Photoinduced DNA Damage Research Articles

(Invited) Impact of Anchors on the Photophysical Processes in Water-Soluble Fullerene Conjugates

Fullerenes are excellent acceptors in both photoinduced electron- and energy-transfer reactions. Many studies on the photophysical processes of fullerenes have been reported in the last decades, especially aiming towards the development of electronic devices. However, only a few studies have been reported on biocompatible fullerene systems, mainly due to the extremely low solubility of C60 in both water and water-miscible solvents. In our previous studies, we reported the synthesis of fullerene-PEG conjugates and demonstrated their ability for photoinduced DNA damage and ROS generation upon visible light irradiation.2 In this work, we investigated the effect of the water-soluble anchors on the photophysical properties of fullerene molecules. The generation of ROSs in water-soluble fullerene-peptide conjugates were tested by electron paramagnetic resonance (EPR) methods in comparison to previously reported fullerene-PEG conjugates above. To better understand the mechanism in ROS generation, a comprehensive investigation was performed by transient absorption spectroscopic data, encompassing the dynamics of photon absorption, exciton formation, and charge transfer mechanisms. Additionally, insights derived from density functional theory (DFT) calculations and in vitro assessments of photocytotoxicity will be discussed in detail. Figure 1

(Invited) Reactive Oxygen Generation from Photoexcited Fullerene-Peptide Conjugates

Fullerenes have been considered as excellent candidates for photosensitizers (PSs) in photodynamic therapy (PDT). Fullerenes are reported to generate reactive oxygen species (ROSs), through energy transfer (singlet oxygen 1O2, Type II reaction) and electron transfer (reduced active oxygen radicals such as superoxide anion radical O2-• and hydroxyl radical •OH, Type I reaction) in an effective way (Figure 1a).1,2 In our previous study, we reported the synthesis of fullerene-PEG conjugates and demonstrated their ability for photoinduced DNA damage and ROS generation upon visible light irradiation.2 In this work, we report the synthesis of a highly water-soluble fullerene-peptide conjugate. The fullerene-peptide conjugates are well-dispersed in aqueous media in comparation to fullerene-PEG conjugates, as indicated by dynamic light scattering (DLS) measurement as well as cryo-TEM microscopy. The ROSs generation capacity of fullerene-peptide conjugate, detected by electron paramagnetic resonance (EPR), DNA damage and in vitro photocytotoxicity tests will also be presented. Figure 1

Development of 4,4′-dibromobinaphthalene analogues with potent photo-inducible DNA cross-linking capability and cytotoxicity towards breast MDA-MB 468 cancer cells

Photoinduced DNA cross-linking process showed advantages of high spatio-temporal resolution and control. We have designed, synthesized, and characterized several 4,4′-dibromo binaphthalene analogues (1a-f) that can be activated by 350 nm irradiation to induce various DNA damage, including DNA interstrand cross-links (ICL) formation, strand cleavages, and alkaline labile DNA lesions. The degree and types of DNA damage induced by these compounds depend on the leaving groups of the substrates, pH value of the buffer solution, and DNA sequences. The DNA ICL products were produced from the carbocations formed via the oxidation of free radicals photo-generated from 1a-f. Most of these compounds alone exhibited minimum cytotoxicity towards cancer cells while 350 nm irradiation greatly improved their anticancer effects (up to 40-fold enhancement) because of photo-induced cellular DNA damage. This work provides guidance for further design of photo-inducible DNA cross-linking agents as potent photo-activated anticancer prodrugs with good control over toxicity and selectivity.

(Invited, Digital Presentation) Aggregation Switchable Fullerene-Peptides Conjugates

Intensive research has been applied on chemical functionalization of fullerenes over the past decades, aiming at utilizing their broad availabilities in chemical biology field. Chemical moieties was induced to improve their water solubility and thus enhance their properties or interaction with biomolecules. In our previous study, we reported the synthesis of fullerene-PEG conjugates and demonstrated their ability for photoinduced DNA damage and ROS generation under visible light irradiation.In this work, we report a water-soluble fullerene derivative by constructing a fullerene-peptide conjugates. The aggregation behavior as well as bio properties has been systematically studied for this series of fullerene-peptide conjugates (Figure 1a). By altering the attached peptides, fullerenes could exist as monomer or micelle in aqueous phase (Figure 1b). Figure 1

Spatial and Temporal Resolution of the Oxygen-Independent Photoinduced DNA Interstrand Cross-Linking by a Nitroimidazole Derivative.

