Hoechst 33258, a synthetic dye containing a bisbenzimidazole moiety, has attracted a great deal of attention due to its selective groove binding interaction with AT-rich DNA sequences. Also, it shows a partially intercalative binding mode with poly(dG-dC)2 and with GC-rich sequences of other types of DNA. The pronounced binding interaction in the AT-rich minor groove of DNA can affect transcription, blocking topoisomerase I and helicases activity. Despite the many studies carried out so far, the behaviour of Hoechst 33258-DNA towards RNA is still not well understood. The dsRNA structure exerts profound regulatory effects on the cell, thereby playing the RNA molecules a crucial role in many key biological processes. Kinetic and thermodynamic measurements contribute to explain the Hoechst33258/RNA interaction. The homopolymer Poly(rA)·Poly(rU) has been used as synthetic RNA. The techniques employed for kinetic experiments, T-jump and conventional spectrophotometer, and for thermodynamic experiments, ITC, DSC, thermal denaturation, CD spectrophotometry and absorbance and fluorescence titrations at different ionic strengths indicate that Hoechst 33258 interacts with Poly(rA)·Poly(rU) by two binding modes, depending on the CD/CP ratio. Thermal denaturation measurements at 25ºC show that triple helix prevails at low CD/CP, whereas the double helix prevails at higher CD/CP values. Kinetic experiments performed at different temperatures and DSC and CD measurements confirm such behaviour.For CD/CP < 0.2, the binding constant has been determined by ITC measurements. The apparent constant Kapp = 1E5 M-1 corresponds to an intercalation process responsible for the formation of the triple helix, in agreement with the reaction: 2[Poly(rA)·Poly(rU) /Hoechst] = Poly(rA)·2Poly(rU)/Hoechst + Poly(rA)/Hoechst Spectrophotometric titrations yielded a binding constant above 1E7 at high dye content, therefore the binding occurs in the minor groove of the duplex and stabilizes the double strand of RNA, shifting the formation of the triple towards higher temperatures.

Hoechst 33258 induces changes from duplex RNA to triplex RNA at different temperatures and concentrations

BIVER, TARITA;SECCO, FERNANDO;VENTURINI, MARCELLA
2013

Abstract

Hoechst 33258, a synthetic dye containing a bisbenzimidazole moiety, has attracted a great deal of attention due to its selective groove binding interaction with AT-rich DNA sequences. Also, it shows a partially intercalative binding mode with poly(dG-dC)2 and with GC-rich sequences of other types of DNA. The pronounced binding interaction in the AT-rich minor groove of DNA can affect transcription, blocking topoisomerase I and helicases activity. Despite the many studies carried out so far, the behaviour of Hoechst 33258-DNA towards RNA is still not well understood. The dsRNA structure exerts profound regulatory effects on the cell, thereby playing the RNA molecules a crucial role in many key biological processes. Kinetic and thermodynamic measurements contribute to explain the Hoechst33258/RNA interaction. The homopolymer Poly(rA)·Poly(rU) has been used as synthetic RNA. The techniques employed for kinetic experiments, T-jump and conventional spectrophotometer, and for thermodynamic experiments, ITC, DSC, thermal denaturation, CD spectrophotometry and absorbance and fluorescence titrations at different ionic strengths indicate that Hoechst 33258 interacts with Poly(rA)·Poly(rU) by two binding modes, depending on the CD/CP ratio. Thermal denaturation measurements at 25ºC show that triple helix prevails at low CD/CP, whereas the double helix prevails at higher CD/CP values. Kinetic experiments performed at different temperatures and DSC and CD measurements confirm such behaviour.For CD/CP < 0.2, the binding constant has been determined by ITC measurements. The apparent constant Kapp = 1E5 M-1 corresponds to an intercalation process responsible for the formation of the triple helix, in agreement with the reaction: 2[Poly(rA)·Poly(rU) /Hoechst] = Poly(rA)·2Poly(rU)/Hoechst + Poly(rA)/Hoechst Spectrophotometric titrations yielded a binding constant above 1E7 at high dye content, therefore the binding occurs in the minor groove of the duplex and stabilizes the double strand of RNA, shifting the formation of the triple towards higher temperatures.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11568/237955
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