Strand Scission Research Articles

In vitro cytotoxcity and interaction of new steroidal oxadiazinanones with calf thymus DNA using molecular docking, gel electrophoresis and spectroscopic techniques

Herein we report synthesis of new steroidal oxadiazinanones from steroidal ketones. After characterization by spectral and analytical data, the interaction studies of compounds (4–6) with DNA were carried out by UV–vis, fluorescence spectroscopy and gel electrophoresis. The compounds bind to DNA preferentially through electrostatic and hydrophobic interactions with Kb; 1.8×104M−1, 2.2×104M−1 and 2.6×104M−1, respectively, indicating the higher binding affinity of compound 6 towards DNA. Gel electrophoresis showed the concentration dependent cleavage activity of compound 6 alone or in presence of Cu (II) causes the nicking of supercoiled pBR322 and it seems to follow the mechanistic pathway involving generation of hydroxyl radicals that are responsible for initiating DNA strand scission. Molecular simulations suggest that compounds binds through minor groove of DNA. MTT assay depicted promising anticancer activity of compound 5 and 6 particularly against HL-60 and MCF-7. The apoptotic degradation of DNA was analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining (comet assay). The results revealed that compound 6 has better prospectus to act as cancer chemotherapeutic candidate which warrants further in vivo anticancer investigations.

Breaking DNA strands by extreme-ultraviolet laser pulses in vacuum.

Ionizing radiation induces a variety of DNA damages including single-strand breaks (SSBs), double-strand breaks (DSBs), abasic sites, modified sugars, and bases. Most theoretical and experimental studies have been focused on DNA strand scissions, in particular production of DNA double-strand breaks. DSBs have been proven to be a key damage at a molecular level responsible for the formation of chromosomal aberrations, leading often to cell death. We have studied the nature of DNA damage induced directly by the pulsed 46.9-nm (26.5eV) radiation provided by an extreme ultraviolet (XUV) capillary-discharge Ne-like Ar laser (CDL). Doses up to 45 kGy were delivered with a repetition rate of 3 Hz. We studied the dependence of the yield of SSBs and DSBs of a simple model of DNA molecule (pBR322) on the CDL pulse fluence. Agarose gel electrophoresis method was used for determination of both SSB and DSB yields. The action cross sections of the single- and double-strand breaks of pBR322 plasmid DNA in solid state were determined. We observed an increase in the efficiency of strand-break induction in the supercoiled DNA as a function of laser pulse fluence. Results are compared to those acquired at synchrotron radiation facilities and other sources of extreme-ultraviolet and soft x-ray radiation.

Rates of chemical cleavage of DNA and RNA oligomers containing guanine oxidation products.

The nucleobase guanine in DNA (dG) and RNA (rG) has the lowest standard reduction potential of the bases, rendering it a major site of oxidative damage in these polymers. Mapping the sites at which oxidation occurs in an oligomer via chemical reagents utilizes hot piperidine for cleaving oxidized DNA and aniline (pH 4.5) for cleaving oxidized RNA. In the present studies, a series of time-dependent cleavages of DNA and RNA strands containing various guanine lesions were examined to determine the strand scission rate constants. The guanine base lesions 8-oxo-7,8-dihydroguanine (OG), spiroiminodihydantoin (Sp), 5-guanidinohydantoin (Gh), 2,2,4-triamino-2H-oxazol-5-one (Z), and 5-carboxamido-5-formamido-2-iminohydantoin (2Ih) were evaluated in piperidine-treated DNA and aniline-treated RNA. These data identified wide variability in the chemical lability of the lesions studied in both DNA and RNA. Further, the rate constants for cleaving lesions in RNA were generally found to be significantly smaller than for lesions in DNA. The OG nucleotides were poorly cleaved in DNA and RNA; Sp nucleotides were slowly cleaved in DNA and did not cleave significantly in RNA; Gh and Z nucleotides cleaved in both DNA and RNA at intermediate rates; and 2Ih oligonucleotides cleaved relatively quickly in both DNA and RNA. The data are compared and contrasted with respect to future experimental design.

