Chapter 4 Effect of varying the number of pendant groups on DNA binding
4.2.3 DNA binding studies performed using UV-Vis spectroscopy
4.2.3.1 Absorption titration method
Ultraviolet-Visible (UV-Vis) absorption spectrophotometry is one of the most frequently used techniques to study the interactions between small molecules and DNA. A number of different individual methods can be used with this particular
(a) (b)
(c) (d)
(e)
DNA alone Ni:DNA 1:1 Ni:DNA 3:1 Ni:DNA 6:1 Ni:DNA 9:1
170 technique. One of the most widely used is the determination of an overall binding constant for drug/DNA interactions by monitoring the effect of adding increasing amounts of DNA on the absorption spectrum of solutions containing drug molecules.[94, 141, 239] For this thesis project, UV-Vis titration experiments were performed in order to determine overall binding constants for the interactions of the nickel complexes with dsDNA. As such experiments can use large quantities of DNA, the studies were carried out using 100 mM NH4OAc solutions containing CT-DNA.
Experiments were performed by monitoring the change in absorbance of an intense band at 280 – 300 nm, and a broad band centred around 380 nm, in the spectra of the nickel complexes. These absorption bands have been assigned to intraligand (IL) electronic transitions and metal to ligand charge transfer (MLCT) transitions, respectively.[94, 97, 239]
The above experiments were performed to provide an indication of the overall strength of binding interactions between the novel nickel complexes and dsDNA, as well as the relative strengths of these binding interactions amongst the nickel complexes. It was hoped that the binding constants obtained might help answer questions arising from differences in binding affinity noted earlier for some complexes, based on results obtained from ESI-MS and CD experiments. The method used to perform the UV-Vis absorption titrations is described in Chapter 2.6.1. The results obtained from a typical absorption titration experiment involving (89) are shown in Figure 4.11, S4.3, whereas those obtained from experiments performed using the other nickel complexes can be found in Figure S4.2, S4.3.
171 Figure 4.11 Effect of addition of 3 mM CT-DNA on the absorption spectrum of 20 M (89).The inset shows the resulting binding isotherm. The arrows indicate the direction of change in absorbance upon addition of CT-DNA.
Adding CT-DNA to solutions containing the nickel complexes resulted in significant hypochromicity for the MLCT bands of every complex and, in some instances, notable changes to the position of the absorption bands (Table 4.7). This absorption band was used for determining binding constants in view of the proximity of the IL band to the absorption band of CT-DNA. The data obtained from these experiments were analysed using Equation 2.1 to provide the binding constants also presented in Table 4.7. The binding constants for the novel nickel complexes with different numbers of pendant groups, and (54), were all very small, and similar to each other. These results therefore suggest that each of these complexes has a relatively low affinity towards dsDNA. This conclusion is consistent with the very low abundances of ions observed in ESI-MS experiments performed using these complexes and D2, as well the relatively small changes to the CD spectrum of D2 observed on most occasions when the metal complexes were added.
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
280 330 380 430 480
Absorbance
Wavelength (nm) Increasing [DNA]
y = 0.0005x + 2E-08 R² = 0.9658
0.000E+00 2.000E-08 4.000E-08 6.000E-08 8.000E-08 1.000E-07 1.200E-07 1.400E-07
0 0.00005 0.0001 0.00015 0.0002 0.00025 [DNA]/(a-f)
[DNA] base pairs (M)
172 Table 4.7 Results obtained from absorption titration experiments performed using nickel Schiff base complexes with different numbers of pendant groups and CT-DNA.
Complexes
max for MLCT band (nm)
max for MLCT band (nm)
Extinction coefficient of complexes (A ×
104 (M-1 cm-1))
A for MLCT band (%)
Mean binding constant (Kb ×
104 (M-1)) a
(54) 366.7 15.5 2.0 ± 0.3 -54.8 24.0 ± 1
(87) 382.2 -4.7 0.7 ± 0.2 -47.3 6.7 ± 0.1
(71) 380.3 -2.2 0.8 ± 0.1 -19.4 5.3 ± 0.3
(77) 377.8 0.0 1.7 ± 0.4 -24.8 1.8 ± 0.1
(89) 385.3 9.2 0.8 ± 0.2 -25.2 2.8 ± 0.2
a Mean binding constants (units = M(base pairs)-1) were obtained from three absorption titration experiments, and are presented here together with standard errors.
