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Updated: Jan 12, 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

Simple in vitro single-stranded linear and circular DNA preparation, functional selection, and validation using

Seyed Vahid Hamidi¹, Vanessa Aguilar-Sánchez², Vincent Héroux²

¹INRS Centre Armand-Frappier Santé Biotechnologie, Laval, Quebec, Canada; Department of Bioengineering, McGill University, Montreal, Quebec, Canada.

The Journal of Biological Chemistry

|October 31, 2025

Summary

This summary is machine-generated.

A new, user-friendly method simplifies circular single-stranded DNA (ssDNA) preparation. This technique enhances DNA library construction and aptamer selection, offering potential for high-throughput applications.

Keywords:

SELEX circular ssDNA lambda exonuclease phosphorothioate modification single-stranded PCR product

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Published on: July 3, 2016

Area of Science:

Molecular Biology
Biochemistry
Synthetic Biology

Background:

Circular single-stranded DNA (ssDNA) is crucial for various molecular biology techniques.
Existing methods for circular ssDNA preparation can be complex and inefficient.
There is a growing need for simple, efficient in vitro methods for generating circular ssDNA libraries.

Purpose of the Study:

To develop a novel, simplified in vitro method for preparing circular ssDNA.
To optimize the ligation reaction for efficient circularization of phosphorylated ssDNA.
To validate the method's performance using aptamer selection against a viral protein.

Main Methods:

Utilized a phosphorylated-phosphorothioated primer to synthesize phosphorylated ssDNA.
Employed a bridging oligonucleotide for ssDNA ligation.
Incorporated an extra thymine nucleotide at the ligation site to enhance efficiency.
Validated the method through SELEX (Systematic Evolution of Ligands by Exponential Enrichment) for aptamer selection.

Main Results:

Successfully generated circular ssDNA using the proposed phosphorylation and ligation strategy.
Optimized ligation conditions, including the addition of a thymine nucleotide, significantly improved circularization efficiency.
Demonstrated the method's efficacy by selecting functional linear and circular aptamers against the Middle East respiratory syndrome coronavirus spike protein.

Conclusions:

The developed method provides a simple, efficient, and user-friendly approach for in vitro circular ssDNA preparation.
This technique has the potential for automation and high-throughput applications in DNA library construction and aptamer development.
The method facilitates broader adoption and exploration of circular ssDNA applications in biotechnology and diagnostics.