Letter pubs.acs.org/NanoLett
Time-Lapse AFM Imaging of DNA Conformational Changes Induced by Daunorubicin Livan Alonso-Sarduy,† Giovanni Longo,*,†,‡ Giovanni Dietler,† and Sandor Kasas†,§ Laboratoire de Physique de la Matière Vivante, École Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland Istituto Superiore di Sanità, Viale Regina Elena 299, I - 00161 Roma, Italy § Département de Biologie cellulaire et de Morphologie, Université de Lausanne (UNIL), CH-1015 Lausanne, Switzerland † ‡
S Supporting Information *
ABSTRACT: Cancer is a major health issue that absorbs the attention of a large part of the biomedical research. Intercalating agents bind to DNA molecules and can inhibit their synthesis and transcription; thus, they are increasingly used as drugs to fight cancer. In this work, we show how atomic force microscopy in liquid can characterize, through time-lapse imaging, the dynamical influence of intercalating agents on the supercoiling of DNA, improving our understanding of the drug’s effect.
KEYWORDS: DNA intercalating agents, daunorubicin, AFM, linking number, time-lapse imaging, drug development
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Escherichia coli, it elicits an increase in supercoiling from negative to positive values.7,8 A wide variety of physical and chemical techniques has been used to study the effect of small binding ligands from thermodynamic and biochemical techniques9−11 to structural methods including NMR spectroscopy12 and X-ray diffraction.13 Recently, very interesting results have been shown using single-molecule investigation techniques such as magnetic,14−18 optical tweezers,19,20 or a combination of the two.21 On the other hand, a direct imaging of the interaction between a single DNA molecule and one of its ligands is a complex, yet extremely rewarding task. Recent works have demonstrated the direct visualization of a single ligand bound to DNA molecules at nanoscale resolution using disparate techniques, including scanning tunneling microscopy,22 electron microscopy,23,24 fluorescence microscopy,25,26 Förster resonance energy transfer,27 and atomic force microscopy (AFM).28−32 This latter technique stands out as one of the most interesting to observe such interactions since it can be used to image DNA strains with subnanometer resolution or to characterize their mechanical properties on a single-molecule level.33−37 Indeed, the imaging capabilities of AFM are still unrivalled and this technique is now routinely used to image
ancer is a major public health issue that absorbs the attention and focus of a large part of the biomedical research. To fight this disease, new drugs are developed, specifically tailored to target biological pathways or peculiar components of the cancerous cells. Particularly interesting in this field is the use of intercalating agents as drugs capable of binding to DNA molecules. Intercalating agents are routinely used in fundamental research as fluorescent tags to determine DNA’s superhelical density but can have also a very important role as anticancer drugs. By wedging between the DNA bases, they affect its structure and prevent polymerase and other binding proteins from functioning properly. The result is inhibition of DNA synthesis and transcription as well as induction of mutations.1−3 Unfortunately, these chemicals lack in specificity and are, therefore, extremely toxic even for healthy cells. An understanding of the manner in which DNA interacts with various chemical agents, especially anticancer drugs, is of paramount importance in this field, especially in chemotherapy. This field is crucial to elucidate the mechanisms whereby the ligands act, which will facilitate the development of new, more specific drugs. For example, daunorubicin (Dau) is a drug used to treat several types of cancers, such as leukemia4 and neuroblastomas.5 This agent intercalates in double-stranded DNA inhibiting the cycle of enzymes (such as topoisomerases) implicated in the replication processes. The intercalation of Dau induces also a local unwinding of the DNA helix, which has been measured and characterized in many studies.6 For instance, when daunorubicin is applied to plasmids of © XXXX American Chemical Society
Received: September 9, 2013 Revised: October 9, 2013
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dx.doi.org/10.1021/nl403361f | Nano Lett. XXXX, XXX, XXX−XXX
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single molecules under near-physiological conditions.32,38,39 Due to this peculiarity, AFM has been used to characterize the effect of the binding of daunorubicin and other well-known intercalating and groove-binding drugs on the supercoiling of single DNA molecules.36,40−42 These works cover a vast quantity of different intercalating and nonintercalating agents including actinomycin, chloroquine, berenil,8 cisplatin,43 echinomycin,44 luzopeptin,45 nogalamycin,46 daunorubicin, doxorubicin, ethidium bromide, and netropsin42,47 but were all performed in air and not in physiological conditions. While the drying procedure ensures an easier measurement and a better quality of the images, it does not allow following dynamically the effect of the interactions. Moreover, the drying procedure prevents the equilibration of the molecule and can result in the projection of a particular 3D conformation onto the surface; this could produce unwanted overlapping and artifacts on the DNA structure. In this work, we present for the first time an AFM characterization of the effect of two intercalating agents on DNA, performed in physiological medium. We monitor the conformational changes induced by the exposure to Dau within the different topological shapes of DNA and, finally, we compare these results with the ones obtained using another known intercalating agent, which is not used as anticancer drug: ethidium bromide (EthBr). Moreover, we use AFM to follow in time-lapse such intercalating-agents-induced transitions, delivering a characterization of the interactions and of the resulting modification in the DNA supercoiling that is unprecedented for its spatial and temporal resolution. Results and Discussion. To perform the AFM imaging we adsorbed highly negatively supercoiled DNA to a flat mica surface (Figure 1a). The strength of such attachment must ensure that the molecules do not detach during imaging; on the other hand, it must permit the interaction between the intercalating agents and the DNA and it must allow the free conformational change of the molecules (for details see Methods section).
We estimated the level of supercoiling of the DNA molecules by measuring their height. Indeed, the mean height of a DNA molecule is a good indication of their supercoiling state; when circular DNA is in a relaxed state we will have no crossings, therefore a low mean height of the molecule, while in the negative or positively supercoiled state DNA crossings will increase the mean height. Thus, the average height of DNA molecules from AFM images can be used as indication of the supercoiling level of DNA molecules (Figures S1 and S2 and their description in the Supporting Information for details on the method used for this calculation). When the negatively supercoiled DNA plasmids are exposed to Dau, the AFM images show that an increase in Dau concentration causes a relaxation of the DNA molecules. The twist of the DNA in negatively supercoiled state is diminished and therefore the population of highly negatively supercoiled DNA plasmids is decreased (the writhe increases from Wr ≪ 0 to Wr ≈ 0, see Figure 1bc). For higher concentrations of Dau (>20 μM), the molecule further increases its writhe and adopts positive supercoiled configurations (Wr > 0, see Figure 1d). As is shown in Figure 2, Dau concentrations in the range of 1.25−10 μM causes a decreasing of the average height of DNA
Figure 2. Concentration dependence of Dau-induced changes in the height of DNA molecules during the uncoiling process. Each bar represents the mean ± sd of height of DNA molecules treated.
molecules (
