Hoechst 33258 aggregation and binding to DNA studied by surface‐enhanced Raman spectroscopy

Abstract

Interactions of Hoechst 33258 (H33258) with two synthetic DNA polynucleotides, poly dA-poly dT and poly dG-poly dC, as well as natural calf thymus (ct) DNA were studied using surface-enhanced Raman spectroscopy (SERS). Given variety of stoichiometry dependent interactions, 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 Â 10 À6 -1 Â 10 À4 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 GC major groove. Spectral characteristics of the dye dominated in the spectra of the complexes with ct-DNA, most likely due to random AT and GC sequences of the long natural nucleic acid, affecting uniform binding of the dye and consequently the adsorption of the H33258/DNA complex onto the enhancing metallic surface.

Single-Cell Analysis with Silver-Coated Pipette by Combined SERS and SICM

“…Orange lines in Figure 3 show the positions of peaks characteristic of Hoechst-980, 1358, 1456, 1610 cm −1 [64], which are not observed in the SERS spectrum of living cells. It is worth noting that these peaks are present in the SERS spectrum of the nucleus obtained from the SERS pipette.…”