Probing the recognition surface of a DNA triplex: binding studies with intercalator-neomycin conjugates

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• DNA triplexes like poly(dA).2poly(dT) are crucial in biological processes.
• Neomycin is an aminoglycoside antibiotic with DNA binding properties.
• Intercalator-neomycin conjugates combine DNA intercalation with neomycin's binding.

Purpose of the Study:

• To investigate the thermodynamic interactions of intercalator-neomycin conjugates with DNA triplexes.
• To synthesize and characterize novel naphthalene diimide-neomycin and anthraquinone-neomycin conjugates.
• To compare the DNA binding affinity and stabilization effects of these conjugates with neomycin.

Main Methods:

• UV denaturation (melting temperature analysis)
• Circular dichroism (CD) titration
• Differential scanning calorimetry (DSC)
• Isothermal titration calorimetry (ITC)
• Competition dialysis
• Fluorescent intercalator displacement assay

Main Results:

• All four intercalator-neomycin conjugates (1-4) significantly stabilized the DNA triplex poly(dA).2poly(dT), increasing melting temperature by over 12°C.
• Neomycin alone showed negligible stabilization under the same conditions.
• Binding affinity followed the order: neomycin < 1 < 3 < 4 < 2, with conjugate 2 exhibiting the highest binding constant (~2.7 x 10^8 M⁻¹).
• Binding was primarily enthalpy-driven with negative DeltaCp values.
• Binding site size for conjugates was slightly larger than for neomycin.

Conclusions:

• Intercalator-neomycin conjugates are potent stabilizers of DNA triplexes.
• The binding affinity is strongly influenced by the intercalator moiety's surface area.
• These conjugates represent promising agents for modulating DNA triplex stability.