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The use of carbon quantum dots (CDs) as trackable nanocarriers for plasmid and gene as hybrid DNA condensates has gained momentum, as evident from the significant recent research efforts. However, the in-depth morphology of the condensates, the energetics of the condensation process, and the photophysical aspects of the CD are not well understood and often disregarded. Herein, for the first time, we covalently attached linearized pUC19 with citric acid and cysteamine-derived CD through the reaction of the surface amine groups of CDs with the 5′-phospho-methyl imidazolide derivative of the plasmid to obtain a 1:1 CD-pUC19 covalent conjugate. The CD-pUC19 conjugates were further transformed into DNA condensates with spermine that displayed a toroidal morphology with a diameter of ∼200 nm involving ∼2–5 CD-pUC19 conjugates in a single condensate. While the interaction of pristine CD to spermine was exothermic, the binding of the CD-pUC19 conjugate with spermine was endothermic and primarily entropy-driven. The condensed plasmid displayed severe conformational stress and deviation from the B-form due to the compact packing of the DNA but better transfection ability than the pristine CD. The CDs in the condensates tend to come close to each other at the core that results in their shielding from excitation. However, this does not prevent them from emanating reactive oxygen species on visible light exposure that compromises the decondensation process and cell viability at higher exposure times, calling for utmost caution in establishing them as nonviral transfecting agents universally.

BackgroundIS26 plays a major role in the dissemination of antibiotic resistance determinants in Gram-negative bacteria.ObjectivesTo determine whether insertion sequence IS26 is able to move alone (simple transposition) or if it exclusively forms cointegrates.MethodsA two-step PCR using outward-facing primers was used to search for circular IS26 molecules. Gibson assembly was used to clone a synthetic IS26 containing a catA1 chloramphenicol resistance gene downstream of the tnp26 transposase gene into pUC19. IS activity in a recA−Escherichia coli containing the non-conjugative pUC19-derived IS26::catA1 construct and the conjugative plasmid R388 was detected using a standard mating-out assay. Transconjugants were screened for resistance.ResultsCircular IS26 molecules that would form with a copy-out route were not detected by PCR. The synthetic IS26::catA1 construct formed CmRTpR transconjugants (where CmR and TpR stand for chloramphenicol resistant and trimethoprim resistant, respectively), representing an R388 derivative carrying the catA1 gene at a frequency of 5.6 × 10−7 CmRTpR transconjugants per TpR transconjugant, which is comparable to the copy-in activity of the unaltered IS26. To test for simple transposition of IS26::catA1 (without the plasmid backbone), 1200 CmRTpR colonies were screened and all were resistant to ampicillin, indicating that the pUC19 backbone was present. Hence, IS26::catA1 had only formed cointegrates.ConclusionsIS26 is unable to move alone and cointegrates are the exclusive end products of the reactions mediated by the IS26 transposase Tnp26. Consequently, when describing the formation of complex resistance regions, simple ‘transposition’ of a single IS26 should not be invoked.

Copper-based binuclear enantiomeric complexes 1S and 1R were synthesized as anticancer chemotherapeutic agents to target G-quadruplex rich region of DNA and thoroughly characterized by various spectroscopic and single X-ray crystal diffraction studies. The structure elucidation of Schiff base ligand LS and complexes 1S & 1R, was carried out by single crystal X-ray studies which showed that ligand crystallized in the monoclinic P21/n space group while complexes 1S and 1R crystallized in triclinic space groups P1̄ and P1, respectively with two copper units connected to each other via an alkoxide bridge to exhibit square planar geometry which is in good agreement with other spectroscopic studies {IR, ESI-MS, EPR and magnetic moment values}. In vitro binding studies of complexes 1S and 1R were carried out with G-quadruplex DNA and CT-DNA which showed higher binding affinity and selectivity toward quadruplex DNA over the duplex DNA. To validate the potential of complexes to act as therapeutic drug candidates, the cleavage studies of complexes 1S and 1R were carried out with G-quadruplex telomeric DNA by PAGE Gel assay which showed sequence selective cleavage of 22G4 via oxidative cleavage pathway. The major cleavage sites identified were G15, T6, G8, G9, G14 for complex 1S whereas for 1R G15, G20, G21, G14 cleavage sites were observed. Furthermore, these complexes were capable of cleaving pUC19 plasmid DNA in double-stranded non-random fashion which is considered to be more potent than single-strand cleavage as a source of lethal DNA lesions. Cellular studies of 1S and 1R were performed on a panel of human cancer cell lines; Huh7, MCF7, BxPC3 and AsPC1, which displayed significant cytotoxicity and differential responses toward different cancer phenotypes.

