Nanopore Analysis as a Tool for Studying Rapid Holliday Junction Dynamics and Analyte Binding

Area of Science:

• Molecular Biology
• Biophysics
• Nanotechnology

Background:

• Holliday junctions (HJs) are critical nucleic acid structures in DNA repair and disease.
• Studying HJ kinetics is vital for therapeutic development but challenging due to rapid timescales.
• Existing methods like smFRET often require modifications (e.g., divalent ions) that alter natural kinetics.

Purpose of the Study:

• To develop a method for studying Holliday junction (HJ) structure fluctuations without divalent ions.
• To characterize the rapid kinetics of HJ conformational changes.
• To create a nanopore-based sensor for detecting DNA strands interacting with HJs.

Main Methods:

• Utilized nanopore sensing in a gating configuration to monitor DNA structure fluctuations.
• Analyzed DNA structure dynamics without the use of divalent ions like Mg2+.
• Developed a selective sensor for short oligonucleotide DNA strands binding to HJ targets.

Main Results:

• Observed Holliday junction (HJ) fluctuations occurring on the time scales of 0.1-10 milliseconds.
• Determined that HJs primarily exist in one conformation with transient shifts to a second.
• Demonstrated nanopore sensing's capability for rapid and selective detection of specific short DNA strands.
• Identified rapid HJ transitions potentially missed by lower-bandwidth techniques.

Conclusions:

• Nanopore sensing offers a novel, ion-free approach to study rapid Holliday junction (HJ) dynamics.
• The observed kinetics suggest HJs undergo faster conformational changes than previously measurable.
• This technique advances our understanding of DNA repair mechanisms and disease-related structures.
• Nanopore sensing provides a versatile platform for developing sensitive diagnostic tools for DNA-binding molecules.