DNA Photosensitization Research Articles

Unprecedented enantio-selective live-cell mitochondrial DNA super-resolution imaging and photo-sensitizing by the chiral ruthenium polypyridyl DNA "light-switch".

Mitochondrial DNA (mtDNA) is known to play a critical role in cellular functions. However, the fluorescent probe enantio-selectively targeting live-cell mtDNA is rare. We recently found that the well-known DNA 'light-switch' [Ru(phen)2dppz]Cl2 can image nuclear DNA in live-cells with chlorophenolic counter-anions via forming lipophilic ion-pairing complex. Interestingly, after washing with fresh-medium, [Ru(phen)2dppz]Cl2 was found to re-localize from nucleus to mitochondria via ABC transporter proteins. Intriguingly, the two enantiomers of [Ru(phen)2dppz]Cl2 were found to bind enantio-selectively with mtDNA in live-cells not only by super-resolution optical microscopy techniques (SIM, STED), but also by biochemical methods (mitochondrial membrane staining with Tomo20-dronpa). Using [Ru(phen)2dppz]Cl2 as the new mtDNA probe, we further found that each mitochondrion containing 1-8 mtDNA molecules are distributed throughout the entire mitochondrial matrix, and there are more nucleoids near nucleus. More interestingly, we found enantio-selective apoptotic cell death was induced by the two enantiomers by prolonged visible light irradiation, and in-situ self-monitoring apoptosis process can be achieved by using the unique 'photo-triggered nuclear translocation' property of the Ru complex. This is the first report on enantio-selective targeting and super-resolution imaging of live-cell mtDNA by a chiral Ru complex via formation and dissociation of ion-pairing complex with suitable counter-anions.

Development and application of metallo-phthalocyanines as potent G-quadruplex DNA binders and photosensitizers

Metallo-phthalocyanines (MPc) are common photosensitizers with ideal photophysical and photochemical properties. Also, these molecules have shown to interact with non-canonical nucleic acid structures, such as G-quadruplexes, and modulate oncogenic expression in cancer cells. Herein, we report the synthesis and characterisation of two metallo-phthalocyanines containing either zinc (ZnPc) or nickel (NiPc) in the central aromatic core and four alkyl ammonium lateral chains. The interaction of both molecules with G-quadruplex DNA was assessed by UV–Vis, fluorescence and FRET melting experiments. Both molecules bind strongly to G-quadruplexes and stabilise these structures, being NiPc the most notable G-quadruplex stabiliser. In addition, the photosensitizing ability of both metal complexes was explored by the evaluation of the singlet oxygen generation and their photoactivation in cells. Only ZnPc showed a high singlet oxygen generation either by direct observation or by indirect evaluation using a DPBF dye. The cellular evaluation showed mainly cytoplasmic localization of ZnPc and a decrease of the IC50 values of the cell viability of ZnPc upon light activation of two orders of magnitude.Graphical abstractTwo metallo-phthalocyanines containing zinc and nickel within the aromatic core have been investigated as G-quadruplex stabilizers and photosensitizers. NiPc shows a high G4 binding but negligible photosensitizing ability while ZnPc exhibits a moderate binding to G-quadruplex together with a high potency to generate singlet oxygen and photocytotoxicity. The interaction with G4s and capacity to be photosensitized is associated with the geometry adopted by the central metal core of the phthalocyanine scaffold.

A DNA and mitochondria dual-targeted photosensitizer for two-photon-excited bioimaging and photodynamic therapy.

Biological substrates and organelle multi-targeted photosensitizers for ultra-efficient cancer treatment through photodynamic therapy (PDT) are highly desirable. Herein, a multiple pyridinium-anchored photosensitizer containing the triphenylamine unit, TPA-2PI, has been designed and utilized for DNA and mitochondria dual-targeted two-photon-excited bioimaging and PDT. TPA-2PI possesses an ultrahigh ABDA consumption rate of 194.84 nmol min-1 in PBS medium, and an ultralow IC50 value of 0.108 μM upon white-light irradiation (80 mW cm-2, 6 min). After irradiation, the abnormal mitochondria have been remarkable occurred in 4T1 cells and further induced cell apoptosis for the efficient PDT efficacy by TPA-2PI. Upon two-photon excitation (λex = 960 nm), the green fluorescence signal of DCFH-DA showed a dramatic turn-on effect. Furthermore, TPA-2PI could penetrate deeper cells (24 μm) and abdominal blood vessels (384 μm). The investigation of the TPA-2PI-triggered PDT effect in vivo further validated the efficacy of cancer cell ablation and significant inhibition of tumor growth. Moreover, the immunofluorescence staining results and blood biochemical parameters after treatment proved the bio-safety of TPA-2PI. The dual-targeted TPA-2PI can serve as a desirable photosensitizer for efficient cancer treatment, and this work sheds light on the development of biological substrate and organelle multi-targeted photosensitizers for future biological applications.