It is important to look closely at the results obtained for (89), in view of the unexpectedly dramatic changes to the CD spectrum of D2 caused by addition of this nickel complex, which were noted in Chapter 4.2.2.6. Whilst the MLCT band of (89) did shift significantly to lower energy when CT-DNA was added, the hypochromicity caused by the binding interaction and the overall binding constant were both relatively minor. Therefore the CD results obtained for this complex in the previous section of this chapter appear to not be consistent with all other evidence concerning the strength of the binding interaction for (89) with duplex DNA. Taking the ESI-MS results presented earlier in this chapter also into consideration, it would appear that (89) has a relatively low affinity towards dsDNA molecules. Of the remaining nickel complexes, the literature compound (54) exhibited the largest hypochromicity and binding constant. The energy of its MLCT band was also the most dramatically affected by addition of CT-DNA. Furthermore, whilst ESI mass spectra of solutions containing (54) and D2 contained ions attributable to non-covalent complexes that were only of low abundance, they were present in greater relative amounts than in spectra of solutions containing the other nickel complexes. This reinforces the view that whilst the binding of (54) to dsDNA may be relatively weak, it is perhaps still slightly stronger than that exhibited by the novel nickel complexes.
173 4.2.3.2 DNA melting studies
It has been well documented that the binding affinity of small molecules towards dsDNA or qDNA can be investigated by using UV spectrophotometry to examine the effect on the thermal behaviour of secondary DNA structures.[127, 141, 239, 240] This technique relies upon the change in absorbance at 260 nm (A260) from the DNA bases, which occurs when dsDNA or G-quadruplex structures denature, or “melt”, as a result of an increase in temperature. Plots of A260 as a function of temperature are often referred to as DNA melting curves, and have a sigmoidal appearance, with the temperature at which 50% of the dsDNA has undergone strand separation being referred to as the melting temperature (Tm).[241]
Small molecules that can bind to and stabilise dsDNA or G-quadruplexes towards this melting process cause a shift in the DNA melting curve to higher temperatures, resulting in an increase in Tm. The magnitude of the change in Tm, referred to as
Tm, is proportional to the ability of the small molecule to bind to and stabilise the DNA secondary structure. Therefore, small molecules which have stronger and/or more effective binding interactions result in higher Tm values. For example, it has been shown that metal complexes that interact with dsDNA via an intercalative binding mode enhance the stability of the double helix and significantly increase Tm.[242, 243] Representative DNA melting profiles for solutions containing D2 alone, or D2 and (71), are shown in Figure 4.12. Figure 4.13 illustrates the effect on Tm of D2 of adding 3 or 6 equivalents of nickel complexes with different numbers of pendant groups, or (54).
174 Figure 4.12 Melting curves for solutions containing 1M dsDNA D2 alone, and a 6:1 ratio of (71) and 1M D2.
Melting temperature experiments were performed using solutions containing a 3:1 or 6:1 ratio of one of the nickel complexes and D2. The melting temperature, Tm, of D2 alone was determined to be 62.6 ± 0.2 °C. Figure 4.13 shows that the Tm of D2 decreased by 1 – 3 °C in the presence of the four novel nickel Schiff base complexes. These results are therefore in accord with the low binding constants reported for the interaction of the nickel complexes and CT-DNA in Chapter 4.2.3.1, and the low abundances of ions observed in ESI mass spectra of solutions containing the nickel complexes and D2 (Chapter 4.2.1). The results obtained from the DNA melting studies not only provide further evidence that the novel nickel Schiff base complexes have a low affinity towards dsDNA, they also suggest that their interactions with the nucleic acid may lead to destabilisation of its secondary structure.
In contrast to the novel nickel Schiff base complexes which are the focus of this chapter, (54) caused a small increase in the Tm of D2 of 2.1 °C under the same conditions. This result is also consistent with those obtained from the absorption titration experiments in particular, as these showed that the overall binding constant
T e m p e r a t u r e (oC )
Normalised Absorbance
4 0 6 0 8 0
0 .0 0 .2 0 .4 0 .6 0 .8 1 .0
D 2
D 2 + ( 7 1 )
175 for interactions between (54) and CT-DNA was slightly larger than for any of the other nickel complexes.
Figure 4.13 Effect of addition of nickel complexes with different numbers of pendant groups on the melting temperature, Tm, of D2. The experiments were performed in triplicate with the error bars showing standard errors. For each nickel complex the left hand value of Tm was obtained from a solution containing a 3:1 ratio of nickel complex:D2. The right hand values were obtained from solutions containing a 6:1 ratio of nickel complex:D2.