Intercalator induced DNA superstructure formation: doxorubicin and a

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Intercalator induced DNA superstructure formation: doxorubicin and a synthetic quinoxaline derivative Tridib Mahata, Jeet Chakraborty, Ajay Kanungo, Dipendu Patra, Gautam Basu, and Sanjay Dutta Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00613 • Publication Date (Web): 31 Aug 2018 Downloaded from http://pubs.acs.org on September 1, 2018

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Intercalator induced DNA superstructure formation: doxorubicin and a synthetic quinoxaline derivative Tridib Mahata,[a] Jeet Chakraborty,[a] Ajay Kanungo,[a],[b] Dipendu Patra,[a],[b] Gautam Basu*[c] and Sanjay Dutta*[a],[b]

[a]

Department of Organic and Medicinal Chemistry, CSIR-Indian Institute of Chemical

Biology, 4, Raja S. C. Mullick Road, Kolkata 700032, WB, India. [b]

Academy of Scientific and Innovative Research (AcSIR), New Delhi, India.

[c]

Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VIIM, Kolkata 700054,

India.

* To whom correspondence should be addressed. Tel: [(+91)33-24995814]; Fax: (+91) 332473-5197; Email: [email protected]

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ABSTRACT Small molecules that intercalate DNA have tremendous therapeutic potential. Typically, DNA intercalators do not alter the overall DNA double helical structure, except locally at the intercalation sites. In a previous report we had shown that a quinoxaline-based intercalator with a mandatory benzyl substitution (1d), induced unusually large CD signal upon DNA binding, suggesting the formation of intercalated-DNA superstructures. However, no detailed structural studies were reported. Using AFM we have probed the nature of the superstructure and report the formation of a plectonemically oversupercoiled structure of pBR322 plasmid DNA by 1d, where close association of distant DNA double helical stretches is the predominant motif. Without the benzyl moiety (1a), no such DNA superstructure was observed. Similar superstructures were also observed with doxorubicin (dox), a therapeutically important DNA intercalator, suggesting that the superstructure is common to some intercalators. The superstructure formation, for both the intercalators, was observed to be GC-specific. Interestingly, at higher concentrations (1d and dox) the DNA superstructure led to DNA condensation – a phenomenon typically associated with polyamines but not intercalators. The superstructure may have important biological relevance in connection to a recent study where dox was shown to evict histone at micromolar concentrations.

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INTRODUCTION DNA regulates a plethora of biological processes. While the role of the canonical double helical DNA structure is well established, we are only beginning to understand the biological role of the more compact DNA superstructures.1 Classic examples of DNA superstructure formation are DNA compaction by histones2 or DNA condensation by polycationic species.3 Proteins such as Hfq or H-NS also participate in DNA condensation and compaction.4 Cationic lipids, designed for gene delivery, can also induce compaction of plasmid DNA.5 DNA intercalation by small molecules can subtly alter the helical conformation of DNA. However, their role in DNA compaction and condensation has not been explored much. For example, DNA intercalating agents like YOYO-1 can cause DNA condensation at acidic pH (5.7), although the mechanism is unclear.6

O N

N

H N

O

OH

O

N

OH

O OH N

NHR O

1a

R=H

1d

R=

O

OH

O

O

Doxorubicin OH H3N

Figure 1. Molecular structures of 1a, 1d and doxorubicin (dox).

In an earlier report7 we had shown that a simple 6-nitroquinoxaline-2,3-diamine derivative (1d, Figure 1) acts as a DNA intercalator and brings about a large DNA conformational change at high 1d concentration. Interestingly, no such conformational change was observed for an analog (1a, Figure 1), lacking a crucial benzyl substituent. The large conformational change in DNA was reflected in the unusually large induced CD (ICD) signal of the ligand, that appeared in a sigmoidal fashion accompanying ligand binding at high ligand:DNA ratio. However, ICD8 only indirectly reports any underlying structural change. In contrast, Atomic Force Microscopy (AFM) can provide direct high-resolution image of single DNA molecules, free or ligand-bound, at nanometer resolution. Here we report the morphological changes in DNA at varying ligand:DNA ratios (1d and 1a) and show that supercoiled plasmid DNA converts into an over-supercoiled plectonemic structure upon increasing 1d:DNA ratio, finally leading to DNA condensation, not observed for 1a. Appearance of the superstructure


