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Restriction Enzymes

Restriction Enzymes

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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Updated: Feb 19, 2026

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

Published on: March 25, 2020

Restriction endonuclease selective inhibition by β-cyclodextrin sulfate.

Bekir C Celikkaya¹, Fraser J Scott¹

¹Department of Pure and Applied Chemistry, Faculty of Science, University of Strathclyde, Thomas Graham Building, 295 Cathedral Street, G1 1XL Glasgow, U.K.

Bioscience Reports

|February 18, 2026

Summary

Beta-cyclodextrin sulfate (β-CDsul) selectively inhibits the EcoRI restriction enzyme, unlike non-selective inhibitors like EDTA. This polyanionic macrocycle offers potential as a biochemical probe for differential endonuclease activity.

Keywords:

DNA restriction endonucleases β-Cyclodextrin sulfate

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Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

Published on: February 7, 2021

Area of Science:

Molecular Biology
Biochemistry

Background:

Restriction endonucleases (REs) are crucial tools in molecular biology for DNA manipulation.
Understanding differential modulation of DNA-binding enzymes, like REs, is key.
β-cyclodextrin sulfate (β-CDsul) was previously identified as a potential non-selective RE inhibitor.

Purpose of the Study:

To investigate the inhibitory activity of β-cyclodextrin sulfate (β-CDsul) against specific restriction enzymes.
To determine if β-CDsul exhibits selective inhibition against different REs.
To explore the potential of polyanionic macrocycles as probes for endonuclease activity.

Main Methods:

Quantification of inhibition of EcoRI, HindIII, and VspI using NdeI-linearised pBR322 DNA.
Comparison of β-CDsul inhibition with EDTA, a known non-selective inhibitor.
Assessment of selective inhibition in a dual-enzyme system (EcoRI and VspI).

Main Results:

β-CDsul demonstrated significantly greater potency in inhibiting EcoRI compared to HindIII and VspI (13- to 27-fold difference in IC50).
EDTA inhibited all three enzymes with comparable potency, indicating non-selective metal ion chelation.
Selective inhibition of EcoRI by β-CDsul was confirmed in a dual-enzyme system, suggesting an enzyme-dependent mechanism.

Conclusions:

β-cyclodextrin sulfate acts as a selective inhibitor of the EcoRI restriction enzyme.
Polyanionic macrocycles show promise as biochemical tools for differentiating endonuclease activities.
This study provides a framework for understanding differential modulation of DNA-binding enzymes.