DNA damage is ubiquitous in nature and is at the basis of emergent treatments such as photodynamic therapy, which is based on the activation of highly oxidative reactive oxygen species by photosensitizing O2. However, hypoxia observed in solid tumors imposes the necessity to devise oxygen-independent modes of action able to induce DNA damage under a low oxygen concentration. The complexity of these DNA damage mechanisms in realistic environments grows exponentially when taking into account light absorption and subsequent excited-state population, photochemical and (photo)-redox reactions, the multiple species involved in different electronic states, noncovalent interactions, multiple reaction steps, and the large number of DNA reactive sites. This work tackles all the intricate reactivity of a photosensitizer based on a nitroimidazole derivative reacting toward DNA in solution under UV light exposition. This is performed through a combination of ground- and excited-state quantum chemistry, classical molecular dynamics, and hybrid QM/MM simulations to rationalize in detail the formation of DNA interstrand cross-links (ICLs) exerted by the noncanonical noncovalent photosensitizer. Unprecedented spatial and temporal resolution of these phenomena is achieved, revealing that the ICL is sequence-specific and that the fastest reactions take place at AT, GC, and GT steps involving either the opposite nucleobases or adjacent Watson–Crick base pairs. The N7 and O6 positions of guanine, the N7 and N3 sites of adenine, the N4 position of cytosine, and the O2 atom of thymine are deemed as the most nucleophile sites and are positively identified to participate in the ICL productions. This work provides a multiscale computational protocol to study DNA reactivity with noncovalent photosensitizers, and contributes to the understanding of therapies based on photoinduced DNA damage at molecular and electronic levels. In addition, we believe the depth understanding of these processes should assist the design of new photosensitizers considering their molecular size, electronic properties, and the observed regioselectivity toward nucleic acids.

Photo-induced telomeric DNA damage in human cancer cells.

Herein we report on the study of novel dinuclear ruthenium(ii) complexes designed to target and to photo-react with G-quadruplex telomeric DNA. Upon irradiation, complexes efficiently generate guanine radical cation sites as photo-oxidation products. The compounds also display efficient cell penetration with localization to the nucleus and show strong photocytotoxicity toward osteosarcoma cells. Thanks to a microscopic-based telomere dysfunction assay, which allows the direct visualization of DNA damage in cells, we brought the first evidence of forming photo-oxidative damage at telomeres in cellulo. This emphasizes interesting prospects for the development of future cancer phototherapies.

Photo-induced mitochondrial DNA damage and NADH depletion by -NO2 modified Ru(II) complexes.

Two mitochondria-localized Ru(ii) complexes with photo-labile ligands were reported to exert one- and two-photon activatable anticancer activity through a dual-function mechanism, i.e. mitochondrial DNA covalent binding after photo-induced ligand dissociation and photo-catalyzed NADH depletion, thus displaying good activity towards cisplatin-resistant cancer cells under both normoxic and hypoxic conditions.

Proton Transfer Reaction Rates in Phenol-Ammonia Cluster Cation.

Proton transfer (PT) in an interaction system of a hydroxyl-amino group (OH-NH) plays a crucial role in photoinduced DNA and enzyme damage. A phenol-ammonia cluster is a prototype of an OH-NH interaction and is sometimes used as a DNA model. In the present study, the reaction dynamics of phenol-ammonia cluster cations, [PhOH-(NH3)n]+ (n = 1-5), following ionization of the neutral parent clusters, were investigated using a direct ab initio molecular dynamics (AIMD) method. In all clusters, PTs from PhOH+ to (NH3)n were found postionization, the reaction of which is expressed as PhOH+-(NH3)n → PhO-H+(NH3)n. The time of the PT was calculated as 43 (n = 1), 26 (n = 2), and 13 fs (n = 3-5), suggesting that the rate of PT increases with an increase in n and is saturated at n = 3-5. The difference in the PT rate originates strongly from the proton affinity of the (NH3)n cluster. In the case of n = 3-5, a second PT was found, the reaction of which is expressed as PhO-H+(NH3)n → PhO-NH3-H+(NH3)n-1, and a third PT occurred at n = 4 and 5. The time of the PT was calculated as 10-13 (first PT), 80-100 (second PT), and 150-200 fs (third PT) in the case of larger clusters (n = 4 and 5). The reaction mechanism based on the theoretical results is discussed herein.