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is sequence dependent (GC-specific). Similar DNA superstructure formation, followed by DNA condensation at high ligand:DNA ratios, was also observed for doxorubicin (dox, Figure 1), a well-known intercalator and also a potent anticancer compound used for the treatment of cancers.9 Thus, the unique sequence-dependent formation of DNA superstructure, along the condensation pathway, could be a common feature of at least some DNA intercalators at high intercalator:DNA ratios. Such superstructure formation could play a role in the recently reported histone eviction property by dox at relatively high concentrations.10

MATERIAL AND METHODS Materials: pBR322 plasmid DNA was purchased from New England Biolabs (UK). pYUB12 plasmid DNA (GC rich) was provided by Prof. Sujoy Kumar Das Gupta (Bose Institute, Kolkata). pARL (AT rich plasmid DNA) was provided by Prof. Alan F. Cowman (The Walter and Eliza Hall institute of Medical Research, Australia). Poly GC [sodium salt], poly AT [sodium salt] were purchased from Sigma- Aldrich in vials of 10 unit. The lyophilized content of the vials were dissolved in 1 mL of water. The molar extinction coefficient in M-1cm-1 of nucleotide units are: Ɛ260 : 6650 for poly [d(A-T)].poly [d(A-T)], Ɛ254 :8400 for poly [d(G-C)] and the concentrations were determined accordingly.11 3aminopropyltriethoxysilane (APTES) was purchased from Sigma-Aldrich. Doxorubicin was purchased from TCI chemicals. ASTM V1 grade Mica was purchased from MICAFAB, Chennai, India. All AFM experiments were performed with nuclease-free water (QIAGEN). Plasmid isolation kit was purchased from MN (Macherey-Nagel). Atomic Force Microscopy (AFM): AFM was performed using Pico plus 5500 ILM AFM (Agilent Technologies USA) operating in AAC mode. AFM image was obtained using micro fabricated silicon cantilevers (resonance frequency of 150-300 kHz and spring constant of 2198 N/m). Images (9 µM x 9 µM) were taken in (256 x 256) pixel resolution with a scan speed of 0.5 line/second. Images were processed using Pico view1.1 version software (Agilent Technologies, USA). Sample preparation: Preparation of APTES modified MICA: MICA was freshly cleaved using tape and modified with 3-aminopropyltriethoxysilane (APTES) using vaporization method.12 In this method 20 µL APTES was kept in small container and the freshly cleaved


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Biochemistry

surface of MICA was exposed in APTES vapor and then incubated in dark for 1 hour. After that the MICA was removed and used for DNA immobilization. Sample preparation of pBR322 and 1d: pBR322 DNA was treated with 1d in 10 mM TrisCl buffer (pH 7.0), 10 mM NaCl at (1d:DNA bp) ratio from 2:1 to 4:1 and incubated for 5 mins. Then the 1d-DNA complexes were diluted in water containing MgCl2 to a final concentration containing 1.75 ng/µL of DNA. A total of 10 µL sample was deposited on APTES treated MICA and kept for 3 min. The sample was rinsed thoroughly with 2 mL of Nuclease free water and dried under vacuum. Sample preparation of pBR322 with 1a and doxorubicin: The sample preparation was similar as described above with 1d. Preparation of sample with pYUB12 and pARL plasmid DNA: Plasmids were amplified in DH5α and purified by plasmid isolation kit (MN). The sample preparation was same as described above for pBR322. Preparation of sample with poly GC and poly AT DNA was same as described above for pBR322 DNA. Circular Dichroism (CD): CD experiments were carried out by a Jasco-815 spectropolarimeter (Jasco, Easton, MD, USA) at 20 ± 0.5 oC using Jasco temperature controller (model PFD425 L/15). A rectangular quartz cuvette with a 1 cm path length was used. CD spectra were collected from 230 to 600 nm at scanning speed of 200 nm/min. In Vitro Single Nucleosome Assembly: Nucleosomes were assembled using Epimark nucleosome assembly kit according to the manufacturer protocol. The assembled single nucleosomes were then treated with increasing concentration of doxorubicin in 50 mM Tris-Cl buffer (pH 7.0). The samples were then analyzed using 6% poly-acrylamide gel electrophoresis. Following electrophoresis the gel was first soaked with EtBr to see the DNA and further silver staining experiment was carried out using PAGE-silver staining kit (Fermentas) in order to visualize the histones.