Present study describes the synthesis of vanadium oxide nanorods (VONRs) via eco-friendly approach by using Tinospora cordifolia leaf extract. The as-synthesized VONRs were characterized by UV-Visible, FT-IR, XRD, EDX, SEM and TGA analysis. The size of VONRs were in the range of 10-40 nm. Further, the XRD data revealed that the as-obtained VONRs has typical orthorhombic phase. The biological activities such as anti-angiogenic, DNA binding and cleaving activity of the as-prepared VONRs were evaluated. Anti-angiogenic activity was carried by shell less Chorio Allantioic Membrane (CAM) assay, DNA binding and cleaving activity were performed using calf thymus DNA (CT-DNA) and supercoiled plasmid (pUC19) DNA, respectively. A marked reduction in the proliferation of capillaries around the zone of application of VONRs were observed in the anti-angiogenic assay, suggesting its biological potentiality. Furthermore, the cleavage of supercoiled DNA confirmed the involvement VONRs in the DNA interaction mechanism.

The cellulase enzymes with high effectiveness under conditions agreeable to the industrial processes necessities are one of the keys for the successful development of chemical and drug synthesis. The soil metagenome is an affluent source for the discovery of new natural products. The objective of the current study was to identify the isolated functional gene(s) of the cellulase enzyme by using metagenomics. The plan of work composed of collection of different soil samples, isolation of total DNA, fragmentation, cloning, and expression of the isolated gene(s) in the suitable host microorganism. The total genomic DNA was extracted using a kit (QIAGEN), and then digested by different restriction enzymes BamHI. The digested fragments ranging from ~300-5000 bp were ligated, cloned into pUC19 vector, and then transformed into Escherichia coli DH5α. The resulting clones were screened as cellulase producers using a qualitative method. The positive clones which showed hydrolysis on the plate were screened once more in Luria-Bertani (LB) medium. The plasmids were isolated and then tested using universal primer (M13), to detect the fragment size and sequence for the Polymerase Chain Reaction (PCR) products. This study establishes an effortless and professional method for cloning of recent cellulase genes through ecological metagenomes. In the outlook, the metagenomic guide approachs may be functional to the elevated selection of novel cellulase from the environment.

The octahedral ruthenium(II) complexes [Ru(Ln)(PPh3)2Cl2] [Ln = biphenyl furanyl pyridine derivatives] were synthesized and characterized using LC–MS, IR spectroscopy, elemental analysis and magnetic measurements. Complexes show enhancement in antibacterial activity compared to free ligands. From the binding mode investigation by absorption titration and viscosity measurement, it is observed that complexes bind to DNA via intercalation and also complexes promote the cleavage of supercoiled pUC19 plasmid DNA. Cytotoxicity analysis shows 100% mortality of Brine shrimp after 48 h. Keywords: Heterocyclic compound, Ruthenium(II) complexes, Nucleic acid intercalating agent, Cytotoxicity

