Quantitative detection of small molecule/DNA complexes employing a force-based and label-free DNA-microarray

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Force-based ligand detection offers label-free characterization of molecular complexes at physiological conditions.
• Conventional methods like atomic force microscopy and optical traps are low-throughput and require specialized equipment, limiting their application.
• There is a need for high-throughput, cost-effective techniques for studying DNA-ligand interactions.

Purpose of the Study:

• To present a novel, low-cost, chip-based assay for high-throughput, force-based detection of double-stranded DNA (dsDNA)-ligand interactions.
• To characterize the DNA binding behavior of artificial pyrrole-imidazole polyamides using this new assay.
• To determine dissociation constants (Kd) of dsDNA-ligand complexes and validate the assay's accuracy.

Main Methods:

• Development of a chip-based comparative unbinding force assay.
• Utilizing fluorescence detection to quantify the fractions of broken target and reference dsDNA duplexes.
• Probing interactions between dsDNA and programmed pyrrole-imidazole polyamides.

Main Results:

• The assay successfully detected dsDNA-ligand interactions with high throughput and ease of fluorescence detection.
• Titration with specific polyamides induced a shift in broken bond fractions, indicating specific binding.
• Dissociation constants in the nanomolar to picomolar range were determined, consistent with DNAase footprinting results.

Conclusions:

• The developed chip-based assay is a cost-effective and high-throughput method for force-based dsDNA-ligand interaction analysis.
• The assay accurately quantifies binding affinities, providing a snapshot of equilibrium distribution.
• This technique advances the study of molecular complexes under physiological conditions.