Preferential Binding of Epirubicin Hydrochloride with Single Nucleotide Mismatched DNA and Subsequent Sequestration by a Mixed Micelle

Abstract

Targeting mismatched base pairs containing DNA using small molecules and exploring the underlying mechanism involved during the binding interactions is one of the fundamental aspects of drug design. These molecules in turn are used in nucleic acid targeted therapeutics and cancer diagnosis. In this work, we systematically delineate the binding of the anticancer drug, epirubicin hydrochloride (EPR) with 20-mer duplex DNA, having both natural nucleobase pairing and thermodynamically least stable non-Watson−Crick base pairing. From the thermal denaturation studies, we observed that EPR can remarkably enhance the thermal stability of cytosine-cytosine (CC) and cytosine-thymine (CT) mismatched (MM) DNA over other 20-mer duplex DNA. From steady-state fluorescence spectroscopy and isothermal titration calorimetry studies, we concluded that EPR binds strongly with the mismatched duplex DNA through the intercalation binding mode. The interaction of EPR and duplex DNA has also been monitored at a single molecular resolution using fluorescence correlation spectroscopy (FCS). Dynamic quantitates such as diffusion coefficients and hydrodynamic radii obtained from an FCS study along with association and dissociation rate constants estimated from intensity time trace analyses further substantiate the stronger binding affinity of EPR to the thermally less stable mismatched DNA, formed by the most discriminating nucleobase (viz. cytosine). Additionally, we have shown that EPR can be sequestered from nucleic acids using a mixed micellar system of an anionic surfactant and a triblock copolymer. From thermal denaturation studies and circular dichroism spectroscopy, we found that the extent of drug sequestration depends on the binding affinity of EPR to the duplex DNA, and this mixed micellar system can be employed for the removal of excess drug in the case of a drug overdose.

Macrocyclic Cavitand β‐Cyclodextrin Inhibits the Alcohol‐Induced Trypsin Aggregation

“…It must be stated here, the probe (here NR) does not show any loss in fluorescence signal during the experiment as evident from Figure S12. Only NR displays a typical single component diffusion with a time constant ∼72 μs as evident from the recorded autocorrelation function trace (Figure 5a), which is well corroborated with a previously reported drug, Epirubicin Hydrochloride, from our group [41] . On the contrary, the longer lag time component of the diffusion arises from the fibrillar binding site as evident from Figure 5a, and Table S5.…”

“…Only NR displays a typical single component diffusion with a time constant ~72 μs as evident from the recorded autocorrelation function trace (Figure 5a), which is well corroborated with a previously reported drug, Epirubicin Hydrochloride, from our group. [41] On the contrary, the longer lag time component of the diffusion arises from the fibrillar binding site as evident from Figure 5a, and Table S5. From Figure 5a, Table 1, and Table S5, it is observed that the longer lag-time component and corresponding relative amplitude increases which attributes the binding of NR to Trypsin Fib.…”

“…K B , T , r H , and η are the Boltzmann constant, absolute temperature, hydrodynamic radius of the fluorescent probe, and viscosity of the medium, respectively. For the elimination of mismatches of refractive index and viscosity of the experimental medium, we have used a ratio method as mentioned in an earlier report [41,43] . To estimate the accurate hydrodynamic radius ( r H ), we used equation (2) with Rhodamine 6G as a standard fluorescent probe having a known diffusion coefficient ( D t =426 μm 2 s −1 ) [42] …”

“…For the elimination of mismatches of refractive index and viscosity of the experimental medium, we have used a ratio method as mentioned in an earlier report. [41,43] To estimate the accurate hydrodynamic radius (r H ), we used equation (2) with Rhodamine 6G as a standard fluorescent probe having a known diffusion coefficient (D t = 426 μm 2 s À 1 ). [42] (2)…”

“…Fluorescence Lifetime Imaging Microscopy (FLIM) images and FCS data were recorded in PicoQuant, MicroTime 200 confocal setup with an inverted optical microscope (Olympus IX-71), the details are mentioned elsewhere. [41,46] Here, NR and ThT were used for FCS and FLIM studies with excitation lasers of 530 nm and 405 nm, respectively. In the case of FCS, a dichroic mirror (DC/470/532 DCXR, Chroma) and a band-pass filter (HQ532lp, Chroma) was used.…”