Copper–obatoclax derivative complexes mediate DNA cleavage and exhibit anti-cancer effects in hepatocellular carcinoma

Obatoclax is an indole–pyrrole compound that induces cancer cell apoptosis through targeting the anti-apoptotic Bcl-2 protein family. Previously, we developed a series of obatoclax derivatives and studied their STAT3 inhibition-dependent activity against cancer cell lines. The obatoclax analog, prodigiosin, has been reported to mediate DNA cleavage in cancer cells by coordinating with copper complexes. To gain an understanding of copper–obatoclax complex activity, we applied obatoclax derivatives to examine their copper–mediated nuclease activity as a means to establish a basis for structure activity relationship. Replacement of the indole ring of obatoclax with furanyl, thiophenyl or Boc-indolyl rings reduced the DNA cleavage ability. The same effect was achieved through the replacement of the obatoclax pyrrolyl ring with thiazolidinedione and thioacetal. Among the compounds tested, we demonstrated that the complex of obatoclax or compound 7 with copper exhibited potent DNA strand scission which correlated with HCC cell growth inhibition.

Rapid RNA Strand Scission Following C2′-Hydrogen Atom Abstraction

C2'-Nucleotide radicals have been proposed as key intermediates in direct strand break formation in RNA exposed to ionizing radiation. Uridin-2'-yl radical (1) was independently generated in single- and double-stranded RNA via photolysis of a ketone precursor. Direct stand breaks result from heterolytic cleavage of the adjacent C3'-carbon-oxygen bond. Trapping of 1 by O2 or β-mercaptoethanol (1 M) does not compete with strand scission, indicating that phosphate elimination is >10(6) s(-1). Uracil loss also does not compete with strand scission. When considered in conjunction with reports that nucleobase radicals produce 1, this chemistry explains why RNA is significantly more susceptible to strand scission by ionizing radiation (hydroxyl radical) than is DNA.

DNA Binding, Cleavage Activity, Molecular Docking, Cytotoxicity and Genotoxicity Studies of Newly Synthesized Copper Based Metal Complexes

The design and synthesis of medicinal chemotherapeutic agents of copper with 2-(benzothiazol-2-yl iminomethyl)-phenol, 2-(benzothiazol-2-yliminomethyl)-valine, cyanoaceto (2-mer- captobenzylidene)-hydrazide, 2-(phenacyl bromide)-aminothiophenol, (2-mercaptobenzaldehyde) thio- semicarbazone, N-(phenacyl bromide)-2- yliminobenzothiazole, 2-aminobenzothiazole, benzothiazol-2-yliminomethyl)-phenol and 2-[(2E¹-aminobenzylidene)- amino]-benzenethiol were synthesized. The characterization was done by FTIR, 1H and 13C NMR, MS, TGA and elemental analysis. Interaction of complexes 8 and 9 with CT DNA was done by using UV-vis and fluorescence spectroscopy depicting the hyperchromic behaviour of complexes. The intrinsic binding constants (Kb) for complex 8 and 9 were 2.35 × 103 M-1 and 2.12 × 103 M-1. The cleavage studies of complex 8 and 9 were done with pBR322 plasmid showing the potential cleaving ability of the complexes at very low concentration. The gel electrophoresis pattern also demonstrated that the complex 8 alone or in presence of Cu (II) causes the nicking of supercoiled pBR322 and it seems to follow the mechanistic pathway involving generation of hydroxyl radicals that are responsible for initiating DNA strand scission. The molecular docking studies showed the minor groove binding behaviour of the complexes 8 and 9 with DNA. During the MTT assay against different cancer cell lines like SW480, HepG2, HT29 and HL60, all the complexes showed potential cytotoxic behaviour by giving effective IC50 close to Cisplatin. The bioactivity score and PASS analysis also depicted the drug like nature of the complexes. During the comet assay, apoptotic degradation of DNA in the presence of complex 8 and 9 was analysed by agarose gel electrophoresis and visualized by ethidium bromide staining.

