engleski

Revealing DNA interactions with Hoechst 33258 by surface-enhanced Raman spectroscopy

Hoechst 33258 (H33258) is a small DNA binding molecule, known as a fluorescent dye. Driven by hydrogen bonding, it selectively binds to adenine- thymine (AT) rich sequences of DNA, though stacking interactions with guanine-cytosine (GC) sequences and electrostatic binding to the nucleic phosphate backbone have been also observed.[1] Besides, at high concentrations the H33258 molecules self-aggregate and can also form aggregates while interacting with DNA.[2] Considering the complexity of interactions between H33258 and DNA and aiming to a complete insight in the small molecule-DNA binding, various methods of structural analysis have been applied. In this work interactions of H33258 with two synthetic DNA polynucleotides, poly dA – poly dT and poly dG – poly dC, and natural calf thymus (ct) DNA were studied using surface-enhanced Raman spectroscopy (SERS). Complexes were prepared in [H33258]/[DNA] molar ratios of 1/1, 1/5 and 1/10 in the silver colloidal suspension. Prior to analysis of binding modes with DNA polynucleotides, aggregation tendency of the dye in the silver colloid was studied in the concentration range 1 × 10–6–1 × 10–4 mol/L. The SERS spectra distinctive of aggregated and monomeric H33258 species were obtained, additionally indicating the form of the molecules bound to DNA. The obtained SERS spectra of H33258 with homo-polymeric polynucleotides implied a deep insertion of the molecules into the AT minor groove, and a partial intercalation between the GC base pairs achieved by the access through the major groove. Spectral characteristics of the dye dominated in the spectra of the complexes with ct-DNA, most likely due to a random AT and GC sequences of the long natural DNA, affecting uniform binding of the dye and consequently the adsorption of the H33258/DNA complex onto the enhancing metallic surface.

DNA ; Hoechst 33258 ; interactions ; SERS

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