Equilibrium studies of ethidium--polynucleotide interactions

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Ethidium is a fluorescent intercalating agent used to study nucleic acid structure.
• Understanding drug-nucleic acid interactions is crucial for developing therapeutics and diagnostics.

Purpose of the Study:

• To investigate the binding of ethidium to various synthetic double-helical polynucleotides.
• To elucidate the influence of base composition and helix geometry on ethidium binding affinity and mechanism.

Main Methods:

• Equilibrium dialysis was employed to quantify ethidium binding.
• Analysis was performed using the neighbor exclusion model, considering cooperativity and structural transitions.

Main Results:

• High ethidium affinity (10^4–10^5 M⁻¹) was observed for DNA and alternating copolymers like poly[d(A-T)] and poly[d(I-C)] in 1 M salt.
• Homopolynucleotides exhibited varied affinities, with poly(rA).poly(rU), poly(rA).poly(dT), and poly(dA).poly(rU) showing high affinity, while others displayed low affinity (≤10³ M⁻¹).
• Neighbor exclusion ranged from two base pairs for DNA (B-family helices) to three for RNA (A-family helices). Poly[d(I-C)] showed a structural switch to a higher-affinity state.

Conclusions:

• Ethidium binding is sequence- and structure-dependent, influenced by base pairing and helix type (A vs. B family).
• Cooperativity and structural plasticity, exemplified by poly[d(I-C)], are key features of ethidium-nucleic acid interactions.
• The study provides insights into the biophysical principles governing intercalator binding to diverse nucleic acid structures.