The Mechanism of Dynamic Interaction between Doxorubicin and Calf Thymus DNA at the Single-Molecule Level Based on Confocal Raman Spectroscopy

Abstract

It is of great fundamental significance and practical application to understand the binding sites and dynamic process of the interaction between doxorubicin (DOX) and DNA molecules. Based on the Confocal Raman spectroscopy, the interaction between DOX and calf thymus DNA has been systemically investigated, and some meaningful findings have been found. DOX molecules can not only interact with all four bases of DNA molecules, i.e., adenine, thymine, cytosine, guanine, and phosphate, but also affect the DNA conformation. Meanwhile, the binding site of DOX and its derivatives such as daunorubicin and epirubicin is certain. Furthermore, the interaction between DOX and DNA molecules is a dynamic process since the intensities of each characteristic peaks of the base, e.g., adenine, cytosine, and phosphate, are all regularly changed with the interaction time. Finally, a dynamic mechanism model of the interaction between DOX and DNA molecules is proposed; that is, there are two kinds of interaction between DOX and DNA molecules: DOX-DNA acts to form a complex, and DOX-DOX acts to form a multimer. The two effects are competitive, as the former compresses DNA molecules, and the latter decompresses these DNA molecules. This work is helpful for accurately understanding and developing new drugs and pathways to improve and treat DOX-induced cytotoxicity and cardiotoxicity.

Keywords: Confocal Raman spectroscopy; binding site; calf thymus DNA; doxorubicin; dynamic process.

Figure 1

(a) The molecular structure of ctDNA; (b) Raman spectra of ctDNA fiber (blue line) and ctDNA solution ($9.8\times {10}^{-2} \mathrm{M}$, black line).

Figure 2

(a) The molecular structure of DOX and (b) Raman spectra of DOX solution ($5\times {10}^{-2} \mathrm{M}$).

Figure 3

(a) Raman spectra of ctDNA solution (blue line) and (b) Raman spectrum of DOX-DNA complex (black line). The molar ratio of base-pair versus drug is two.

Figure 4

The molecular structure of (a) DAU and (b) EPI.

Figure 5

Raman spectra of (a) DOX (black line), (b) DAU (red line), and (c) EPI (blue line).

Figure 6

Raman spectra of (a) DNA (black line), (b) DOX-DNA complex (red line), (c) DAU-DNA complex (blue line), and (d) EPI-DNA complex (green line).

Figure 7

The diagram of the interaction sites of DOX and DNA.

Figure 8

Raman spectra of DOX interacting with DNA at different times. (a–f) Raman spectrum of the band at 726, 785, 1089, 1450, 1578, and 1657${ \mathrm{cm}}^{-1}$, respectively.

Figure 9

Line charts of Raman intensity change with incubation time at various wave numbers $({\mathrm{cm}}^{-1}$): (a) 726, (b) 785, (c) 1089, (d)1450, (e) 1578, and (f) 1657.

Figure 10

(a) Absorption spectra of the DOX-DNA complex with different incubating times; (b) absorption peak values of DOX-DNA complex around 258 nm at various incubation times.

Figure 11

Schematic diagram of the DNA configuration changing process interacted by DOX. (a–e) Graphics of DOX interaction with DNA at different times. The insets in the middle indicate the detailed process of forming a DOX trimer. (1) Free two DOX molecules are attracted to the DOX-DNA complex; (2) A trimer is formed; (3) The trimer leaves the DOX-DNA complex and other doxorubicin molecules will interact with DNA.