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RESULTS AND DISCUSSION AFM and CD of 1d with pBR322 plasmid DNA

Figure 2. AFM images and CD spectra of pBR322 in presence of 1d and 1a. AFM images (scale 50 nm) of pBR322 plasmid DNA, in absence of 1d (A), in presence of 1d (B-E) and in presence of 1a (F) at varying ligand:DNA bp ratios (indicated in each panel). (G) Height (DNA) versus [1d]/[DNA] obtained from AFM images. The heights (and standard deviations) were obtained from four independent DNA images with six points per molecule (total 24 points). (H) CD spectra of pBR322 (10 µM) with increasing concentrations of 1d. (I) Integrated ellipticity of the 320 nm and 450 nm band (panel H) as a function of [1d].

Figure 2A shows the AFM image of pBR322 supercoiled plasmid DNA in absence of 1d. The supercoiled DNA unwinds at 2:1 1d/DNA bp ratio (Figure 2B) with a slight increase in DNA height (see Figure 2G for height versus 1d/DNA bp ratio; representative images containing AFM height data for all 1d/pBR322 combinations are given in Figures S1-S3). Figure 2C shows the same DNA at 2.5:1 1d/DNA bp ratio, where looping of DNA and formation of nodes are seen. Upon further increase of 1d/DNA bp ratio (to 3:1), the structure changes predominantly to plectonemic oversupercoiled DNA (Figure 2D); the height of the DNA (Figure 2G) also increases. Figure 2E shows that 4:1 1d/DNA bp ratio induces compaction and condensation of DNA. A variety of 1d-induced condensed structures of pBR322 were observed at 4:1 1d/DNA bp ratio (Figure S4). The plasmid pBR322 did not show any such plectonemic oversupercoiling or DNA condensation in presence of 1a (nonbenzyl analog of 1d) even at a 6:1 1a/DNA bp ratio (Figure 2F). As a control, pBR322 condensation was also followed with spermine, a known polyamine DNA condensing agent (Figure S5). Interestingly, spermine-induced condensation occurred at much higher ligand:DNA ratio (~1000) than 1d-induced condensation. Overall, the AFM data clearly point towards a unique DNA superstructure, induced by 1d, along the path to condensation. It


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Biochemistry

also showed the indispensability of the benzyl group in DNA superstructure formation and condensation. Circular dichroism (CD) is an important technique to probe binding and structural changes in DNA induced by small molecules. Compound 1d was reported earlier7 to exhibit ICD with calf thymus DNA (ctDNA) in a sigmoidal fashion, whereas for 1a there was no ICD. With pBR322, 1d also exhibited ICD similar to seen earlier with ctDNA (Figure 2H). The intensities of the 320 nm and the 450 nm band (Figure 2I) showed a sigmoidal transition with increasing [1d], which correlated with the concentration dependent changes in height of DNA in AFM (Figure 2G). At both wavelengths, the ICD started to appear around 2.5:1.0 1d/DNA bp ratio where superstructures started nucleating in AFM image (Figure 2C) and reached a plateau around 4.0:1.0 1d/DNA bp ratio where DNA condensation was observed in AFM (Figure 2E). While ICD8 originates from close asymmetric interaction between 1d molecules, the AFM data showed that ICD appeared only when DNA superstructure formed. Together, the AFM and the CD data, suggest that DNA superstructure formation is associated with close asymmetric interaction between 1d molecules.