Label-free surface-enhanced Raman spectroscopy with artificial neural network technique for recognition photoinduced DNA damage

Taking advantage of surface-enhanced Raman scattering (SERS) methodology with its unique ability to collect abundant intrinsic fingerprint information and noninvasive data acquisition we set up a SERS-based approach for recognition of physically induced DNA damage with further incorporation of artificial neural network (ANN). As a proof-of-concept application, we used the DNA molecules, where the one oligonucleotide (OND) was grafted to the plasmonic surface while complimentary OND was exposed to UV illumination with various exposure doses and further hybridized with the grafted counterpart. All SERS spectra of entrapped DNA were collected by several operators using the portable spectrometer, without any optimization of measurements procedure (e.g., optimization of acquisition time, laser intensity, finding of optimal place on substrate, manual baseline correction, etc.) which usually takes a significant amount of operator's time. The SERS spectra were employed as input data for ANN training, and the performance of the system was verified by predicting the class labels for SERS validation data, using a spectra dataset, which has not been involved in the training process. During that phase, accuracy higher than 98% was achieved with a level of confidence exceeding 95%. It should be noted that utilization of the proposed functional-SERS/ANN approach allows identifying even the minor DNA damage, almost invisible by control measurements, performed with common analytical procedures. Moreover, we introduce the advanced ANN design, which allows not only classifying the samples but also providing the ANN analysis feedback, which associates the spectral changes and chemical transformations of DNA structure.

Light exposure during growth increases riboflavin production, reactive oxygen species accumulation and DNA damage in Ashbya gossypii riboflavin-overproducing strains.

The overproduction of riboflavin (vitamin B2) by Ashbya gossypii, one of the most distinctive traits of this filamentous hemiascomycete, has been proposed to act as an ecological defense mechanism, since it is triggered by environmental stress. The interaction of endogenous riboflavin with light generates reactive oxygen species (ROS) and induces oxidative DNA damage in mammalian cells, but exogenous riboflavin was shown to protect A. gossypii spores against ultraviolet light. Envisioning a better understanding of this biotechnologically relevant trait, here we investigated the putative genotoxic effects associated with the overproduction of riboflavin by A. gossypii. For assessing that we developed the Ashbya Comet Assay, which was able to reproducibly measure oxidative (H2O2/menadione-mediated) and non-oxidative (camptothecin-mediated) DNA damage in A. gossypii. Using this protocol, we determined that exposure to sunlight-mimicking light during growth significantly increased the DNA damage accumulation in riboflavin-overproducing cells, but not in non-overproducing ones. The exposure of overproducing cells to light induced the intracellular accumulation of ROS and increased the production of riboflavin 1.5-fold. These results show that riboflavin-overproducing strains are highly susceptible to photo-induced oxidative DNA damage and draw attention for the importance of controlling the exposure to light of biotechnological riboflavin production processes with A. gossypii.

Photoactive platinum(II) complexes of nonsteroidal anti-inflammatory drug naproxen: Interaction with biological targets, antioxidant activity and cytotoxicity

The effect on the therapeutic efficacy of Pt(II) complexes on combining non-steroidal anti-inflammatory drugs (NSAIDs) is an attractive strategy to circumvent chronic inflammation mediated by cancer and metastasis. Two square-planar platinum(II) complexes: [Pt(dach)(nap)Cl] (1) and [Pt(dach)(nap)2] (2), where dach = (1R,2R)-dichloro(cyclohexane-1,2-diamine) and NSAID drug naproxen (nap), have been designed for studying their biological activity. The naproxen bound to the Pt(II) centre get released upon photoirradiation with low-power UV-A light as confirmed by the significant enhancement in emission intensities of the complexes. The compounds were evaluated for their photophysical properties, photostability, reactivity with 5′-guanosine monophophosphate (5′-GMP), interactions with CT-DNA and BSA, antioxidant activity and reactive oxygen species mediated photo-induced DNA damage properties. ESI-MS studies demonstrated the formation of bis-adduct with 5′-GMP and the formation of PtII-DNA crosslinks by gel electrophoretic mobility shift assay and ITC studies. The interaction of the complexes 1 and 2 with the CT-DNA exhibits potential binding affinity (Kb ∼ 104 M−1, Kapp∼ 105 M−1), implying intercalation to CT-DNA through planar naphthyl ring of the complexes. Both the complexes also exhibit strong binding affinity towards BSA (KBSA∼ 105 M−1). The complexes exhibit efficient DNA damage activity on irradiation at 365 nm via formation of singlet oxygen (1O2) and hydroxyl radical (•OH) under physiological conditions. Both the complexes were cytotoxic in dark and exhibit significant enhancement of cytotoxicity upon photo-exposure against HeLa and HepG2 cancer cells giving IC50 values ranging from 8 to 12 μM for 1 and 2. The cellular internalization data showed cytosolic and nuclear localization of the complexes in the HeLa cells.

