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Kinetic Screening of Nuclease Activity using Nucleic Acid Probes
Published on: November 1, 2019
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A Novel Fluorescent Probe-Based High-Throughput Screening Approach for Nicotinamide Cofactor Biomimetics-Dependent
Huiru Wang1, Jieyu Zhou1, Xiangyuan Gu1
1Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China.
Analytical Chemistry
|November 17, 2025
Summary
A new fluorescent probe enables high-throughput screening of nicotinamide cofactor biomimetics (NCBs)-dependent oxidoreductases. This sensitive assay enhances enzyme engineering, leading to improved catalytic efficiency in enzymes like aldehyde dehydrogenase.
Area of Science:
- Biochemistry
- Enzyme Engineering
- Analytical Chemistry
Background:
- Nicotinamide cofactor biomimetics (NCBs) are crucial in redox reactions.
- Existing assays for NCB-dependent oxidoreductases often lack sensitivity.
- Development of sensitive, high-throughput screening methods is needed for enzyme discovery and engineering.
Purpose of the Study:
- To develop a high-throughput screening platform for NCB-dependent oxidoreductases.
- To adapt a fluorescent probe for real-time monitoring of enzymatic reactions involving NCBs.
- To engineer an aldehyde dehydrogenase for enhanced activity with NCB analogs.
Main Methods:
- Adaptation of a fluorescent probe originally designed for NAD(P)H detection.
- Real-time monitoring of enzymatic activity using fluorescence.
- Engineering of *Sp*ALDH2 using the developed fluorescent probe-based screening assay.
- Validation of results using High-Performance Liquid Chromatography (HPLC).
Main Results:
- A sensitive fluorescent probe-based assay with a >10-fold higher molar extinction coefficient than UV-based assays was developed.
- The assay enabled real-time monitoring of reactions involving NCBs like 1-benzyl-3-carbamoylpyridin-1-ium (BNA+).
- Engineered *Sp*ALDH2 exhibited a 17.6-fold increase in catalytic efficiency with BNA+ analogs.
- HPLC validation confirmed a strong correlation between fluorescence intensity and product yield.
Conclusions:
- The developed fluorescent probe-based assay is a versatile and reliable platform for NCB-related research.
- The assay facilitates enzyme screening, kinetic analysis, and enzyme engineering.
- This method significantly improves upon conventional UV-based activity assays for detecting NCB-dependent oxidoreductases.
