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Development of a His-Tag-mediated pull-down and quantification assay for G-quadruplex containing DNA sequences
Enrico Cadoni1, Hanne Moerman1, Annemieke Madder1
1Organic and Biomimetic Chemistry Research Group, Department of Organic and Macromolecular Chemistry, Ghent University Krijgslaan 281-S4 9000 Gent Belgium Annemieke.madder@ugent.be Enrico.cadoni@ugent.be.
RSC Chemical Biology
|December 5, 2024
Summary
We developed a simple pull-down assay using peptide nucleic acids (PNAs) with His-Tag and G-quadruplex (G4) ligand for DNA sequence detection. This method enhances recovery and selectivity for G4-forming DNA, offering a novel quantification strategy.
Area of Science:
- Biochemistry
- Molecular Biology
- Analytical Chemistry
Background:
- G-quadruplex (G4) DNA structures are increasingly recognized for their roles in various biological processes.
- Accurate detection and quantification of G4 DNA are crucial for understanding their functions.
- Existing methods for G4 DNA analysis often face challenges in specificity and efficiency.
Purpose of the Study:
- To develop a novel, simple, and highly selective pull-down assay for G-quadruplex (G4) DNA sequences.
- To enable efficient recovery and accurate quantification of G4-forming DNA.
- To validate the assay's performance and adaptability for high-throughput applications.
Main Methods:
- A pull-down assay was designed utilizing peptide nucleic acids (PNAs) functionalized with a His-Tag and a G-quadruplex (G4) ligand.
- Magnetic Ni-NTA beads were employed for efficient capture of His-tagged PNAs and bound G4 DNA.
- Quantification was achieved by leveraging the enzyme-like activity of the G4/hemin complex.
- High-Performance Liquid Chromatography (HPLC) was used for assay validation.
- The assay was adapted to a 96-well plate format.
Main Results:
- Optimized buffer conditions and magnetic Ni-NTA beads facilitated efficient and specific recovery of G4 DNA targets.
- The His-Tag and Ni-NTA magnetic bead system demonstrated superior recovery compared to biotin-streptavidin purification.
- Incorporating a G4-ligand as an additional selectivity handle significantly improved both target recovery and assay specificity.
- The G4/hemin complex's catalytic activity provided a reliable method for G4 DNA quantification.
- The assay was successfully validated and adapted for a 96-well plate format, indicating its potential for high-throughput screening.
Conclusions:
- The developed PNA-based pull-down assay offers a simple, efficient, and selective method for G4 DNA recognition and quantification.
- The combination of His-Tag/Ni-NTA beads and a G4-ligand enhances recovery and specificity, outperforming conventional purification methods.
- The assay's adaptability to a 96-well format makes it a valuable tool for G4 DNA analysis in various research and diagnostic applications.