Heteroleptic monometallic and trimetallic ruthenium(II) complexes incorporating a π-extended dipyrrin ligand: Light-activated reactions with the A549 lung cancer cell line

A heteroleptic monometallic ruthenium(II) and a heteroleptic trimetallic ruthenium(II) complex have been synthesized and characterized. Both complexes have an overall 3+ charge, with the charge density greater for the monometallic complex. The electronic spectra of the monometallic ruthenium(II) complex exhibits intense π-π* transitions associated with the bipyridyl groups along with overlapping metal to ligand charge transfer (MLCT) and ligand centered π-π* transitions ranging from 520nm to approximately 600nm. The trimetallic ruthenium(II) complex, on the other hand, displays more well defined transitions with the expected π-π* transition of the bipyridyl groups at 294nm and Ru(dπ) to bpy(π*) MLCT transitions at 355nm and 502nm. In addition to these absorption bands an intense transition, 578nm, resulting from overlapping dipyrrin (π-π*) and Ru(dπ) to dipyrrin(π*) transitions is observed. Electrochemical and spectroelectrochemical experiments were used to help in assigning these transitions. Irradiation of the complexes in the presence of plasmid DNA within the photodynamic therapy window (600nm to 850nm) reveal, using electrophoresis, that both complexes are capable of causing photo-damage to the DNA backbone. The trimetallic ruthenium(II) complex; however, also shows the ability to generate photoinduced DNA damage in the absence of oxygen, suggesting a photo-oxidative process. Studies of the complexes toward lung cancer cells (A549 cell line) in the absence of light indicate little cytotoxicity up to 50μM. Upon irradiation of the cells with a low power 420nm light source the trimetallic complex showed considerably greater photo-cytotoxicity compared to the monometallic analog. A dose-dependent response curve gives an IC50 of 92μM for complex B.

Ruthenium(II) complexes of saccharin with dipyridoquinoxaline and dipyridophenazine: Structures, biological interactions and photoinduced DNA damage activity

Ruthenium complexes trans-[Ru(sac)2(dpq)2] (1) and trans-[Ru(sac)2(dppz)2] (2) where sac is artificial sweetener saccharin (o-sulfobenzimide; 1,2-benzothiazole-3(2H)-one1,1-dioxide (Hsac)), dpq = dipyrido[3,2-d:2′,3′-f]quinoxaline and dppz = dipyrido[3,2-a:2′,3′-c]phenazine have been synthesized and thoroughly characterized using various analytical and spectral techniques. Saccharin known to act as carbonic anhydrase IX (CA IX) inhibitor which is a biomarker for highly aggressive and proliferative tumor in hypoxic stress, so inhibition of CA IX is a potential strategy for anticancer chemotherapy. The solid state structures, photophysical properties, photostability, DNA and protein binding affinity, and DNA photocleavage activity were explored. The structural analysis revealed Ru(II) centre is in discrete mononuclear, distorted octahedral {RuN6} coordination geometry with two monoanionic nitrogen donor saccharinate ligands and two neutral bidentate nitrogen donors ligands dpq and dppz. cis-[Ru(sac)2(dppz)2] (cis-2) geometrical isomer was also isolated and structurally characterized by X-ray crystallography. The photo-induced dissociation of monodentate saccharin ligand is observed when irradiated at UV-A light of 365 nm. The complexes show significant binding affinity to the calf thymus DNA (Kb ∼ 105 M−1) through significant intercalation through planar dpq and dppz ligands. Interaction of complexes 1 and 2 with bovine serum albumin (BSA) showed remarkable tryptophan emission quenching (KBSA ∼105 M−1). The complexes showed appreciable photoinduced DNA cleavage activity upon irradiation of low power UV-A light of 365 nm from supercoiled (SC) to its nicked circular (NC) form at micromolar complex concentrations. Photocleavage mechanistic studies in presence of O2 reveals involvement of reactive oxygen species (ROS) mediated through ligand-centered 3ππ* and/or 3MLCT excited states generated upon photoactivation leads to nicking of supercoiled DNA to nicked circular form. In absence of O2, we also observed photocleavage of DNA through formation of photoinduced ligand dissociated Ru-DNA complex involving PACT pathway.

Dual-Sensitized Luminescent Europium(ΙΙΙ) and Terbium(ΙΙΙ) Complexes as Bioimaging and Light-Responsive Therapeutic Agents.