In Vitro DNA-Binding, Cleavage Activity with pBR322, Molecular Docking and Antiproliferative Studies of Newly Synthesized Steroidal Imidazolidine Hybrids

New steroidal imidazolidine derivatives (7-9) were synthesized by reacting steroidal thiosemicarbazones (4-6) with ethyl-2-chloroacetate in absolute ethanol. After characterization by spectral and analytical data, the interaction studies of compounds (7-9) with DNA were carried out by UV-vis, luminescence spectroscopy, circular dichroism and gel electrophoresis. The compounds bind to DNA preferentially through electrostatic and hydrophobic interactions with Kb; 2.07 × 104 M-1, 2.1 × 104 M–1 and 1.9 × 104 M–1, respectively indicating the higher binding affinity of compound 8 towards DNA. Gel electrophoresis demonstrated that the compound 8 show strong interaction with DNA and during its cleavage activity with pBR322 DNA, it seems to follow the mechanistic pathway, involving singlet oxygen and superoxide anion to generate ROS responsible for initiating DNA strand scission. The docking study suggested the intercalation of imidazolidine moiety of steroid derivative in minor groove of DNA. In MTT assay, compounds 7-9 revealed potential toxicity against different human cancer cells especially compound 8 against A549 cells. Genotoxicity of compounds (7-9) was checked by comet assay. In western blotting, the expressions of relevant apoptotic markers depicted an apoptosis by steroidal imidazolidines in A549 cells.

Synthetic access to the chemical diversity of DNA and RNA 5′-aldehyde lesions.

Hydrogen atom abstraction from the C5′-position of nucleotides in DNA results in direct strand scission by generating alkali-labile fragments from the oxidized nucleotide. The major damage consists in a terminus containing a 5′-aldehyde as part of an otherwise undamaged nucleotide. Moreover it is considered as a polymorphic DNA strand break lesion since it can be borne by any of the four nucleosides encountered in DNA. Here we propose an expeditious synthesis of oligonucleotides (ON) ending with this 5′-aldehyde group (5′-AODN). This straightforward and cheap strategy relies on Pfitzner–Moffatt oxidation performed on solid support followed by a transient protection of the resulting aldehyde function. This method is irrespective of the 5′-terminal nucleobase and most interestingly can be directly extended to RNA to produce the corresponding 5′-AORN. We also report preliminary results on recognition of 5′-AODN by base excision repair (BER) enzymes.

Amplified detection of bleomycin based on an electrochemically driven recycling strategy

The present work demonstrates a rapid and sensitive method for bleomycin (BLM) detection, taking advantage of an electrochemically driven recycling strategy. DNA probes, the substrate for BLM, are first immobilized on a gold electrode surface. Complexation of BLM with Fe2+ ions and oxygen may result in the production of the BLM·Fe3+ complex and free radicals, which may trigger an irreversible cleavage of DNA probes. Cyclic voltammetry is applied as a stimulus to regenerate the BLM·Fe2+ complex from Fe3+ species, subsequently initiating a new cleavage event. The electrochemically driven recycling of the BLM·Fe2+ complex may lead to the continuous strand scission of a large quantity of DNA probes, which can be transduced into significantly amplified electrochemical signals. With [Fe(CN)6]3−/4− as the redox species, BLM can be detected down to 0.74 pM within 10 min. Further studies reveal that the proposed method can also exhibit satisfactory performance in serum samples, and thus may provide a promising tool for the BLM assay in clinical diagnosis.

English

The characterization of sweet cherry (Prunus avium L.) genetic resources in Turkey may help to increase their use in breeding programmers worldwide, as Turkey is the centre of origin of sweet cherry. In the Malatya region, cherry leaves are gathered in April and May, fresh and dried used for stuffing. Components of cherry trees have been used as traditional herbal remedies for various diseases. These components are known to possess antioxidative effects. However, the mechanisms underlying cherry tree component-mediated antioxidative effects remain largely unknown. This study focused on cherry 0-900 Ziraat (Malatya Dalbastı) leaves methanol extract (CLME) and examined antioxidant capacity and DNA damage protecting activity. The antioxidant capacity of  these extracts were evaluated using different antioxidant tests, including reducing power, free radical scavenging, hydroxyl radical scavenging, hydrogen peroxide scavenging, 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ABTS radical scavenging, deoxyribose assay, β-Carotene bleaching assay and metal chelating activities. The content of total phenolic compounds in CLME was determined using Folin-Ciocalteus reagent and compared with standard antioxidants butylated hydroxytoluene (BHT), Trolox and α-tocopherol. CLME showed a concentration-dependent free radical scavenging capacity and protective DNA strand scission by OHË™ on pBR322 super coiled plasmid DNA (95 to 97%). Total phenolic effect on DNA cleavage CLME at 400 µg/ml exhibited significant protecting activity against the compounds in the CLME and was determined as gallic acid equivalent 132.17 meq/g dried leaves. Key words: Cherry 0-900 ziraat (Malatya dalbastı), antioxidant activity, radical scavenging ability, DNA damage, phenolic compounds.