AFM and CD of dox with pBR322 plasmid DNA DNA intercalators such as dox also condense DNA.13 However, intermediates along the dox-induced DNA condensation pathway have not been characterized. As shown in Figure 3A, at 0.1:1 dox/DNA bp ratio, the pBR322 plasmid DNA is unwound with a slight increase in the average height of DNA (see Figure 3F for height versus dox/DNA bp ratio;; see Figures S6-S8 for AFM height data for representative dox/pBR322 combinations). Further increasing the dox/DNA bp ratio to 0.25:1 (Figure 3B) caused positive supercoiling and looping with substantial increase in the average height of DNA (Figure 3F). At dox/DNA bp ratio of 0.5:1, oversupercoiled plectonemic structures started to appear (Figure 3C); the average height of DNA increases in a sigmoidal fashion, reaching a plateau at this dox/DNA bp ratio (Figure 3F). At further dox/DNA bp ratio (1:1) formation of toroidally supercoiled DNA was observed (Figure 3D). Eventually increasing the dox/DNA bp ratio to 4:1 induced DNA condensation (Figure 3E).The AFM data clearly showed that dox, similar to 1d, also induces DNA superstructure formation along the condensation pathway. The common sigmoidal trend in height increase, for both 1d and dox treatment, also point towards a common phenomenon. Both in presence of dox and 1d, the AFM pictures of pBR322 showed


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the appearance of nodes at the junction of overlapping strands. The nodes represent clustering of intercalated ligands, bring the strands of DNA together.

Figure 3. AFM images and CD spectra of pBR322 in presence of dox. (A-E) AFM images (scale 50 nm) of pBR322 plasmid DNA in presence of dox at varying ligand:DNA bp ratios (indicated in each panel). (F) Height (DNA) versus [dox]/[DNA] obtained from AFM images. The heights (and standard deviations) were obtained from four independent DNA images with six points per molecule (total 24 points). (G) Concentration dependent CD spectra of dox in presence and absence of pBR322 (20 µM). (H) λmax shift and intensity difference (doxDNA - dox) of the 450 nm band as a function of [dox].

Unlike 1d, dox is associated with intrinsic CD signal which can render the interpretation of DNA-dox titration CD data complicated. Nevertheless, titration of pBR322 with dox was monitored by CD spectroscopy (Figures S9-S10) upto 6:1 dox:DNA ratio, much beyond the completion of intercalation (~ 0.3:1 dox:DNA ratio).14 For the sake of noninterference by DNA CD signal, here we focus on the 450 nm band of dox (Figure 3G), whose change in intensity (as well as λmax) was found to be different in absence and in presence of DNA. As summarized in Figure 3H, the λmax (dox only) showed a sigmoidal hypsochromic transition with ~ 35 µM as the midpoint, reflecting self-association of dox.15 Interestingly, in presence of DNA, a similar sigmoidal transition in λmax, albeit bathochromic, was also observed at the same transition midpoint. This probably corresponds to an altered mode of dox aggregation in presence of DNA. This was followed by a sigmoidal hypsochromic transition, almost identical to what was seen for pure dox except a shift in the transition midpoint by about 35 µM (new midpoint ~ 70 µM). The same transition was also reflected in the intensity difference (dox-DNA - dox) of the 450 nm band. This transition reflects dox self-association, after DNA condensation. The initial intercalation event (which


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Biochemistry

is over by ~ 0.3:1 dox:DNA ratio) is reflected in the initial bathochromic shift of the 450 nm band (~ 5 nm), as was reported earlier in the 280 nm ICD band.14 Thus, the DNA structural changes observed in AFM correlated with unique CD signatures in the 450 nm band. The appearance of new inter-molecular interactions of dox in presence of DNA, yielding new CD signatures, correlated with the appearance of nodes observed in the AFM data.

