Absorption and fluorescence spectra of the probe Hoechst 33258

Institute of Macromolecular Compounds, Russian Academy of Sciences, St. Petersburg Russian Federation
Journal of Photochemistry and Photobiology A Chemistry (Impact Factor: 2.5). 09/1994; 83(1):39-47. DOI: 10.1016/1010-6030(94)03797-3

ABSTRACT The steric and electronic structure and absorption spectra of the fluorophore Hoechst 33258 (I) and a number of model compounds in various protonated forms were calculated by the semi-empirical quantum chemical methods PM3 and CNDO/S. The low-frequency transition was shown to provide an increase in electron affinity of the imidazole rings. The fluorescence quenching of I in neutral aqueous solutions was suggested to result from the keto form (or bipolar structure), derived via proton transfer in the excited state of the dimer associate of I. The polar (approximately 10 D) fluorescence state of I due to intramolecular charge transfer is markedly sensitive to the polarity of the environment.

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    ABSTRACT: The groove binding dye Hoechst 33258 was investigated both in aqueous solution and in the adsorbed form on silver colloids and island films. Fourier transform Raman, resonance Raman and surface enhanced resonance Raman spectra were used together with the results of a normal coordinate analysis for a comprehensive assignment of the rich vibrational spectrum. Relative band intensities in the SERRS spectra indicate that the molecule is adsorbed with its long axis oriented along the silver surface and with the planes of the benzimidazole rings approximately parallel to the surface. The molecule is bound to the surface as a cation protonated at a piperazine nitrogen atom and is desorbed on complete deprotonation.
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    ABSTRACT: The complexes of Hoechst 33258 with poly[d(A-T)2], poly[d(I-C)2], and poly[d(G-C)2], and poly[d(G-m5C)2] were studied using linear dichroism, CD, and fluorescence spectroscopies. The Hoechst-poly[d(I-C)2] complex, in which there is no guanine amino group protruding in the minor groove, exhibits spectroscopic properties that are very similar to those of the Hoechst-poly[d(A-T)2] complex. When bound to both of these polynucleotides, Hoechst exhibits an average orientation angle of near 45 degrees relative to the DNA helix axis for the long-axis polarized low-energy transition, a relatively strong positive induced CD, and a strong increase in fluorescence intensity--leading us to conclude that this molecule also binds in the minor groove of poly[d(I-C)2]. By contrast, when bound to poly[d(G-C)2] and poly[d(G-m5C)2], Hoechst shows a distinctively different behavior. The strongly negative reduced linear dichroism in the ligand absorption region is consistent with a model in which part of the Hoechst chromophore is intercalculated between DNA bases. From the low drug:base ratio onset of excitonic effects in the CD and fluorescence emission spectra, it is inferred that another part of the Hoechst molecule may sit in the major groove of poly[d(G-C)2] and poly[d(G-m5C)2] and preferentially stacks into dimers, though this tendency is strongly reduced for the latter polynucleotide. Based on these results, the importance of the interactions of Hoechst with the exocyclic amino group of guanine and the methyl group of cytosine in determining the binding modes are discussed.
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    ABSTRACT: To increase the binding affinity and/or the sequence selectivity of the chemical nuclease manganese-(III) tetrakis(4-N-methylpyridiniumyl)porphyrin, we synthesized a conjugate molecule by associating a tris(4-N-methylpyridiniumyl)metalloporphyrin motif to Hoechst 33258 (H33258), a DNA minor groove binding dye known for its selective affinity for A-T tracts. Selected double-stranded (ds) 35-mer oligodeoxyribonucleotides have been used to probe DNA chain breakages induced by the managanese derivative of the conjugate after activation by potassium monopersulfate. Gel electrophoresis analyses show that DNA cuts were generated by the metalloporphyrin moiety of the hybrid molecule, with the H33258 entity interacting in two different possible orientations, upstream or downstream, with its preferred affinity site inside the minor groove. Also studied was the cleavage of a ds 29-mer oligodesoxyribonucleotide containing two stretches of A.T basepair (bp) which clearly showed that the hybrid can occupy the binding region at least in four preferred ways. These cleavage experiments support the strong and selective interaction of the metalloporphyrin-dye hybrid with DNA and allow the estimate of 10 bp as an average size for the affinity site of an isolated conjugate molecule. Further studies by UV-visible spectroscopy, DNA melting temperature determinations, and DNase I footprinting showed, for higher concentrations of H33258 conjugate, a preferential interaction of only the H33258 moiety with DNA (estimated binding site size 6-7 bp) with the porphyrin entity pushed out of the groove and, for the highest concentrations, self-aggregation of the H33258 conjugate all along the DNA strand in a nonselective mode.
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