Single-molecule views of MutS on mismatched DNA.

Base-pair mismatches that occur during DNA replication or recombination can reduce genetic stability or conversely increase genetic diversity. The genetics and biophysical mechanism of mismatch repair (MMR) has been extensively studied since its discovery nearly 50 years ago. MMR is a strand-specific excision-resynthesis reaction that is initiated by MutS homolog (MSH) binding to the mismatched nucleotides. The MSH mismatch-binding signal is then transmitted to the immediate downstream MutL homolog (MLH/PMS) MMR components and ultimately to a distant strand scission site where excision begins. The mechanism of signal transmission has been controversial for decades. We have utilized single molecule Forster Resonance Energy Transfer (smFRET), Fluorescence Tracking (smFT) and Polarization Total Internal Reflection Fluorescence (smP-TIRF) to examine the interactions and dynamic behaviors of single Thermus aquaticus MutS (TaqMutS) particles on mismatched DNA. We determined that TaqMutS forms an incipient clamp to search for a mismatch in ~1 s intervals by 1-dimensional (1D) thermal fluctuation-driven rotational diffusion while in continuous contact with the helical duplex DNA. When MutS encounters a mismatch it lingers for ~3 s to exchange bound ADP for ATP (ADP→ATP exchange). ATP binding by TaqMutS induces an extremely stable clamp conformation (~10 min) that slides off the mismatch and moves along the adjacent duplex DNA driven simply by 1D thermal diffusion. The ATP-bound sliding clamps rotate freely while in discontinuous contact with the DNA. The visualization of a train of MSH proteins suggests that dissociation of ATP-bound sliding clamps from the mismatch permits multiple mismatch-dependent loading events. These direct observations have provided critical clues into understanding the molecular mechanism of MSH proteins during MMR.

In vitro antioxidant potential and deoxyribonucleic acid protecting activity of CNB-001, a novel pyrazole derivative of curcumin

Background: Free radicals are underpinned to initiate cascade of toxic events leading to oxidative stress and resultant cell death in many neurodegenerative disorders. Now-a-days antioxidants have become mandatory in the treatment of various diseases apart from the drug's modes of action. CNB-001, a novel hybrid molecule synthesized by combining curcumin and cyclohexyl bisphenol A is known to possess various biological activities, but the antioxidative property of the compound has not yet been elucidated. Aim: The present study is aimed to analyze various free radicals scavenging by employing in vitro antioxidant assays and to evaluate the deoxyribonucleic acid (DNA) protecting the ability of CNB-001 against hydroxyl radicals. Materials and methods: The in vitro antioxidant potential of CNB-001 was evaluated by analyzing its ability to scavenge DPPH, ABTS, nitric oxide, superoxide, hydrogen peroxide, superoxide anion, hydroxyl, hydrogen peroxide radicals and reducing power using spectroscopic method. The DNA protecting activity of CNB-001 was also evaluated on pUC19 plasmid DNA subjected to hydroxyl radicals using standard agarose gel electrophoresis. Results: From the assays, it was observed that CNB-001 scavenged free radicals effectively in a dose dependent manner. CNB-001 scavenged 2,2-diphenyl-1-picrylhydrazyl (IC50 = 44.99 μg/ml), 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (IC50 = 17.99 μg/ml), nitric oxide (IC50 = 1.36 μg/ml), superoxide radical (IC50 = 77.17 μg/ml), hydrogen peroxide (IC50 = 492.7 μg/ml), superoxide (IC50 = 36.92 μg/ml) and hydroxyl (IC50 = 456.5 μg/ml) radicals effectively and the reducing power was found to be 11.53 μg/ml. CNB-001 showed considerable protecting activity against plasmid DNA (pUC19) strand scission by •OH at dose dependent manner. Conclusion: Results from these assays concluded that CNB-001 has a good antioxidant potential by reducing reactive oxygen and reactive nitrogen radicals and it showed significant protecting activity against DNA scission by hydroxyl radicals. Hence, CNB-001 can be further developed as potential drug for free radical induced neurodegenerative disorders.