Remote Photodamaging of DNA by Photoinduced Energy Transport.

Local DNA photodamaging by light is well‐studied and leads to a number of structurally identified direct damage, in particular cyclobutane pyrimidine dimers, and indirect oxidatively generated damage, such as 8‐oxo‐7,8‐hydroxyguanine. Similar damages have now been found at remote sites, at least more than 105 Å (30 base pairs) away from the site of photoexcitation. In contrast to the established mechanisms of local DNA photodamaging, the processes of remote photodamage are only partially understood. Known pathways include those to remote oxidatively generated DNA photodamages, which were elucidated by studying electron hole transport through the DNA about 20 years ago. Recent studies with DNA photosensitizers and mechanistic proposals on photoinduced DNA‐mediated energy transport are summarized in this minireview. These new mechanisms to a new type of remote DNA photodamaging provide an important extension to our general understanding to light‐induced DNA damage and their mutations.

Formulation induces direct DNA UV-A photooxidation. Part II. Pro-oxidant effect of formulated Vitamin E via generation of singlet oxygen

The study focuses on the photosensitization of DNA in nanoemulsion-type systems containing Vitamin E under UVA radiation. Cetyltriethylammonium bromide (CTEAB) surfactant was first combined with Vitamin E to form a CTEAB-Vitamin E nanoemulsion to complex the plasmid pBR322, in TRIS/HCl buffer. The self-organization properties of the CTEAB-Vitamin E system were studied by complementary physico-chemical characterizations (tensiometry, pyrene fluorimetry and scattering experiments). It formed a nanoemulsion composed of CTEAB micelles (that can incorporate Vitamin E) as well as larger Vitamin E droplets. This nanoemulsion was then contacted with DNA and the resulting formulated CTEAB-Vitamin E-DNA lipoplex was irradiated under UVA radiation (λ ≥ 335 nm). The presence of Vitamin E generated a significant enhancement of the DNA photodamage as shown by agarose gel electrophoresis and quantified by photodensitometry. It underlines the pro-oxidant role of the Vitamin E in this self-assembled system. A mechanistic study based on the use of specific scavengers reveals that the singlet oxygen is responsible for the UVA DNA photooxidation in the CTEAB-Vitamin E-DNA lipoplex.

Photoinduced intersystem crossing in DNA oxidative lesions and epigenetic intermediates.

The propensity of 5-formyluracil and 5-formylcytosine, i.e. oxidative lesions and epigenetic intermediates, in acting as intrinsic DNA photosensitizers is unraveled by using a combination of molecular modeling, simulation and spectroscopy. Exploration of potential energy surfaces and non-adiabatic dynamics confirm a higher intersystem crossing rate for 5-formyluracil, whereas the kinetic models evidence different equilibria in the excited states for both compounds.

Unprecedented Enantiospecific Live-cell Nuclear DNA Targeting and Photosensitizing of the Chiral DNA Light-Switch Ru(II) Cationic Complex via Simple Ion-Pairing with Chlorophenolic Counter-anions

Argopecten nucleus is a small size pectinid, whose farming and hatchery technology have been developed at experimental and pilot scales. In order to assess the effectiveness of biotechnological tools for the improvement of productive traits, the effects of triploidy induction on its growth and survival rates at different developmental stages were analyzed. Thus, zygotes that underwent cold-shock treatment (12 °C for 15 min) and untreated zygotes were cultured in the laboratory during their embryonic, larval and post-larval stages, while juvenile and adults were farmed in sea-based suspended systems. Ploidy condition was verified by cytofluorometry. Growth was similar in induced to triploid and untreated larvae and post-larvae. Similarly, no differences were found for shell growth and wet weight in confirmed triploid juveniles and adults, in comparison to diploid individuals. In addition, although no effects of the triploidy induction treatment was observed on the survival of larvae and post-larvae, triploid juveniles and adults exhibited a lower survival than diploids. Thus, the triploid condition did not to confer advantages in terms of growth; instead it caused a decrease on the survival of juveniles and adults, negatively impacting the biomass yield of this pectinid species.

