

ReliabilityVSMeasurement precision

Solution Approach 1:

The patent applies parameter changes by modifying the DHFR protein structure through insertion of heterologous protein domains at specific positions (particularly positions 20-27). This structural modification changes the binding parameters of DHFR for NADPH, enhancing both affinity and specificity. The insertion of domains such as T4 lysozyme or other heterologous proteins alters the conformational dynamics of DHFR, creating a more effective NADPH-binding interface while maintaining the essential catalytic function.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent employs composite materials by creating chimeric proteins that combine DHFR with heterologous protein domains. These composite proteins integrate the NADPH-binding capability of DHFR with the structural stability and conformational properties of the inserted domains. The resulting hybrid protein structure exhibits enhanced NADPH-binding characteristics that neither component possesses alone, thereby improving sensor reliability and measurement precision simultaneously.

Inventive Principle:

Principle #40Composite materials

2Measurement precision

If a heterologous protein domain is inserted into DHFR to enhance NADPH affinity, then binding specificity improves, but protein structure complexity increases

Engineering Contradiction:

ImproveNADPH binding specificityVSAvoidprotein structure complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The patent applies segmentation by dividing the enhanced DHFR protein into distinct functional modules: the original DHFR core domain responsible for catalytic activity, and inserted heterologous domains positioned at specific regions (N-terminal, C-terminal, or internal positions 20-27). This modular segmentation allows each domain to perform its specialized function while maintaining overall protein stability. The segmented structure enables independent optimization of binding specificity without compromising the simplicity of the overall design.

Inventive Principle:

Principle #1Segmentation

3Measurement precision

If DHFR is modified with inserted domains to improve NADPH-dependent affinity, then sensor sensitivity increases, but ease of manufacture decreases

Engineering Contradiction:

Improvesensor sensitivityVSAvoidprotein production simplicity

Core Design Contradiction:

Measurement precisionVSEase of manufacture

Solution Approach 1:

The patent applies universality by designing the modified DHFR protein to maintain its essential catalytic function while adding enhanced NADPH-binding capability. The heterologous domains are selected and positioned to be compatible with standard protein expression systems, allowing the modified protein to be produced using conventional recombinant DNA technology. This multi-functional design ensures that the protein can be manufactured with relative ease while achieving high sensor sensitivity through improved NADPH affinity and specificity.

Inventive Principle:

Principle #6Universality (Multi-functionality)

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

The sensor achieves sensitive and selective detection of NADPH, enabling quantitative analysis in complex bodily fluids, such as serum and blood, with improved affinity and specificity, suitable for point-of-care testing and enzymatic assays.

Implementation Method 1

Methodology Applied

Scientific EffectResonance energy transfer (RET):

Implementation Method 2

allowing for the detection of NADPH through fluorescence or bioluminescence resonance energy transfer (FRET or BRET) mechanisms

Methodology Applied

Scientific EffectFluorescence resonance energy transfer (FRET):

Implementation Method 3

Bioluminescence Resonance Energy Transfer (BRET)

Methodology Applied

Scientific EffectBioluminescence: Bioluminescence

Data Source

PatentUS11959121B2Sensors, methods and kits for detecting NADPH based on resonance energy transfer

Publication Date: 2024.04.16 MAX PLANCK GESELLSCHAFT ZUR FOERDERUNG DER WISSENSCHAFTEN EV

AI summary

The invention relates to the detection of the cofactor reduced nicotinamide adenine dinucleotide phosphate (NADPH). Provided is a sensor molecule for the resonance energy transfer (RET)-based detection of NADPH, the sensor comprising a segment A connected via a linker to a segment B, wherein each of segment A and segment B comprises a member of a RET pair comprising a donor moiety and an acceptor moiety, further characterized in that (i) segment A comprises a binding protein (BP) for NADPH, the BP being dihydrofolate reductase (DHFR; EC 1.5.1.3) or a functional homolog, fragment, derivative or variant thereof, showing the desired NADPH binding properties, and wherein the BP comprises a heterologous protein domain inserted at or replacing at least part of the region corresponding to positions (20) to (27) of E. coli DHFR, said heterologous protein domain comprising the member of the RET pair; (ii) segment B comprises a ligand (L) capable of intramolecular binding to said BP only in the presence of NADPH; such that the donor moiety and the acceptor moiety are in a suitable juxtaposition to yield a RET signal when L is bound to BP, and wherein NADPH-induced binding of L to BP results in an increase in RET efficiency.