AFM of 1d and dox with GC-rich, AT-rich plasmids.

Figure 4. AFM images of pYUB12: (A) control, (B) with 1d(2.5:1 1d/DNA bp), (C) with dox(0.5:1 dox/DNA bp). AFM images of pARL: (D) control, (E) with 1d(2.0:1 1d/DNA bp), (F) with dox(0.5:1 dox/DNA bp). AFM images of polyGC:(G) control, (H) with 1d(4:1 1d/DNA bp), (I) with 1d (6:1 1d/DNA bp). AFM images of polyAT: (J) control, (K) with 1d (4:1 1d/DNA bp), (L) with 1d (6:1 1d/DNA bp). Scale 50 nm.

Sequence specificity on DNA structural changes induced by 1d and dox was studied using a GC-rich (pYUB12 with 65% GC-containing pAL5000 region) and a AT-rich (pARL with 67 % AT-content) plasmid. At 2.5:1.0 1d/DNA bp ratio, the GC-rich plasmid (see Figures 4A and 4D for AFM pictures of ligand free GC-rich and AT-rich plasmids) formed


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nodes and looping of oversupercoiled DNA leading to DNA compaction (Figure 4B). At comparable 1d/DNA bp ratio (2.0:1.0) the AT-rich plasmid showed negligible compaction (Figure 4E). In presence of dox(0.5:1.0 DNA bp ratio) the GC-rich plasmid also showed formation of loops and nodes (Figure 4C). However, at identical ligand/DNA bp ratio, dox showed more surface-binding than inducing structural change or compaction with AT-rich plasmid (Figure 4F). The AFM data clearly shows that DNA compaction and structural changes brought about by 1d or dox in DNA plasmids is strongly sequence dependent and is favored in GC-rich sequences. The observed sequence-specificity led us to investigate interactions of 1d with polyGC and polyAT DNA sequences. AFM images of polyGC (see Figures 4G and 4J for AFM pictures of ligand free polyGC and polyAT DNA), at 4:1 1d/DNA bp ratio (Figure 4H), exhibited intramolecular interaction leading to looping from one end. There was also the formation of “tennis racquet shapes”, reported in GC-rich regions as a pathway to DNA condensation.16 Indeed, when the 1d/DNA bp ratio was increased to 6:1, DNA condensation was observed (Figure 4I). In contrast, AFM images of polyAT at 4:1 1d/DNA bp ratio (Figure 4K) showed intermolecular interaction between different AT strands and clustering of DNA. Unlike polyGC, DNA condensation was not observed at higher 1d/DNA bp ratios (Figure 4L), rather there enhanced surface binding and intermolecular interactions were observed.

CD of polyGC/AT DNA and GC-/AT-rich plasmids with 1d Our earlier observation7 of correlation between AFM and CD data prompted us to measure ICD of 1d in presence of the polyGC, polyAT, GC-rich and AT-rich plasmids. In presence of polyGC, 1d yielded negative ICD bands at 320 nm and 440 nm (Figure 5A). In presence of polyAT the 320 nm band was slightly red shifted. The 440 nm band showed exciton coupling giving rise to a negative peak ~ 450 nm and a positive peak ~ 380 nm (Figure 5B). As shown in Figure 5C, in presence of polyGC the ICD440 of 1d showed the expected sigmoidal negative growth (see Figure S11 for full spectra with polyGC and polyAT), as seen earlier for the pBR322 plasmid (Figure 2H). In contrast, in presence of polyAT, the ICD450 began with a sigmoidal negative growth but soon the signal strength decreases, slowly approaching zero with increasing [1d]. This decrease is correlated with a growth in ICD380 (exciton coupling). Interestingly, a sudden burst in growth of ICD380 was


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Biochemistry

observed after about 150 µM of [1d] (data not shown), roughly after ICD450 approached zero, indicating surface binding. Clearly the binding modes of 1d to polyAT and polyGC sequences are different, with more surface binding of 1d to polyAT, as was also observed in AFM.

