The Hoechst 33258 Covalent Dimer Covers a Total Turn of the Double-stranded DNA

Abstract

With the goal to design ligands recognizing extended regions on dsDNA, a covalent dimer of the fluorescent dye Hoechst 33258 [bis-HT(NMe)] composed of two dye molecules linked via the phenol oxygen atoms with a (CH₂)₃-N+H(CH₃)-(CH₂)₃ fragment was constructed using computer modeling and then synthesized. Its interactions with the double-stranded DNA (dsDNA) were studied by fluorescent and UV-Vis spectroscopy and circular (CD) and linear dichroism (LD). Based on variations in the affinity to the dsDNA, it was shown that complexes of three types are formed. The first type complexes result from binding of a bis- HT(NMe) monomer in the open conformation; in this case the ligand covers the total dsDNA turn and is located in the minor groove according to the positive value of CD at 370 nm. In addition, the ability to form bis-HT(NMe)-bridges between two dsDNA molecules, i.e., each of the two bis-HT(NMe) ends binds to two different dsDNA molecules, was demonstrated for the first type complexes. Spectral characteristics (maximal absorption at 362 nm, positive sign, and maximal value of CD at 370 nm) of the first type complexes conform to those of the specific Hoechst 33258 complex with poly[d(A-T)] · poly[d(A-T]. The second type complexes correspond to the bis-HT(NMe) sandwich (as an inter- or intramolecular) binding to dsDNA with stoichiometry ≥ 5 bp. Thereby, a negative LD at 360 nm and the location of bis- HT(NMe) sandwiches in the minor groove of B form dsDNA seems contradictory. Spectral characteristics (maximal positive CD at 345 nm, a dramatic decrease in fluorescence intensity and the shift of its maximum to 490 nm) of these complexes favor a suggestion that this binding correlates to the formation of nonspecific dimeric Hoechst 33258 complex with dsDNA. The third type complexes are characterized by stoichiometry of one bis-HT(NMe) molecule per ≈ 2 bp and the tendency to zero of LD values at 270 and 360 nm. We assume that in these complexes bis-HT(NMe) sandwich dimers are formed on dsDNA. The complexes of this type conform to the aggregation type complex of Hoechst 33258 with dsDNA.

The ability of bis-HT(NMe) to cover the whole dsDNA turn or form bridges with two dsDNA upon the formation of the first type complexes essentially distinguishes it from Hoechst 33258, which can only occupy 5 bp and does not form such bridges. This specific property of bis-HT(NMe) may support new biological activities.

Key words:

• bis-Hoechst 33258
• Dye-dsDNA complexes of three types
• UV-Vis absorbance
• Fluorescence
• Circular and linear dichroism