Dual-photosensitized stable EuΙΙΙ and TbΙΙΙ complexes, namely [Eu(dpq)(tfnb)3 ] (1) and [Tb(dpq)(tfnb)3 ] (2), in which dpq=dipyrido[3,2-d:2',3'-f]quinoxaline and Htfnb=4,4,4-trifluoro-1-(2-napthyl)-1,3-butanedione, were designed as bioimaging and light-responsive therapeutic agents. Their X-ray structures, photophysical properties, biological interactions, photoinduced DNA damage, photocytotoxicity, and cellular uptake properties were studied. Discrete mononuclear complexes adopt an eight-coordinated {LnN2 O6 } distorted square antiprism geometry with bidentate N,N-donor dpq and O,O-donor tfnb ligands. The designed probes have the advantage of dual-sensitizing antennae (dpq, Htfnb) to modulate their desirable optical properties for cellular imaging and light-responsive intracellular damage. The remarkable photostability, absence of inner-sphere water (q<1), and longer excited-state lifetimes of the complexes make them suitable as cellular-imaging probes. The dpq 3 T state is well located energetically to allow efficient energy transfer (ET) to the emissive 5 D0 and 5 D4 states of EuΙΙΙ and TbΙΙΙ . This leads to higher quantum yields (φ=0.15-0.20) in aqueous media and makes these compounds suitable cellular-imaging probes. The complexes display significant binding ability toward DNA and bovine serum albumin (K≈105 m-1 ). They effectively cleave supercoiled DNA to its nicked circular form at λ=365 nm through photoredox pathways. The cellular internalization studies showed cytosolic and nuclear localization. The remarkable photocytotoxicity of these probes offers a strategy towards developing photoresponsive LnΙΙΙ probes as cellular-imaging and phototherapeutic agents.

Anticancer potential of a photoactivated transplatin derivative containing the methylazaindole ligand mediated by ROS generation and DNA cleavage.

The limitations associated with the clinical utility of conventional platinum anticancer drugs have stimulated research leading to the design of new metallodrugs with improved pharmacological properties, particularly with increased selectivity for cancer cells. Very recent research has demonstrated that photoactivation or photopotentiation of platinum drugs can be one of the promising approaches to tackle this challenge. This is so because the application of irradiation can be targeted exclusively to the tumor tissue so that the resulting effects could be much more selective and targeted to the tumor. We show in this work that the presence of 1-methyl-7-azaindole in trans-[PtCl2(NH3)(L)] (L = 1-methyl-7-azaindole, compound 1) markedly potentiated the DNA binding ability of 1 when irradiated by UVA light in a cell-free medium. Concomitantly, the formation of cytotoxic bifunctional cross-links was markedly enhanced. In addition, 1, when irradiated with UVA, was able to effectively cleave the DNA backbone also in living cells. The incorporation of 1-methyl-7-azaindole moiety had also a profound effect on the photophysical properties of 1, which can generate singlet oxygen responsible for the DNA cleavage reaction. Finally, we found that 1, upon irradiation with UVA light, exhibited a pronounced dose-dependent decrease in viability of A2780 cells whereas it was markedly less cytotoxic if the cells were treated in the absence of light. Hence, it is possible to conclude that 1 is amenable to photodynamic therapy.

Photosensitized samarium(iii) and erbium(iii) complexes of planar N,N-donor heterocyclic bases: crystal structures and evaluation of biological activity

A series of Sm(iii) and Er(iii) complexes of N,N-donor heterocyclic bases were studied for their crystal structures, luminescence properties, binding with biomolecules and photo-induced DNA damage activity.

Monitoring one-electron photo-oxidation of guanine in DNA crystals using ultrafast infrared spectroscopy.

To understand the molecular origins of diseases caused by ultraviolet and visible light, and also to develop photodynamic therapy, it is important to resolve the mechanism of photoinduced DNA damage. Damage to DNA bound to a photosensitizer molecule frequently proceeds by one-electron photo-oxidation of guanine, but the precise dynamics of this process are sensitive to the location and the orientation of the photosensitizer, which are very difficult to define in solution. To overcome this, ultrafast time-resolved infrared (TRIR) spectroscopy was performed on photoexcited ruthenium polypyridyl-DNA crystals, the atomic structure of which was determined by X-ray crystallography. By combining the X-ray and TRIR data we are able to define both the geometry of the reaction site and the rates of individual steps in a reversible photoinduced electron-transfer process. This allows us to propose an individual guanine as the reaction site and, intriguingly, reveals that the dynamics in the crystal state are quite similar to those observed in the solvent medium.