Abasic and Oxidized Abasic Site Reactivity in DNA: Enzyme Inhibition, Cross-Linking, and Nucleosome Catalyzed Reactions

Abasic lesions are a family of DNA modifications that lack Watson-Crick bases. The parent member of this family, the apurinic/apyrimidinic lesion (AP), occurs as an intermediate during DNA repair, following nucleobase alkylation, and from random hydrolysis of native nucleotides. In a given day, each cell produces between 10000 and 50000 AP lesions. A variety of oxidants including γ-radiolysis produce oxidized abasic sites, such as C4-AP, from the deoxyribose backbone. A number of potent, cytotoxic antitumor agents, such as bleomycin and the enediynes (e.g., calicheamicin, esperamicin, and neocarzinostatin) also lead to oxidized abasic sites in DNA. The absence of Watson-Crick bases prevents DNA polymerases from properly determining which nucleotide to incorporate opposite abasic lesions. Consequently, several studies have revealed that (oxidized) abasic sites are highly mutagenic. Abasic lesions are also chemically unstable, are prone to strand scission, and possess electrophilic carbonyl groups. However, researchers have only uncovered the consequences of the inherent reactivity of these electrophiles within the past decade. The development of solid phase synthesis methods for oligonucleotides that both place abasic sites in defined positions and circumvent their inherent alkaline lability has facilitated this research. Chemically synthesized oligonucleotides containing abasic lesions provide substrates that have allowed researchers to discover a range of interesting chemical properties of potential biological importance. For instance, abasic lesions form DNA-DNA interstrand cross-links, a particularly important family of DNA damage because they block replication and transcription absolutely. In addition, bacterial repair enzymes can convert an interstrand cross-link derived from C4-AP into a double-strand break, the most deleterious form of DNA damage. Oxidized abasic lesions can also inhibit DNA repair enzymes that remove damaged nucleotides. DNA polymerase β, an enzyme that is irreversibly inactivated, is vitally important in base excision repair and is overproduced in some tumor cells. Nucleosome core particles, the monomeric components that make up chromatin, accentuate the chemical instability of abasic lesions. In experiments using synthetic nucleosome core particles containing abasic sites, the histone proteins catalyze strand cleavage at the sites that incorporate these lesions. Furthermore, in the presence of the C4-AP lesion, strand scission is accompanied by modification of the histone protein. The reactivity of (oxidized) abasic lesions illustrates how seemingly simple nucleic acid modifications can have significant biochemical effects and may provide a chemical basis for the cytotoxicity of the chemotherapeutic agents that produce them.

Structure, Dynamics, and Interactions of a C4′-Oxidized Abasic Site in DNA: A Concomitant Strand Scission Reverses Affinities

Apurinic/apyrimidinic (AP) sites constitute the most frequent form of DNA damage. They have proven to produce oxidative interstrand cross-links, but the structural mechanism of cross-link formation within a DNA duplex is poorly understood. In this work, we study three AP-containing d[GCGCGCXCGCGCG]·d[CGCGCGKGCGCGC] duplexes, where X = C, A, or G and K denotes an α,β-unsaturated ketoaldehyde derived from elimination of a C4'-oxidized AP site featuring a 3' single-strand break. We use explicit solvent molecular dynamics simulations, complemented by quantum chemical density functional theory calculations on isolated X:K pairs. When X = C, the K moiety in the duplex flips around its glycosidic bond to form a stable C:K pair in a near-optimal geometry with two hydrogen bonds. The X = A duplex shows no stable interaction between K and A, which contrasts with AP sites lacking a strand scission that present a preferential affinity for adenine. Only one, transient G:K hydrogen bond is formed in the X = G duplex, although the isolated G:K pair is the most stable one. In the duplex, the stable C:K pair induces unwinding and sharp bending into the major groove at the lesion site, while the internal structure of the flanking DNA remains unperturbed. Our simulations also unravel transient hydrogen bonding between K and the cytosine 5' to the orphan base X = A. Taken together, our results provide a mechanistic explanation for the experimentally proven high affinity of C:K sites in forming cross-links in DNA duplexes and support experimental hints that interstrand cross-links can be formed with a strand offset.

