Syntheses and DNA-binding studies of a series of unsymmetrical cyanine dyes: structural influence on the degree of minor groove binding to natural DNA.

Twelve crescent-shaped unsymmetrical dyes have been synthesized and their interactions with DNA have been investigated by spectroscopic methods. A new facile synthetic route to this type of cyanine dyes has been developed, involving the preparation of 6-substituted 2-thiomethyl-benzothiazoles in good yields. The new dyes are analogues to the minor groove binding unsymmetrical cyanine dye, BEBO, recently reported by us. In this dye, the structure of the known intercalating cyanine dye BO was extended with a 6-methylbenzothiazole substituent. Herein we further investigate the role of the extending benzazole heterocycle, as well as of the pyridine or quinoline moiety of the cyanine chromophore, for the binding mode of these crescent-shaped dyes to calf thymus DNA. Flow LD and CD studies of the 12 dyes show that the extent of minor groove binding to mixed sequence DNA varies significantly between the dyes. We find that hydrophobicity and size are the crucial parameters for recognition of the minor groove. The relatively high fluorescence quantum yield of many of these cyanines bound to DNA, combined with their absorption at long wavelengths, may render them useful in biological applications. In particular, two of the benzoxazole containing dyes BOXTO and 2-BOXTO show a high degree of minor groove binding and quantum yields of 0.52 and 0.32, respectively, when bound to DNA.

Groove-binding unsymmetrical cyanine dyes for staining of DNA: syntheses and characterization of the DNA-binding.

Two new crescent-shaped unsymmetrical cyanine dyes have been synthesised and their interactions with DNA have been investigated by different spectroscopic methods. These dyes are analogues to a minor groove binding unsymmetrical cyanine dye, BEBO, recently reported by us. In this dye, the structure of the known intercalating cyanine dye BO was extended with a benzothiazole substituent. To investigate how the identity of the extending heterocycle affects the binding to DNA, the new dyes BETO and BOXTO have a benzothiazole group and a benzoxazole moiety, respectively. Whereas BEBO showed a heterogeneous binding to calf thymus DNA, linear and circular dichroism studies of BOXTO indicate a high preference for minor groove binding. BETO also binds in the minor groove to mixed sequence DNA but has a contribution of non-ordered and non-emissive species present. A non-intercalative binding mode of the new dyes, as well as for BEBO, is further supported by electrophoresis unwinding assays. These dyes, having comparable spectral properties as the intercalating cyanine dyes, but bind in the minor groove instead, might be useful complements for staining of DNA. In particular, the benzoxazole substituted dye BOXTO has attractive fluorescence properties with a quantum yield of 0.52 when bound to mixed sequence DNA and a 300-fold increase in fluorescence intensity upon binding.

Groove-binding unsymmetrical cyanine dyes for staining of DNA: dissociation rates in free solution and electrophoresis gels.

The rates of dissociation of three non-intercalative unsymmetrical cyanine dyes, BEBO, BETO and BOXTO from mixed-sequence DNA have been studied with the DNA either free in solution or in confining porous agarose gels. The properties of the new dyes were compared to the related intercalating dyes BO, BO-PRO, TO-PRO and YO-PRO. With DNA in solution, BEBO dissociates more slowly than the monovalent BO and interestingly also more slowly than the divalent dye BO-PRO. Similarly, both BETO and BOXTO exhibit considerably slower dissociation than TO-PRO. The new dyes show biexponential dissociation kinetics in mixed-sequence DNA. The average rate of dissociation increases with increasing ionic strength, but the salt dependence of the dissociation is weaker than for the corresponding intercalating dye. The rate of dye-dissociation decreases by a factor of about 10(5) in the gel. The rates for the dyes generally follow the pattern that we observe with the DNA in free solution, however a more accentuated stabilization was seen for intercalators than for groove-bound dyes. The results show that, in particular, BOXTO is a promising candidate as a preferentially groove-bound DNA-stain with a large enhancement of the fluorescence quantum yield upon binding to DNA, and which exhibits slow and salt-insensitive dissociation compared to corresponding intercalative dyes.

A new minor groove binding asymmetric cyanine reporter dye for real-time PCR.

The minor groove binding asymmetric cyanine dye 4-[(3-methyl-6-(benzothiazol-2-yl)-2,3-dihydro- (benzo-1,3-thiazole)-2-methylidene)]-1-methyl-pyridin ium iodide (BEBO) is tested as sequence non- specific label in real-time PCR. The fluorescence intensity of BEBO increases upon binding to double-stranded DNA allowing emission to be measured at the end of the elongation phase in the PCR cycle. BEBO concentrations between 0.1 and 0.4 micro M generated sufficient fluorescence signal without inhibiting the PCR. A comparison with the commonly used reporter dye SYBR Green I shows that the two dyes behave similarly in all important aspects.

Synthesis and DNA binding studies of a new asymmetric cyanine dye binding in the minor groove of [poly(dA-dT)]2.

A new asymmetric cyanine dye has been synthesised and its interaction with different DNA has been investigated. In this dye, BEBO, the structure of the known intercalating cyanine dye BO has been extended with a benzothiazole substituent. The resulting crescent-shape of the molecule is similar to that of the well-known minor groove binder Hoechst 33258. Indeed, comparative studies of BO illustrate a considerable change in binding mode induced by this structural modification. Linear and circular dichroism studies indicate that BEBO binds in the minor groove to [poly (dA-dT)](2), but that the binding to calf thymus DNA is heterogeneous, although still with a significant contribution of minor groove binding. Similar to other DNA binding asymmetric cyanine dyes, BEBO has a large increase in fluorescence intensity upon binding and a relatively large quantum yield when bound. The minor groove binding of BEBO to [poly (dA-dT)](2) affords roughly a 180-fold increase in intensity, which is larger than to that of the commonly used minor groove binding probes DAPI and Hoechst 33258.