Figure 5. (A) CD spectra of 15 µM polyGC in presence of 1d (70 µM). (B) CD spectra of 15 µM polyAT in presence of 1d (100 µM). (C) Integrated ellipticity of 450 nm and 380 nm ICD bands of 1d in presence of polyAT and polyGC. (D) Integrated ellipticity of 450 nm and 380 nm ICD bands of 1d in presence of pYUB12 and pARL.

The ICD spectrum in presence of AT-rich plasmid was slightly different than ICD in presence of GC-rich plasmid (Figure 5D). The intensities of the 440/450 and the 380 nm bands (Figure 5D) were also monitored as a function of [1d] in presence of GC-rich and ATrich plasmids (see Figure S12-S13 for full spectra). Like polyGC, the 450 nm band for GCrich plasmid showed a negative sigmoidal growth. The AT-rich plasmid too showed a similar sigmoidal transition but a lower value of [1d]. ICD450 started to diminish after the transition was over. This correlated with the growth of ICD380, which remained undetectable until the end of the sigmoidal transition. The small proportion of GC stretches present in the AT-rich plasmid is responsible for the initial sigmoidal growth of the negative ICD450. However, once saturated, surface binding of 1d occurs at the AT-regions and the ICD signals reproduce the trend observed for polyAT DNA. A model for 1d-induced DNA superstructure formation We showed that both 1d and dox induce DNA superstructure formation at high concentrations, ultimately leading to DNA condensation. Since charge neutralization of


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negative phosphate groups of DNA is a prerequisite for DNA condensation and both 1d and dox are positively charged at neutral pH, one could argue that it is the positive charge rather than intercalation property that is responsible for condensation. However, 1a, also positively charged, did not induce condensation. Lacking a benzyl group but otherwise identical to 1d, 1a does not intercalate DNA. Therefore we can conclude that 1d and dox induces superstructure formation and condensation by the virtue of their positive charges and intercalation property. But how would intercalated 1d or dox induce DNA superstructure formation? AFM data showed the DNA superstructures to be associated with inter-strand interactions through nodes, representing self-associated intercalator molecules. Benzyl-benzyl self-association was earlier proposed to be responsible for DNA structural change by 1d.7 Interestingly, variants of 1d with a more polar aromatic moiety in place of the benzyl group (pyridine or furan) abolished DNA condensation or DNA superstructure formation under comparable ligand:DNA ratio (unpublished data). Without a benzyl group, 1a was also incapable of forming DNA superstructure or induce DNA condensation. This suggests that benzyl-benzyl interaction of DNA-intercalated 1d, possibly driven by hydrophobicity, is responsible for DNA superstructure formation. The superstructure condenses at even higher chargeneutralized state. This is shown schematically in Figure 6. A similar phenomenon may be operable in dox-DNA interaction at high dox:DNA ratios. An earlier analysis of dox binding to ctDNA17 showed that at higher drug content, after all intercalation sites are occupied, the intercalated dox partially overhangs, serving as a scaffold to form aggregates. Selfassociation of the overhung dox is possibly the key to inducing the DNA superstructures by dox.

Figure 6. A proposed model of intercalation-induced DNA condensation by 1d with plectonemic DNA superstructure as the on-pathway intermediate.