Repair effects of exogenous SOD on Bacillus subtilis against gamma radiation exposure

Superoxide dismutase (SOD) is an enzyme that removes free radicals from cells in many organisms. In order to further characterize these repair effects and their mechanism when subjected to radiation, Bacillus subtilis cells were exposed to gamma radiation and the cell survival rate, intracellular SOD activity, and DNA double-strand breakage were investigated. Vegetative cells of B. subtilis were irradiated by 60Co gamma radiation at varying doses and subsequently exposed to varying levels of exogenous SOD. Standard plate-count, xanthine oxidase, and pulsed-field gel electrophoresis (PFGE) methods were employed to investigate the repair effects. The results showed that the exogenous SOD could significantly improve cell survival rate and intracellular SOD activity after gamma radiation. The cell survival rate was elevated 30–87 times above levels observed in control samples. Adding exogenous SOD into gamma irradiated cells may dramatically increase intracellular SOD activity (p < 0.01), while percentage of DNA release (PR) values may decrease significantly when cells are treated with SOD. The repair effects were observed to vary with the gamma radiation dose and SOD concentration. These findings suggest that exogenous SOD may have the ability to repair vegetative B. subtilis cell damage after irradiated by gamma radiation. DNA strand scission may also be prevented by addition of SOD. This research contributes to better understanding of protection from the effects of free radicals and their mechanisms, an ongoing process in many organisms that involves the cellular response to gamma radiation, which occurs naturally in soil and water, as well as in unusual cases of high-dosage exposure.

DNA binding, docking studies, artificial nuclease activity and in vitro cytotoxicity of newly synthesized steroidal 1H–pyrimidines

Abstract A new series of steroidal pyrimidines (7–9) has been synthesized by reacting steroidal thiosemicarbazones (4–6) with ethyl cyanoacetate. The compounds were characterized by IR, 1H NMR, 13C NMR, MS and analytical data. The interaction studies of compounds 7–9 with DNA were carried out by UV–vis and luminescence spectroscopy. Compounds (7–9) bind to DNA preferentially through electrostatic and hydrophobic interactions, with Kb values found to be 6.56 × 103 M−1, 1.54 × 104 M−1 and 9.34 × 103 M−1, respectively, indicating the higher binding affinity of compound 8 towards DNA. Gel electrophoresis pattern demonstrated that compound 8 shows strong interaction with DNA and that, during its cleavage activity with pBR322 DNA, it seems to follow the mechanistic pathway involving the generation of singlet oxygen and a superoxide anion, which are responsible for initiating DNA strand scission. The docking study suggested that the intercalation of compounds in between the nucleotide base pairs is due to the presence of a pyrimidine moiety in the steroid molecule. MTT assay was carried out to check the toxicity of new compounds 7–9 against the different human cancer as well as non-cancer cell lines A545, MCF-7, HeLa, HL-60, SW480, HepG2, HT-29, A549, 184B5, MCF10A, NL-20, HPC, and HPLF. Apoptotic degradation of DNA in the presence of steroidal pyrimidines 7–9 was analysed by agarose gel electrophoresis and visualized by ethidium bromide staining (comet assay).

A transfection reporter for the prevention of false-negative results in molecular beacon experiments

We previously developed a molecular beacon-type probe to detect the strand scission in cellular base excision repair and found that the phosphodiester linkages in the fluorophore/quencher linkers were cleaved. This reaction was applied to a transfection reporter, which contained the unmodified phosphodiester in the linker to another type of fluorophore. After cotransfection of cells with the probe and the reporter, the signals were used to detect the incision and to confirm the proper transfection, respectively. This method will contribute to the prevention of false-negative results in experiments using molecular beacon-type probes.

