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Related Concept Videos

Restriction Enzymes01:11

Restriction Enzymes

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Restriction enzymes are bacterial enzymes used to cut DNA in a sequence-specific manner. To cleave DNA, they bind to specific palindromic sequences called restriction sites. Such palindromic DNA sequences or inverted repeats are commonly found in regions of functional significance, such as the origin of replication, gene operator sites, and regions containing transcription termination signals.
The host bacteria protect their own genomic DNA from these enzymes by methylating these sites. Some...
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Related Experiment Video

Updated: Mar 18, 2026

Author Spotlight: Characterizing Novel Enzymes from Extremophiles and Common Pathogens to Understand DNA Repair and Replication
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Ultrasensitive quantification and comprehensive evaluation of nicking endonuclease efficiency.

Yi Zhang1, Chieko Ishiwata2, Noriko Kudo3

  • 1SUGAR Program, X-star, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, 237-0061, Japan.

Analytical Biochemistry
|March 16, 2026
PubMed
Summary

Nicking endonucleases (NEs) are crucial biotechnological tools. A new single-molecule counting method reveals NEs achieve high nicking efficiencies, outperforming restriction enzymes and offering insights for enzyme development.

Keywords:
Cas9 nickaseDigital protein synthesisDropletNicking efficiencyNicking endonucleaseT5 exonuclease

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A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases
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Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Enzymology

Background:

  • Sequence-specific nicking endonucleases (NEs) are vital for biotechnology, cleaving only one DNA strand.
  • Fundamental understanding of NE reaction efficiencies is limited by assay method sensitivity.

Purpose of the Study:

  • To develop an ultrasensitive method for quantifying nicking endonuclease efficiencies.
  • To compare the efficiencies of various NEs, including canonical and programmable types.

Main Methods:

  • Utilized a single-molecule counting approach for ultrasensitive quantification.
  • Employed sequence-optimized DNA substrates for parallel measurements.
  • Enabled digital quantification of nicking efficiency via fluorescent protein synthesis in droplet reactors.

Main Results:

  • Established a novel, ultrasensitive assay for NE nicking efficiency.
  • Demonstrated NEs achieve a median nicking efficiency of 99.6%.
  • Showed NEs generally outperform Type II restriction enzymes in efficiency and consistency.

Conclusions:

  • The developed method provides a comprehensive view of NE reaction efficiencies.
  • NEs exhibit superior performance compared to Type II restriction enzymes.
  • This study offers tools and insights for advancing enzyme-based biotechnology and biological research involving NEs.