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Correlation between superstructure formation and histone eviction by dox The anticancer drug dox causes a topological change in DNA and blocks DNA topoisomerse II18 giving rise to its anticancer activity. Recently it was shown10 that at higher concentrations (~20 µM), close to the peak plasma level in dox-treated patients,19 dox causes histone eviction, which could be a cause of its cardiotoxicity. As a possible mechanism, it was also suggested that the amino sugar of dox, present in the minor groove of DNA-bound dox crystal structure20, competes with an important H4 Arg residue (responsible for stabilization of nucleosome structure) for the same minor groove. If indeed this is the mechanism then histone eviction should occur at lower concentrations too, where intercalation occurs. To check this hypothesis, we performed a concentration-dependent histone eviction experiment with dox. Histone eviction experiments were performed by running gel electrophoresis of in vitro assembled single nucleosomes, treated with increasing amounts of dox (or 1d/1a; see below). The resulting gel was visualized with EtBr (for visualising DNA in nucleosome) or silver-staining (for visualising histone protein). Simultaneous disappearance of the nucleosome band (EtBr staining) and the histone band (silver staining) would indicate histone eviction. As shown in Figure 7A, it was found that histone eviction by dox is not operative at lower concentrations. In fact, the observed concentration-dependent histone eviction followed a trend that is strongly correlated with dox-induced DNA superstructure formation. Both histone eviction and DNA superstructure formation occurs at dox:DNA ratio of 0.6:1, much beyond that required for simple intercalation (0.1:1). This suggests that DNA superstructue formation could be the mechanism that triggers histone eviction by dox. Interestingly, an analogue of dox lacking the amino sugar moiety is incapable of histone eviction. We believe that the amino sugar moiety is important for dox-induced superstructure formation by playing a role in selfassociation. Histone eviction experiments were also performed with 1d since it also formed DNA superstructures. As shown in Figure 7B, it was found that 1d also evicts histone in a concentration dependent manner, at a much higher concentration than dox, as expected from the higher 1d concentrations required to bring about large scale DNA conformational changes in AFM. It should be noted that while DNA superstructure formation occurred at ~ 3:1 1d:DNA ratio, histone eviction required much higher 1d:DNA (~ 10:1). This could have arisen due to different sized DNA used in AFM and the histone eviction experiment. Also, the histone eviction experiment required high salt concentration (~60 mM NaCl) that is ACS Paragon Plus Environment

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known to require higher 1d concentrations for DNA binding (data not shown). Histone eviction experiments were also performed with 1a, identical to 1d except for an important benzyl moeity and incapable of superstructure formation. As shown in Figure 7B, no histone eviction was observed for 1a, even at 12.5:1 1a/DNA ratio. Histone eviction experiments with 1d, capable of DNA superstructure formation, and 1a, incapable of DNA superstructure formation, clearly show that histone eviction and DNA superstructure formation are correlated. Therefore, our observation of histone eviction by dox and its correlation with DNA superstructure formation may be a general phenomenon and not necessarily restricted to dox-DNA interaction.

Figure 7. A. EtBr-stained native polyacrylamide gel electrophoresis (PAGE) of in vitro assembled single nucleosomes treated with dox. B. Silver-stained gel of panel A (histone visualization).10 C. EtBr-stained native polyacrylamide gel electrophoresis (PAGE) of in vitro assembled single nucleosomes treated with 1d (with dox and 1a for comparison). D. Silver-stained gel of panel C (histone visualization).10 DNA base-pair concentration was 32 µM for all panels (N and C stand for nucleosome and unbound DNA, respectively).

CONCLUSION In summary, here we report a new phenomenon of DNA superstructure formation induced by two intercalators, 1d and dox, at high ligand:DNA ratios. The superstructures lie along the pathway of DNA condensation induced by 1d or dox. We also show that the superstructure formation may be responsible for the earlier reported histone eviction by dox.


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DNA superstructure formation by 1d and dox and its correlation with histone eviction need to be investigated further with other known intercalators.

SUPPLEMENTARY DATA Supporting information contains Figures (S1-S13).

ACKNOWLEDGEMENT We thank Sujoy Dasgupta and Alan F. Cowman for gifting us pYUB12 and pARL plasmids, T. Muruganandan assisted the AFM experiments. SD acknowledges CSIR (Grant No. BSC0120) and DBT (Grant No. BT/PR6922/BRB/10/1144/2012) for financial support.

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Intercalator induced DNA superstructure formation: doxorubicin and a

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