Radio- and photosensitization of plasmid DNA by DNA binding ligand propidium iodide: Investigation of Auger electron induction and detection of Cherenkov-emission

We investigated whether propidium iodide (PI) enhances DNA damaging effects of ionizing and non-ionizing radiation species (X-rays, alpha-, beta-, auger electron emission and light of various wavelengths, respectively). This biophysical experimental setting allowed us, furthermore, to investigate whether Cherenkov emission can be detected by photodynamic effects and increased DNA damage. Conformation changes of plasmid DNA were detected and quantified by gelelectrophoresis and fluorescence imaging. Hydrogen peroxide, stannous dichloride, and dimethylsulfoxide were used as chemical modulators, Tc-99m, Re-188, Ra-223, and x-ray (32 kV and 200 kV) reflected radiotoxicity and light (λ = 254 nm, 366 nm and 530-575 nm) induced phototoxicity. Radiotracers and x-rays induced dose dependent DNA damage. PI did not serve as radiosensitizer in radioisotopes, while a low effect was detected in X-rays. The phototoxicity was dependent on the wavelengths of light. Light with a wavelength range of 530-575 nm in combination with PI resulted in direct DNA damage. The yield of Cherenkov emission was far below the photon emission of light irradiation and not distinguishable from general radiotoxicity. PI binds to plasmid DNA, is not chemotoxic, and increases radiotoxicity only to minor extent. Phototoxicity and its stimulation by PI is dependent on the wavelength of the light. No kind of energy deposition was capable of inducing an Auger electron cascade. Furthermore, no increase in DNA damage induced by photodynamic effects from Cherenkov emission was detectable.

Generation of the Thymine Triplet State by Through-Bond Energy Transfer.

Benzophenone (BP) and drugs containing the BP chromophore, such as the non-steroidal anti-inflammatory drug ketoprofen, have been widely reported as DNA photosensitizers through triplet-triplet energy transfer (TTET). In the present work, a direct spectroscopic fingerprint for the formation of the thymine triplet (3 Thy*) by through-bond (TB) TTET from 3 BP* has been uncovered. This has been achieved in two new systems that have been designed and synthesized with one BP and one thymine (Thy) covalently linked to the two ends of the rigid skeleton of the natural bile acids cholic and lithocholic acid. The results shown here prove that it is possible to achieve triplet energy transfer to a Thy unit even when the photosensitizer is at a long (nonbonding) distance.

Resolving the Singlet Excited State Manifold of Benzophenone by First-Principles Simulations and Ultrafast Spectroscopy.

Accurate characterization of the high-lying excited state manifolds of organic molecules is of fundamental importance for the interpretation of the rich response detected in time-resolved nonlinear electronic spectroscopies. Here, we have characterized the singlet excited state manifold of benzophenone (BP), a versatile organic photoinitiator and a well-known DNA photosensitizer. Benchmarks of various multiconfigurational/multireference (RASSCF/PT2) and time-dependent density functional theory (TD-DFT) approaches allowed assignments of experimental linear absorption signals of BP in the ultraviolet (UV) region, with unprecedented characterization of ground state absorptions in the far UV. Experimental transient absorption spectra obtained by UV-vis pump-probe spectroscopy at very short time delays are shown to be directly comparable to theoretical estimates of excited state absorptions (from the low-lying nOπ* and ππ* singlet states) in the Franck-Condon region. Multireference computations provided reliable interpretation of the PP spectra, with TD-DFT results yielding a fair agreement as long as electronic transitions featuring double excitations contributions are not involved. These results lay the groundwork for further computational studies and interpretation of experimental nonlinear electronic spectra of benzophenone in more complex systems, such as BP/DNA adducts.

