Enzyme-linked immunosorbent assays for Z-DNA

Area of Science:

• Molecular Biology
• Biochemistry
• Immunology

Background:

• Z-DNA is a left-handed helical conformer of DNA that plays roles in gene regulation and genome stability.
• Sensitive and specific detection methods are crucial for studying Z-DNA's biological functions.
• Current methods for Z-DNA detection often involve radioactivity or lack sufficient sensitivity and specificity.

Purpose of the Study:

• To develop and validate sensitive, non-radioactive enzyme-linked immunosorbent assays (e.l.i.s.a.) for Z-DNA detection.
• To establish methods for screening Z-DNA-specific antibodies.
• To quantify Z-DNA in various DNA contexts, including synthetic polynucleotides and supercoiled plasmids.

Main Methods:

• Dot blot and transblot e.l.i.s.a. formats were developed using alkaline phosphatase and horseradish peroxidase enzyme conjugates.
• Z-DNA and B-DNA forms were immobilized on nitrocellulose or diazophenylthioether (DPT) paper.
• Detection involved specific antibodies against Z-DNA, followed by enzyme-conjugated secondary antibodies and appropriate substrates.

Main Results:

• Alkaline phosphatase dot blot e.l.i.s.a. allowed quantification of Z-DNA [brominated poly(dG-dC).poly(dG-dC)] in the range of 5-250 ng.
• Horseradish peroxidase transblot e.l.i.s.a. detected Z-DNA within supercoiled plasmid DNAs immobilized on DPT paper.
• Transblot e.l.i.s.a. demonstrated specificity for Z-DNA containing plasmids after absorption of cross-reactive antibodies, detecting as little as 16 base pairs of alternating G-C residues.

Conclusions:

• Dot blot and transblot e.l.i.s.a. provide sensitive, non-radioactive alternatives for Z-DNA detection and antibody screening.
• These assays are effective for analyzing Z-DNA in both free polynucleotides and supercoiled plasmid DNA.
• The developed methods enhance the ability to study Z-DNA's biological significance in various DNA structures.