The chemical stage of the Monte Carlo track-structure (MCTS) code Geant4-DNA was extended for its use in DNA strand break (SB) simulations and compared against published experimental data. Geant4-DNA simulations were performed using pUC19 plasmids (2686 base pairs) in a buffered solution of DMSO irradiated by 60Co or 137Cs γ-rays. A comprehensive evaluation of SSB yields was performed considering DMSO, DNA concentration, dose and plasmid supercoiling. The latter was measured using the super helix density value used in a Brownian dynamics plasmid generation algorithm. The Geant4-DNA implementation of the independent reaction times method (IRT), developed to simulate the reaction kinetics of radiochemical species, allowed to score the fraction of supercoiled, relaxed and linearized plasmid fractions as a function of the absorbed dose. The percentage of the number of SB after •OH + DNA and H• + DNA reactions, referred as SSB efficiency, obtained using MCTS were 13.77% and 0.74% respectively. This is in reasonable agreement with published values of 12% and 0.8%. The SSB yields as a function of DMSO concentration, DNA concentration and super helix density recreated the expected published experimental behaviors within 5%, one standard deviation. The dose response of SSB and DSB yields agreed with published measurements within 5%, one standard deviation. We demonstrated that the developed extension of IRT in Geant4-DNA, facilitated the reproduction of experimental conditions. Furthermore, its calculations were strongly in agreement with experimental data. These two facts will facilitate the use of this extension in future radiobiological applications, aiding the study of DNA damage mechanisms with a high level of detail.

In cyanobacteria DNA supercoiling varies over the diurnal light/dark cycle and is integrated with the circadian transcription program and (Woelfle et al. [2007], Vijayan et al. [2009], PNAS). Specifically, Woelfle et al. have reported that DNA supercoiling of an endogenous plasmid became progressively higher during prolonged dark phases in Synechococcus elongatus PCC 7942. This is counterintuitive, since higher levels of negative DNA supercoiling are commonly associated with exponential growth and high metabolic flux. Vijayan et al. then have reverted the interpretation of plasmid mobility on agarose gels supplemented with chloroquine diphosphate (CQ), but not further discussed the differences. Here, we set out to clarify this open issue in cyanobacterial DNA supercoiling dynamics. We first re-capitulate Keller’s band counting method (1975, PNAS) using CQ instead of ethidium bromide as the intercalating agent. A 500x–1000x higher CQ concentration is required in the DNA relaxation reaction (topoisomerase I) than in the agarose gel buffer to quench all negative supercoiling of pUC19 extracted from Escherichia coli. This is likely due to the dependence of both, the DNA binding affinity of CQ and the induced DNA unwinding angle, on the ionic strength of the buffer. Lower levels of CQ were required to fully relax in vivo pUC19 supercoiling than were used by Woelfle et al. Next, we analyzed the in vivo supercoiling of endogenous plasmids of Synechocystis sp. PCC 6803, at two different CQ concentrations. These experiments indicate that negative supercoiling of plasmids does not increase but decreases in the dark phase, and progressively decreases further in prolonged darkness.

Endonucleases play a crucial role as reagents in laboratory research and diagnostics. Here, metagenomics was used to functionally screen a fosmid library for endonucleases. A fosmid library was constructed using metagenomic DNA isolated from soil sampled from the unique environment of the Kogelberg Nature Reserve in the Western Cape of South Africa. The principle of acquired immunity against phage infection was used to develop a plate-based screening technique for the isolation of restriction endonucleases from the library. Using next-generation sequencing and bioinformatics tools, sequence data were generated and analysed, revealing 113 novel open reading frames (ORFs) encoding putative endonuclease genes and ORFs of unknown identity and function. One endonuclease designated Endo52 was selected from the putative endonuclease ORFs and was recombinantly produced in Escherichia coli Rosetta™ (DE3) pLysS. Endo52 was purified by immobilised metal affinity chromatography and yielded 0.437 g per litre of cultivation volume. Its enzyme activity was monitored by cleaving lambda DNA and pUC19 plasmid as substrates, and it demonstrated non-specific endonuclease activity. In addition to endonuclease-like genes, the screen identified several unknown genes. These could present new phage resistance mechanisms and are an opportunity for future investigations.