Decoding the Molecular Basis for the Population Mechanism of the Triplet Phototoxic Precursors in UVA Light-Activated Pyrimidine Anticancer Drugs.

The photosensitization of DNA by thionucleosides is a promising photo-chemotherapeutic treatment option for a variety of malignancies. DNA metabolization of thionated prodrugs can lead to cell death upon exposure to a low dose of UVA light. The exact mechanisms of thionucleoside phototoxicity are still not fully understood. In this work, we have combined femtosecond broadband transient absorption experiments with state-of-the-art molecular simulations to provide mechanistic insights into the ultrafast and efficient population of the triplet state in the UVA-activated pyrimidine anticancer drug 4-thiothymine. The triplet state is thought to act as a precursor to DNA lesions and the reactive oxygen species responsible for 4-thiothymine photocytotoxicity. The electronic-structure and mechanistic results presented in this contribution reveal key molecular design criteria that can assist in developing alternative chemotherapeutic agents that may overcome some of the primary deficiencies of classical photosensitizers.

From non-covalent binding to irreversible DNA lesions: nile blue and nile red as photosensitizing agents.

We report a molecular modeling study, coupled with spectroscopy experiments, on the behavior of two well known organic dyes, nile blue and nile red, when interacting with B-DNA. In particular, we evidence the presence of two competitive binding modes, for both drugs. However their subsequent photophysical behavior is different and only nile blue is able to induce DNA photosensitization via an electron transfer mechanism. Most notably, even in the case of nile blue, its sensitization capabilities strongly depend on the environment resulting in a single active binding mode: the minor groove. Fluorescence spectroscopy confirms the presence of competitive interaction modes for both sensitizers, while the sensitization via electron transfer, is possible only in the case of nile blue.

The (6–4) Dimeric Lesion as a DNA Photosensitizer

AbstractThe front cover artwork is provided by Miguel A. Miranda and co‐workers at Instituto de Tecnología Química (UPV‐CSIC). The image shows that (6–4) photoproducts, primary UVB dimeric lesions, act as photosensitizers of cyclobutane pyrimidine dimers that are produced as secondary DNA lesions. Read the full text of the article at 10.1002/cphc.201600154.

Cover Picture: The (6–4) Dimeric Lesion as a DNA Photosensitizer (ChemPhysChem 13/2016)

Cover Picture: The (6–4) Dimeric Lesion as a DNA Photosensitizer (ChemPhysChem 13/2016)

The (6-4) Dimeric Lesion as a DNA Photosensitizer.

Based on our previous investigations into the photophysical properties of the 5-methyl-2-pyrimidone (Pyo) chromophore, we now extend our studies to the photobehavior of the dimeric (6-4) thymine photoproducts (6-4 PP) to evaluate their capability to act as instrinsic DNA photosensitizers. The lesion presents significant absorption in the UVB/UVA region, weak fluorescence emission, a singlet-excited-state energy of approximately 351 kJ mol(-1) , and a triplet-excited-state energy of 297 kJ mol(-1) . Its triplet transient absorption has a maximum at 420-440 nm, a lifetime of around 7 μs, and a high formation quantum yield, ΦISC =0.86. This species is efficiently quenched by thymidine. Its DNA photosensitizing properties are demonstrated by a series of experiments run on a pBR322 plasmid. The lesion photoinduces both single-strand breaks and the formation of cyclobutane thymine dimers. Altogether, these results show that, the substitution of the pyrimidone ring at C4 by a 5-hydroxy-5,6-dihydrothymine does not cancel out the photosensitization properties of the chromophore.

Hydrogen abstraction by photoexcited benzophenone: consequences for DNA photosensitization.

We report a computational investigation of the hydrogen abstraction (H-abstraction) induced by triplet benzophenone ((3)BP) on thymine nucleobase and backbone sugar. The chemical process is studied using both high level multiconfigurational perturbation and density functional theory. Both methods show good agreement in predicting small kinetic barriers. Furthermore the behavior of benzophenone in DNA is simulated using molecular dynamics and hybrid quantum mechanics/molecular mechanics methods. The accessibility of benzophenone to the labile hydrogens within B-DNA is demonstrated, as well as the driving force for this reaction. We evidence a strong dependence of the H-abstraction with the non-covalent BP-DNA interaction mode, and a reaction that is less favorable when embedded in DNA than for the isolated system.