Fine-tuning recA expression in Staphylococcus aureus for antimicrobial photoinactivation: importance of photo-induced DNA damage in the photoinactivation mechanism.

Bacterial cell envelope is generally accepted as the primary target for a photo-induced oxidative stress. It is plausible that DNA damage occurs during the antimicrobial photoinactivation. Here we investigate the correlation between DNA damage and photoinactivation by evaluating the level of RecA-based DNA repair system in Staphylococcus aureus. By using exogenous photosensitizers (new methylene blue (NMB), toluidine blue O (TBO), 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin tetra(p-toluenesulfonate) (TMPyP), zinc phthalocyanine (ZnPc), Rose Bengal (RB)) and ALA-induced endogenous porphyrin-dependent blue light (405 nm), several outcomes were observed: (i) an increase of DNA damage (from gel electrophoresis in DNA damage assay), (ii) an increase of recA expression (luminescence assay in recA-lux strain), and (iii) an increase of RecA protein level (Western blotting). When recA expression was repressed by novobiocin, or abolished by deleting the gene, S. aureus susceptibility towards photoinactivation was increased at approximately a hundred-fold. The absence of RecA increases DNA damage to yield bactericidal effect. In novobiocin-resistant mutant (gyrB), as opposed to wild type, neither RecA protein level nor cell’s susceptibility was affected by photoinactivation (when novobiocin is present). This is to suggest that GyrB-dependent inhibition mediated recA repression. Therefore, we have established the role of RecA in DNA damage during photoinactivation. With the use of rifampicin mutation frequency and Ames tests, we demonstrated that photoinactivation did not increase S. aureus mutagenesis and potentially is not mutagenic toward eukaryotic cells. The results suggest that the treatment is considered safe. In conclusion, we provide an evidence that recA inhibitor may serve as therapeutic adjuvant for antimicrobial photoinactivation. Clinical relevance of our findings warrants further investigations.

Photoinduced DNA damage and cytotoxicity by a triphenylamine-modified platinum-diimine complex

Many planar photosensitizers tend to self-aggregate via van der Waals interactions between π-conjugated systems. The self-aggregation of the photosensitizer may reduce the efficiency of the photosensitizer to generate singlet oxygen, thereby diminishing its photodynamic activity. Efforts have been made to improve the photodynamic activity of bis-(o-diiminobenzosemiquinonato)platinum(II) which has planar geometry by the introduction of the sterically hindered triphenylamine moiety into the ligand. Herein we report the photoinduced DNA damage and cytotoxicity by a triphenylamine-modified platinum-diimine complex in red light studied by fluorescence spectra, agarose gel assay and cell viability assay. The results suggest that the triphenylamine-modified platinum-diimine complex has better capability to generate singlet oxygen than bis-(o-diiminobenzosemiquinonato)platinum(II), and it can induce DNA damage in red light, causing high photocytotoxicity in HepG-2 cells in vitro.

Interaction of 6 Mercaptopurine with Calf Thymus DNA – Deciphering the Binding Mode and Photoinduced DNA Damage

DNA is one of the major intracellular targets for a wide range of anticancer and antibiotic drugs. Elucidating the binding between small molecules and DNA provides great help in understanding drug-DNA interactions and in designing of new and promising drugs for clinical use. The ability of small molecules to bind and interfere with DNA replication and transcription provides further insight into how the drugs control the expression of genes. Interaction of an antimetabolite anticancer drug 6mercaptopurine (6MP) with calf thymus DNA was studied using various approaches like UV-visible spectroscopy, fluorescence spectroscopy, CD, viscosity and molecular docking. UV-visible spectroscopy confirmed 6MP-DNA interaction. Steady state fluorescence experiments revealed a moderate binding constant of 7.48×103 M−1 which was consistent with an external binding mode. Competitive displacement assays further confirmed a non-intercalative binding mode of 6MP which was further confirmed by CD and viscosity experiments. Molecular docking further revealed the minimum energy conformation (−119.67 kJ/mole) of the complex formed between DNA and 6MP. Hence, the biophysical techniques and in-silico molecular docking approaches confirmed the groove binding/electrostatic mode of interaction between 6MP and DNA. Further, photo induced generation of ROS by 6MP was studied spectrophotometrically and DNA damage was assessed by plasmid nicking and comet assay. There was a significant increase in ROS generation and consequent DNA damage in the presence of light.