DNA conformation and base number simultaneously determined in a nanopore.

Area of Science:

• Biophysics
• Nanotechnology
• Molecular Biology

Background:

• Electrophoresis through nanopores is a technique used to analyze DNA molecules.
• The event-charge-deficit (ecd) is a measure of the ionic current blocked during DNA translocation.
• Existing methods may struggle to differentiate DNA conformation and base number simultaneously.

Purpose of the Study:

• To investigate the relationship between DNA conformation, base number, and nanopore translocation measurements.
• To determine if nanopore analysis can simultaneously assess DNA conformation and base number.

Main Methods:

• Electrophoresis of double-stranded DNA (dsDNA) polymers of varying lengths and conformations (linear, circular relaxed, supercoiled) through a voltage-biased silicon nitride nanopore.
• Measurement of the time integral of blocked ionic current (event-charge-deficit, ecd) for each DNA translocation event.
• Analysis of current blockages to identify characteristic differences among various DNA forms.

Main Results:

• The ecd was identical for dsDNA polymers with the same number of base pairs, irrespective of their form (linear, circular relaxed, supercoiled).
• The ecd strongly depended on and accurately predicted the number of base pairs for DNA polymers with different lengths.
• Current blockages showed distinct patterns differentiating between single-stranded linear, double-stranded linear, circular relaxed, and supercoiled DNA forms.

Conclusions:

• A single nanopore assay can simultaneously determine both the DNA conformation and the number of base pairs.
• This finding offers a powerful, integrated approach for comprehensive DNA analysis using nanopore technology.