PP075: Development of near-infrared nanoscissor for down regulation of target cancer genes

Purpose Altered gene expression is involved not only in the initiation of cancers, but also in tumor progression and treatment response. Cancer gene therapy is an emerging concept for advanced malignant disease management. However, most of current approaches in gene silencing act in the mRNA levels such as siRNA, ribozyme and anti-sense strategies. These approaches only gave partial and temporal gene expression suppression. Besides, delivery system of such approaches is also challenging. This study aimed to develop a Near-Infrared, Artificial, Targeted, Light-Activated Nanoscissor (NIR-ATLANS) to perform gene sequence specific double strand scission to permanently silence target cancer genes. Materials and methods The NIR-ATLANS comprised gold nanorod core with a monolayer of cypate-modified triplex-forming oligonucleotides (TFOs) that targets EGFP as a model system. The NIR-ATLANS activation specific wavelength is around 808 nm. After laser exposure at 808 nm peak wavelength, we used real-time polymerase chain reaction to check the gene cleavage efficiency. Results We discovered that the NIR-ATLANS could selectively target gene through electrophoretic mobility shift assay. When activated the nanoscissor by near-infrared laser, we observed decreased EGFP expression at both mRNA and protein level in the NIR-ATLANS combined lasing group, while not as significant in all other groups. Conclusions Our results showed a permanent gene silencing at the genomic level by combined ATLANS and laser activation. The gold nanorod could act as a quencher to prevent non-specific NIR-ATLANS cutting through free radical attack, thus protects non-target DNA from collateral damage. In our study, NIR-ATLANS successfully down regulate EGFP expression. Both reported gene and functional gene were able to be down regulated in cancer cell through our approach. We anticipate such technology hold a great potential in the future cancer gene therapy.

Steroidal pyrimidines: Synthesis, characterization, molecular docking studies with DNA and in vitro cytotoxicity

A series of new steroid pyrimidines (7–9) were synthesized by reacting steroidal thiosemicarbazones (4–6) with diethyl malonate. The new compounds were characterized by IR, 1H NMR, 13C NMR, MS and analytical data. The interaction studies of compounds (7–9) with DNA were carried out by employing gel electrophoresis, UV–vis and fluorescence spectroscopy. The acting force between the compounds (7–9) and DNA was mainly hydrophobic while the other interactions like van der Waals, hydrogen bonding cannot be ruled out. The gel electrophoresis pattern also demonstrated that the compound 7 alone or in presence of Cu (II) causes the nicking of supercoiled pBR322 and it seems to follow the mechanistic pathway involving generation of hydroxyl radicals that are responsible for initiating DNA strand scission. The docking study of compounds (7–9) suggested that the intercalation of compounds in between the nucleotide base pairs might be due to the presence of pyrimidine moiety in steroid molecule. MTT assay was carried out to check the toxicity of new compounds (7–9) against the different human cancer as well as non-cancer cell lines A545, MCF-7, HeLa, HL-60, SW480, HepG2, HT-29, A549, 184B5, MCF10A, NL-20, HPC and HPLF. Apoptotic degradation of DNA in presence of steroidal pyrimidines (7–9) was analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining (comet assay).

Histone Modification via Rapid Cleavage of C4′-Oxidized Abasic Sites in Nucleosome Core Particles

The C4'-oxidized abasic site is produced in DNA by a variety of oxidizing agents, including potent cytotoxic antitumor agents. Independent generation of this alkali-labile lesion at defined positions within nucleosome core particles reveals that the histone proteins increase strand scission between 130- and 550-fold. Strand scission proceeds via a Schiff base intermediate, but the DNA-protein cross-links are unstable. The oxidized abasic site is removed in its entirety from the DNA and transferred to the lysine-rich tail region of the proximal histone protein in the form of a lactam. The modification is distributed over several residues within the amino-terminal tail of the proximal histone. Transfer of DNA damage to histones could affect gene regulation.