Triple helix formation on plasmid DNA determined by a size-exclusion chromatographic method

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Triple-helix-forming oligodeoxynucleotides show promise for in vivo gene inhibition.
• Development is hindered by poor triple helix formation under physiological conditions.
• Accurate assays are needed to quantify oligodeoxynucleotide binding to target sequences.

Purpose of the Study:

• To develop a novel, rapid method for detecting and quantifying triple helix formation.
• To enable the study of oligodeoxynucleotide binding under various experimental conditions.
• To facilitate the selection of high-affinity oligodeoxynucleotides for gene targeting.

Main Methods:

• Radiolabeling of oligodeoxynucleotides.
• Incubation with target plasmid DNA.
• Separation of unbound from bound oligodeoxynucleotides using rapid gel filtration spun columns.

Main Results:

• Demonstrated triple helix formation between plasmids and various oligodeoxynucleotides.
• Analyzed temperature, sequence, and ionic dependencies.
• Characterized kinetics of association for different oligodeoxynucleotide-target pairs.

Conclusions:

• The novel method allows rapid detection and quantification of triple helix formation.
• It is adaptable to various experimental conditions, including physiological ones.
• This technique facilitates the identification of optimal oligodeoxynucleotides for specific gene inhibition.