Force spectroscopy unravels the role of ionic strength on DNA-cisplatin interaction: Modulating the binding parameters

In the present work we have gone a step forward in the understanding of the DNA-cisplatin interaction, investigating the role of the ionic strength on the complexes formation. To achieve this task, we use optical tweezers to perform force spectroscopy on the DNA-cisplatin complexes, determining their mechanical parameters as a function of the drug concentration in the sample for three different buffers. From such measurements, we determine the binding parameters and study their behavior as a function of the ionic strength. The equilibrium binding constant decreases with the counterion concentration ([Na]) and can be used to estimate the effective net charge of cisplatin in solution. The cooperativity degree of the binding reaction, on the other hand, increases with the ionic strength, as a result of the different conformational changes induced by the drug on the double-helix when binding under different buffer conditions. Such results can be used to modulate the drug binding to DNA, by appropriately setting the ionic strength of the surrounding buffer. The conclusions drawn provide significant new insights on the complex cooperative interactions between the DNA molecule and the class of platinum-based compounds, much used in chemotherapies.

Figure 1

Chemical structure of the cisplatin molecule. In aqueous solutions, the two chloride ions dissociate and the compound incorporates two water molecules, reaching its bivalent (2+) active state, which binds to DNA.

Figure 2

Persistence length $A$ of the DNA-cisplatin complexes as a function of the drug concentration in the sample $C_T$ for [Na] = 150 mM. Observe that for such ionic strength the persistence length decreases as a function of the drug concentration, and the data present a sigmoidal shape, indicating that significant cooperativity is present in the interaction at such situation. In addition, the relevant concentration range in which the persistence length changes is 0 $<C_T<$ 100 $\mu \mathrm{M}$.

Figure 3

Persistence length $A$ of the DNA-cisplatin complexes as a function of the drug concentration in the sample $C_T$ for [Na] = 10 mM. The sigmoidal shape of the persistence length curve is much less evident, indicating that a considerable decrease in the cooperativity degree of the binding reaction has occurred. The inset evidences the low concentration range. The relevant concentration range in which the persistence length changes is 0 $<C_T<$ 20 $\mu \mathrm{M}$.

Figure 4

Persistence length $A$ of the DNA-cisplatin complexes as a function of the drug concentration in the sample $C_T$ for [Na] = 1 mM. In this case, the persistence length strongly decreases even for very small drug concentrations, and there is no evidence of sigmoidal shape on the decay curve. The relevant concentration range in which the persistence length changes is 0 $<C_T<$ 3 $\mu \mathrm{M}$.

Figure 5

Equilibrium binding constant $K$ as a function of the counterion concentration. A fitting to Eq. (3) (dashed line) is also shown. Observe that the Record-Lohman model explains well our experimental data, and from the fitting we obtain $z$ = (1.5 $\pm$ 0.1), which is relatively close to the expected value of the cisplatin charge (+2).

Figure 6

Persistence length at saturation $A_1$ (blue squares) and Hill exponent $n$ (black circles) as a function of the counterion concentration. The dashed lines presented in the figure are only guides to the eyes.

Figure 7

Normalized contour length $L/L_0$ of the DNA-cisplatin complexes as a function of the drug concentration in the sample $C_T$ for [Na] = 150 mM. Observe that this mechanical property here decreases as a function of the drug concentration. Such behavior is related to the DNA compaction process induced by the formation of crosslinks and loops on the double-helix upon drug binding, which decreases the apparent contour length measured at the low force entropic regime.

Figure 8

Normalized contour length $L/L_0$ of the DNA-cisplatin complexes as a function of the drug concentration in the sample $C_T$ for [Na] = 10 mM. Here an effective increase on the DNA apparent contour length was obtained, which is probably related to DNA unwinding and/or local strand breaks induced by cisplatin binding. Such changes on the double-helix structure become evident when the drug does not compact the DNA.

Figure 9

Normalized contour length $L/L_0$ of the DNA-cisplatin complexes as a function of the drug concentration in the sample $C_T$ for [Na] = 1 mM. The behavior here is very similar to the one found for [Na] = 10 mM.

Figure 10

Exemplifying force-extension curves of some DNA-cisplatin complexes obtained for [Na] = 150 mM. Black circles: bare DNA; Blue squares: 40 $\mu \mathrm{M}$ of cisplatin, i.e., an intermediate drug concentration; Red diamonds: 100 $\mu \mathrm{M}$ of cisplatin, i.e., a drug concentration corresponding to the saturation regime.

Figure 11

Exemplifying force-extension curves of some DNA-cisplatin complexes obtained for [Na] = 10 mM. Black circles: bare DNA; Blue squares: 4 $\mu \mathrm{M}$ of cisplatin, i.e., an intermediate drug concentration; Red diamonds: 10 $\mu \mathrm{M}$ of cisplatin, i.e., a drug concentration corresponding to the saturation regime.

Figure 12

Exemplifying force-extension curves of some DNA-cisplatin complexes obtained for [Na] = 1 mM. Black circles: bare DNA; Blue squares: 2 $\mu \mathrm{M}$ of cisplatin, i.e., a drug concentration corresponding to the saturation regime.