Quantitative analysis of monovalent counterion binding to random-sequence, double-stranded DNA using the replacement...

Area of Science:

• Biophysical Chemistry
• Molecular Biology
• Analytical Chemistry

Background:

• Understanding monovalent cation interactions with double-stranded DNA (dsDNA) is crucial for various biological processes.
• Previous methods for measuring cation-DNA binding have limitations.
• The existence of strongly correlated, tightly bound ions near dsDNA has been postulated.

Purpose of the Study:

• To develop and validate a novel method, the replacement ion (RI) method, for measuring monovalent cation binding to random-sequence dsDNA.
• To investigate the binding affinities of several monovalent cations (Tris+, NH4+, Li+, Na+, K+) to dsDNA.
• To assess the potential competition of Tris buffer ions with other monovalent cations for dsDNA binding.

Main Methods:

• Development of the replacement ion (RI) method, a variation of affinity capillary electrophoresis.
• Maintaining constant ionic strength by gradually replacing a non-binding ion with a binding ion.
• Measuring analyte mobility as a function of binding ion concentration to determine apparent dissociation constants.

Main Results:

• The RI method was validated by accurately measuring Li+ ion binding to adenosine nucleotides, yielding comparable results to literature values.
• Apparent dissociation constants for counterion binding to a 26-bp dsDNA oligomer ranged from 71 mM (Tris+) to 173 mM (Na+, K+).
• Positively charged Tris buffer ions were found to compete with other monovalent cations for binding to dsDNA.

Conclusions:

• The replacement ion method provides a reliable approach for quantifying monovalent cation binding to dsDNA.
• Tris buffer ions can significantly influence monovalent cation binding equilibria with dsDNA.
• The findings support the hypothesis of tightly bound ion classes near dsDNA surfaces and indicate that cation binding is distinct from site-specific interactions.