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Related Experiment Videos

Phosphorothioate substrates for the SfiI restriction endonuclease

T J Nobbs1, S A Williams, B A Connolly

  • 1Department of Biochemistry, University of Bristol, UK.

Biological Chemistry
|June 17, 1998
PubMed
Summary

Phosphorothioate modifications at the SfiI endonuclease cleavage site affect DNA recognition and cleavage. Rp diastereomers show limited cleavage, while Sp diastereomers are resistant, impacting SfiI activity on plasmids.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Enzymology

Background:

  • Oligodeoxynucleotides are crucial for studying DNA-protein interactions.
  • Phosphorothioate modifications alter DNA backbone properties and nuclease interactions.
  • SfiI endonuclease is a restriction enzyme with specific DNA recognition requirements.

Purpose of the Study:

  • To synthesize and characterize SfiI recognition sequence oligodeoxynucleotides with phosphorothioate modifications at the cleavage site.
  • To investigate the impact of Rp and Sp phosphorothioate diastereomers on SfiI endonuclease activity.
  • To evaluate the effect of these modified oligonucleotides on SfiI-mediated plasmid cleavage.

Main Methods:

  • Synthesis of oligodeoxynucleotides with phosphorothioate linkages at the SfiI cleavage site.

Related Experiment Videos

  • Separation of Rp and Sp diastereomers using High-Performance Liquid Chromatography (HPLC) with L-cysteine mobile phase.
  • Enzymatic cleavage assays using SfiI endonuclease with Mg2+ and Mn2+ cofactors, and assessment of plasmid cleavage inhibition.

    • Main Results:

    • Rp phosphorothioate duplexes exhibited slow cleavage with Mg2+ and efficient cleavage with Mn2+.
    • Sp phosphorothioate duplexes showed no significant cleavage with either Mg2+ or Mn2+.
    • Both Rp and Sp modified duplexes inhibited SfiI plasmid cleavage, unlike control duplexes which enhanced it.

    Conclusions:

    • Stereochemistry of phosphorothioate linkages at the SfiI cleavage site critically determines susceptibility to enzymatic cleavage.
    • SfiI endonuclease activity is sensitive to the stereochemical configuration of the phosphodiester bond.
    • Modified oligonucleotides can act as inhibitors of SfiI activity, highlighting the requirement for four intact phosphodiester bonds for SfiI function.