Sepiapterin Reductase Sensor for NAD Detection

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Solution Overview

Problem

Current methods for measuring nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) concentrations and ratios in biological samples are not sensitive enough and are prone to interference, especially in complex samples like lysates or serum, and cannot accurately measure enzyme activity in clinical tests.

Innovation Solution

Development of a sensor molecule based on sepiapterin reductase (SPR) with a ligand capable of intramolecular binding, which changes spectroscopic properties upon binding to the oxidized form of NAD or NADP, allowing for fluorescence or luminescence-based detection of these cofactors and their ratios through resonance energy transfer (RET) or bioluminescence resonance energy transfer (BRET).

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If absorbance-based methods are used to measure NAD(P) concentrations, then the measurement can be performed directly, but the sensitivity is low and interference from other molecules occurs

Engineering Contradiction:

Improvemeasurement accuracyVSAvoidinterference from other molecules

Core Design Contradiction:

Measurement precisionVSObject-affected harmful factors

Solution Approach 1:

The patent introduces an enzyme (alcohol dehydrogenase or glycerol kinase) as an intermediary that specifically catalyzes reactions producing NADH or NADPH from NAD+. This enzyme mediator enables indirect measurement with high specificity, eliminating interference from other light-absorbing molecules while maintaining direct measurement capability

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent replaces the direct absorbance measurement mechanism with an enzyme-catalyzed reaction mechanism followed by absorbance measurement. Instead of directly measuring NAD(P)+ absorbance, the system uses enzymatic conversion to NADH/NADPH, which has stronger and more specific absorbance characteristics at 340 nm

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Measurement precision

If HPLC separation followed by mass spectrometry is used, then specificity is improved, but the device complexity and measurement time increase

Engineering Contradiction:

ImprovespecificityVSAvoidcomplexity of separation and detection system

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The patent extracts only the essential measurement function by using enzyme-cycling assays that specifically amplify NAD(P)+ signals through controlled biochemical reactions. This eliminates the need for complex HPLC separation and mass spectrometry equipment while maintaining high specificity through enzyme substrate specificity

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent changes the measurement parameter from direct NAD(P)+ absorbance to NADH/NADPH absorbance after enzymatic conversion. This parameter transformation enables highly specific detection using simple spectrophotometry, avoiding complex separation and mass spectrometry systems

Inventive Principle:

Principle #35Parameter changes

3Ease of manufacture

If enzyme cycling assays are used, then the measurement can be performed without complex equipment, but the measurement is not direct and is prone to interference

Engineering Contradiction:

Improvesimplicity of assay systemVSAvoidmeasurement accuracy

Core Design Contradiction:

Ease of manufactureVSMeasurement precision

Solution Approach 1:

The patent implements a cycling assay mechanism where the product of the first enzyme reaction becomes the substrate for the second reaction, creating a feedback loop that amplifies the signal. The NADH produced by alcohol dehydrogenase is regenerated back to NAD+ by glyceraldehyde-3-phosphate dehydrogenase, enabling continuous cycling and signal amplification for highly sensitive detection

Inventive Principle:

Principle #23Feedback

Solution Approach 2:

The patent replaces direct measurement mechanisms with enzyme-catalyzed conversion mechanisms. By substituting direct NAD(P)+ detection with indirect detection through NADH production via specific enzyme reactions, the system achieves both simplicity and high measurement accuracy

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

4Productivity

If existing fluorescent sensors based on Rossman domain proteins are used, then real-time detection is possible, but they cannot measure NADP(H) or NAD(P)+ and only measure total concentrations

Engineering Contradiction:

Improvereal-time detection capabilityVSAvoidability to measure different cofactor forms

Core Design Contradiction:

ProductivityVSAdaptability or versatility

Solution Approach 1:

The patent creates a universal sensing platform using alcohol dehydrogenase and glycerol kinase that can measure all four cofactor forms (NAD+, NADH, NADP+, NADPH) through the same basic enzymatic cycling mechanism. By selecting different substrate pairs, the system can specifically target any cofactor form, providing versatile real-time detection capability

Inventive Principle:

Principle #6Universality (Multi-functionality)

Solution Approach 2:

The patent changes the detection parameter from total NAD(P) concentration to specific cofactor form concentrations by controlling the substrate and product ratios in the enzymatic cycling reactions. This enables selective measurement of oxidized vs. reduced forms and allows calculation of oxidation-reduction ratios in real-time

Inventive Principle:

Principle #35Parameter changes

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 provides a highly sensitive, specific, and pH-independent method for measuring NAD+ and NADP+ concentrations and their ratios in complex samples, suitable for enzymatic assays and clinical tests, with improved kinetics for high-throughput screening and live cell measurements.

Implementation Method 1

allowing for fluorescence or luminescence-based detection of these cofactors and their ratios through resonance energy transfer (RET) or bioluminescence resonance energy transfer (BRET)

Methodology Applied

Scientific EffectResonance energy transfer (RET):

Implementation Method 2

allowing for fluorescence or luminescence-based detection of these cofactors and their ratios through resonance energy transfer (RET) or bioluminescence resonance energy transfer (BRET)

Methodology Applied

Scientific EffectBioluminescence: Bioluminescence

Implementation Method 3

changes spectroscopic properties upon binding to the oxidized form of NAD or NADP, allowing for fluorescence or luminescence-based detection

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Data Source

PatentEP3259365B1Sensors, methods and kits for detecting nicotinamide adenine dinucleotides

Publication Date: 2020.04.01 MAX PLANCK GESELLSCHAFT ZUR FOERDERUNG DER WISSENSCHAFTEN EV

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AI summary

The invention relates to the in vitro and in cellulo detection of the cofactors nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). Provided is a sensor molecule for fluorescence- or luminescence-based detection of a nicotinamide adenine dinucleotide analyte, in particular for detecting the concentrations of NAD+, NADP+ and/or the ratios of the concentrations of NAD+/NADH and NADP+/NADPH, the sensor comprising (i) a binding protein (BP) for the nicotinamide adenine dinucleotide analyte, the BP being derived from sepiapterin reductase (SPR; EC 1.1.1.153) (ii) an SPR ligand (SPR-L) capable of intramolecular binding to said BP in the presence of the oxidized form of said analyte; and (iii) at least one fluorophore.