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Related Experiment Videos

DOTAP cationic liposomes prefer relaxed over supercoiled plasmids.

S Even-Chen1, Y Barenholz

  • 1Laboratory of Membrane and Liposome Research, Department of Biochemistry, The Hebrew University - Hadassah Medical School, P.O. Box 12272, 91120, Jerusalem, Israel.

Biochimica Et Biophysica Acta
|December 19, 2000
PubMed
Summary

Cationic liposomes bind DNA to form lipoplexes, with SYBR Green I and SYBR Gold dyes offering superior sensitivity for quantifying DNA binding. Helper lipids influence binding, and DOTAP liposomes preferentially bind relaxed DNA over supercoiled DNA.

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Area of Science:

  • Biochemistry
  • Materials Science
  • Molecular Biology

Background:

  • Cationic liposomes and DNA form complexes called lipoplexes through electrostatic interactions.
  • DNA binding isotherms quantify the efficiency of DNA binding to cationic lipids under various conditions.
  • Accurate quantification of DNA forms and binding ratios is crucial for understanding lipoplex formation.

Purpose of the Study:

  • To develop a sensitive assay for quantifying DNA binding to cationic liposomes.
  • To compare the sensitivity of SYBR Green I and SYBR Gold dyes with ethidium bromide for DNA detection.
  • To investigate the influence of lipid composition and DNA form on lipoplex formation.

Main Methods:

  • Development of a quantitative assay using agarose gel electrophoresis and fluorescent cyanine DNA dyes (SYBR Green I, SYBR Gold).

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  • Comparison of the developed assay with ethidium bromide staining.
  • Analysis of DNA binding isotherms at various cationic lipid:DNA molar ratios and medium conditions.

    • Main Results:

    • SYBR Green I and SYBR Gold demonstrate significantly higher sensitivity than ethidium bromide for detecting double-stranded and single-stranded DNA in solution and in gels.
    • No significant difference in DNA binding was observed between monocationic DOTAP and polycationic DOSPA liposomes.
    • Helper lipids, such as DOPE and cholesterol, affect DNA binding efficiency, with DOTAP/DOPE showing higher binding than DOTAP/cholesterol.
    • Cationic liposomes exhibit a preference for binding nicked-relaxed DNA over supercoiled DNA, particularly DOTAP formulations.

    Conclusions:

    • SYBR Green I and SYBR Gold are superior fluorescent dyes for quantifying DNA binding to cationic liposomes.
    • Lipid composition, specifically helper lipids, plays a role in the DNA binding capacity of liposomes.
    • The preference for binding relaxed DNA suggests differential binding affinities and potential implications for lipoplex stability and DNA release.