Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

CRISPR/Cas12a-Enhanced Cascade Amplification for Ultra-sensitive DNA Ligase Detection.

Analytical chemistry·2026
Same author

Quality changes in plum puree based on anti-Browning treatment: Nonvolatile and volatile compounds analysis.

Food chemistry·2026
Same author

Transcription factor ID3 promotes fibroblast differentiation and proliferation in lung fibrosis through augmenting the TGF-β signaling pathway.

Molecular immunology·2026
Same author

Ultrasound-guided dynamic needle tip positioning versus conventional palpation for improving the success rate of radial artery catheterization: A randomized controlled trial.

The journal of vascular access·2026
Same author

A Glycyrrhiza Glabra-derived pH/ROS Dual-Responsive Phytomedicine Nanoplatform for Combatting Triple-Negative Breast Cancer via Amplified Mitochondrial Damage.

Advanced healthcare materials·2026
Same author

Fabrication and characterization of bio-polymer-based flavonoid nanoemulsion/TiO<sub>2</sub> nanoparticles bilayer composite films and its application in mutton preservation.

International journal of biological macromolecules·2026

Related Experiment Video

Updated: Jan 11, 2026

Author Spotlight: Characterizing Novel Enzymes from Extremophiles and Common Pathogens to Understand DNA Repair and Replication
05:33

Author Spotlight: Characterizing Novel Enzymes from Extremophiles and Common Pathogens to Understand DNA Repair and Replication

Published on: July 5, 2024

1.3K

Double-stranded endonuclease activity exploration through scanning substrate assay and its biosensing application.

Zhen Zhang1, Guowen Li2, Ruiming Luo2

  • 1The First Affiliated Hospital, and College of Clinical Medicine of Henan University of Science and Technology, Luoyang, 471003, China.

Talanta
|November 12, 2025
PubMed
Summary

This study reveals that optimizing DNA substrates enhances double-stranded endonuclease (DEN) activity, improving biosensing applications. Researchers found non-canonical substrates significantly boost DEN catalytic efficiency for enzymes like APE1.

Keywords:
Apurinic/apyrimidinic endonuclease 1Cell lysateDouble-stranded endonucleaseEndonuclease activityFluorescent detection

More Related Videos

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes
06:52

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Published on: November 1, 2019

8.6K
A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases
06:10

A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases

Published on: December 23, 2013

5.6K

Related Experiment Videos

Last Updated: Jan 11, 2026

Author Spotlight: Characterizing Novel Enzymes from Extremophiles and Common Pathogens to Understand DNA Repair and Replication
05:33

Author Spotlight: Characterizing Novel Enzymes from Extremophiles and Common Pathogens to Understand DNA Repair and Replication

Published on: July 5, 2024

1.3K
Kinetic Screening of Nuclease Activity using Nucleic Acid Probes
06:52

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Published on: November 1, 2019

8.6K
A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases
06:10

A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases

Published on: December 23, 2013

5.6K

Area of Science:

  • Biochemistry and Molecular Biology
  • Enzymology
  • Biosensing Technologies

Background:

  • Double-stranded endonucleases (DENs) are crucial tools in molecular biology, with their activity traditionally linked to well-defined substrates.
  • The precise regulation of DEN catalytic activity by substrate sequence, particularly near the cleavage site, remains an area for exploration.

Purpose of the Study:

  • To investigate the impact of substrate sequence, specifically the number of complementary base pairs near the cleavage site, on DEN catalytic activity.
  • To optimize substrates for enhanced DEN activity and explore the potential for widespread application of this strategy in biosensing.

Main Methods:

  • Enzyme kinetics assays using fluorescence measurements to quantify cleavage rates of APE1 with various substrates.
  • Systematic scanning of substrate sequences to identify optimal base pair configurations near the AP site.
  • Application of the optimized substrate strategy to five other representative endonucleases (Nt.BsmAI, Nt. BstNBI, Nt. BbvCI, BsmAI, and Lba Cas12a).
  • Evaluation of APE1 detection performance in buffer and cell lysate samples using optimized substrates for biosensing.

Main Results:

  • An approximately tenfold increase in APE1 cleavage activity was achieved with optimized substrates compared to canonical ones.
  • Catalytic activity of APE1 was found to be highly sensitive to the number of base pairs downstream of the AP site.
  • Five other tested endonucleases also exhibited enhanced activity with non-canonical substrates, suggesting broad applicability.
  • APE1 detection sensitivity was improved fivefold, reaching 0.01 U/mL, enabling differentiation of APE1 levels in normal and cancer cell lysates.

Conclusions:

  • Substrate design is a critical factor in modulating DEN catalytic activity, challenging the traditional view of 'well-defined' substrates.
  • The developed strategy of optimizing substrates offers a powerful approach to enhance DEN efficiency for various enzymes.
  • Optimized substrates significantly improve the sensitivity and applicability of DENs in biosensing, with potential for distinguishing biological states like cancer.