5-Bromo-2'-deoxycytidine-a potential DNA photosensitizer.

A double-stranded oligonucleotide, 80 base pairs in length, was multiply labeled with 5-bromo-2'-deoxycytidine (BrdC) using polymerase chain reaction (PCR). The modified oligonucleotide was irradiated with 300 nm photons and its damage was assayed by employing DHPLC, LC-MS and denaturing polyacrylamide gel electrophoresis (PAGE). Two types of damage were demonstrated, namely, single strand breaks (SSBs) and intrastrand cross-links (ICLs); the ICLs were in the form of d(G^C) and d(C^C) dimers. The former species are probably formed due to photoinduced electron transfer between the photoexcited BrdC and the ground state 2'-deoxyguanosine (dG), whereas the latter is a result of a cycloaddition reaction. Since SSBs and ICLs are potentially lethal to the cell, BrdC could be considered as a nucleoside with possible clinical applications.

DNA Photosensitization by an “Insider”: Photophysics and Triplet Energy Transfer of 5‐Methyl‐2‐pyrimidone Deoxyribonucleoside

The main chromophore of (6-4) photoproducts, namely, 5-methyl-2-pyrimidone (Pyo), is an artificial noncanonical nucleobase. This chromophore has recently been reported as a potential photosensitizer that induces triplet damage in thymine DNA. In this study, we investigate the spectroscopic properties of the Pyo unit embedded in DNA by means of explicit solvent molecular-dynamics simulations coupled to time-dependent DFT and quantum-mechanics/molecular-mechanics techniques. Triplet-state transfer from the Pyo to the thymine unit was monitored in B-DNA by probing the propensity of this photoactive pyrimidine analogue to induce a Dexter-type triplet photosensitization and subsequent DNA damage.

Mechanisms of Damage to DNA Labeled with Electrophilic Nucleobases Induced by Ionizing or UV Radiation.

Hypoxia--a hallmark of solid tumors--makes hypoxic cells radioresistant. On the other hand, DNA, the main target of anticancer therapy, is not sensitive to the near UV photons and hydrated electrons, one of the major products of water radiolysis under hypoxic conditions. A possible way to overcome these obstacles to the efficient radio- and photodynamic therapy of cancer is to sensitize the cellular DNA to electrons and/or ultraviolet radiation. While incorporated into genomic DNA, modified nucleosides, 5-bromo-2'-deoxyuridine in particular, sensitize cells to both near-ultraviolet photons and γ rays. It is believed that, in both sensitization modes, the reactive nucleobase radical is formed as a primary product which swiftly stabilizes, leading to serious DNA damage, like strand breaks or cross-links. However, despite the apparent similarity, such radio- and photosensitization of DNA seems to be ruled by fundamentally different mechanisms. In this review, we demonstrate that the most important factors deciding on radiodamage to the labeled DNA are (i) the electron affinity (EA) of modified nucleoside (mNZ), (ii) the local surroundings of the label that significantly influences the EA of mNZ, and (iii) the strength of the chemical bond holding together the substituent and a nucleobase. On the other hand, we show that the UV damage to sensitized DNA is governed by long-range photoinduced electron transfer, the efficiency of which is controlled by local DNA sequences. A critical review of the literature mechanisms concerning both types of damage to the labeled biopolymer is presented. Ultimately, the perspectives of studies on DNA sensitization in the context of cancer therapy are discussed.

Radio- and photosensitization of DNA with compounds containing platinum and bromine atoms

Irradiations of plasmid DNA by both X-rays and UV light in the presence and absence of compounds containing platinum and bromine atoms were performed in order to asses the sensitization potential of these compounds. Plasmid DNA pBR322 was incubated with platinum (II) bromide, hydrogen hexabromoplatinate (IV), hydrogen hexahydroxyplatinate (IV) and sodium hexahydroxyplatinate (IV). Incubation was followed by X-ray or UV irradiations. It was found that amongst the sensitizers tested, during irradiations carried out in the presence of platinum (II) bromide, the highest levels of double strand breaks formation upon X-ray treatment were recorded. In contrast much less damage was induced by UV light. Data presented here suggests that this compound may be a promising radiosensitizer for cancer treatment.