A study on endonuclease BspD6I and its stimulus-responsive switching by modified oligonucleotides.

Area of Science:

• Molecular Biology
• Biochemistry
• Biotechnology

Background:

• Nicking endonucleases (NEases) are crucial tools in molecular biology, but their DNA interactions are not fully understood.
• The large subunit of BspD6I (Nt.BstD6I) functions as a NEase, with its specific DNA interactions requiring further investigation.

Purpose of the Study:

• To investigate the interaction between Nt.BstD6I and modified DNA containing azobenzene insertions.
• To design and develop stimulus-responsive oligonucleotide inhibitors for Nt.BstD6I activity regulation.

Main Methods:

• Modification of DNA with non-nucleotide insertions containing azobenzene moieties.
• Design of oligonucleotide inhibitors with azobenzene or triethylene glycol residues.
• Assessing the effect of modified oligonucleotides on Nt.BstD6I DNA cleavage activity under varying temperatures (20-25°C and 45°C).

Main Results:

• Modified DNA with azobenzene insertions influenced Nt.BstD6I interactions.
• Developed oligonucleotide inhibitors effectively modulated Nt.BstD6I activity.
• Inhibitors blocked T7 phage DNA cleavage at 20-25°C and activity was restored upon heating to 45°C.

Conclusions:

• Temperature-responsive regulation of Nt.BstD6I activity is achievable using designed oligonucleotide inhibitors.
• This study provides a foundation for developing platforms to control NEase activity with external signals in vitro and in vivo.