Metal complexes to promote oxidative DNA cleavage by H2O2 are desirable as anticancer drugs. A dicopper(II) complex of known p-cresol-derived methylene-tether ligand Hbcc [Cu2(bcc)]3+ did not promote DNA cleavage by H2O2. Here, we synthesized a new p-cresol-derived amide-tether one, 2,6-bis(1,4,7,10-tetrazacyclododecyl-1-carboxyamide)-p-cresol (Hbcamide). A dicopper(II) complex of the new ligand [Cu2(μ-OH)(bcamide)]2+ was structurally characterized. This complex promoted the oxidative cleavage of supercoiled plasmid pUC19 DNA (Form I) with H2O2 at pH 6.0-8.2 to give Forms II and III. The reaction was largely accelerated in a high pH region. A μ-1,1-hydroperoxo species was formed as the active species and spectroscopically identified. The amide-tether complex is more effective in cytotoxicity against HeLa cells than the methylene-tether one.

In the present study, a donor plasmid derived from pUC19 and two recipient plasmids, which had been modified from the donor plasmid and contained the red fluorescence protein gene mCherry as a reporter gene downstream of the hybrid tac promoter with the -35 region deletion mutation, were constructed. The complete genome sequence of coxsackievirus A10 downstream of the T7 promoter was divided into 7 fragments and synthesized by overlap extension PCR and the DNAworks program. Using the Golden Gate cloning strategy, the 7 fragments were then cloned into the donor plasmid and transferred to the recipient plasmid upstream of the deletion mutation tac promoter in a defined order and orientation without any deletions or insertions at the junction sites. Because the -35 region of the tac promoter was introduced into the 3' end of the last fragment during construction, the hybrid promoter was reconstructed to promote expression of mCherry, which facilitated the selection of colonies with the complete genome of coxsackievirus A10 to generate an infectious cDNA clone via reverse genetic engineering.

Motivation The Oxford Nanopore sequencing enables to directly detect methylation states of bases in DNA from reads without extra laboratory techniques. Novel computational methods are required to improve the accuracy and robustness of DNA methylation state prediction using Nanopore reads. Results In this study, we develop DeepSignal, a deep learning method to detect DNA methylation states from Nanopore sequencing reads. Testing on Nanopore reads of Homo sapiens (H. sapiens), Escherichia coli (E. coli) and pUC19 shows that DeepSignal can achieve higher performance at both read level and genome level on detecting 6 mA and 5mC methylation states comparing to previous hidden Markov model (HMM) based methods. DeepSignal achieves similar performance cross different DNA methylation bases, different DNA methylation motifs and both singleton and mixed DNA CpG. Moreover, DeepSignal requires much lower coverage than those required by HMM and statistics based methods. DeepSignal can achieve 90% above accuracy for detecting 5mC and 6 mA using only 2× coverage of reads. Furthermore, for DNA CpG methylation state prediction, DeepSignal achieves 90% correlation with bisulfite sequencing using just 20× coverage of reads, which is much better than HMM based methods. Especially, DeepSignal can predict methylation states of 5% more DNA CpGs that previously cannot be predicted by bisulfite sequencing. DeepSignal can be a robust and accurate method for detecting methylation states of DNA bases. Availability and implementation DeepSignal is publicly available at https://github.com/bioinfomaticsCSU/deepsignal. Supplementary information Supplementary data are available at bioinformatics online.

Rifampicin, an important member of ansamycin family, exhibits various biological activities. It is frequently used for the treatment of tuberculosis and leprosy. Recently, its interaction with protein is evidenced. But, its interaction with DNA is still unknown. Whether, exhibition of anti-cancer activity of rifampicin is associated with DNA-cleavage activity is also unknown. In this study, an attempt has been taken to understand these two unknown aspects. Spectroscopic studies indicated that rifampicin binds to CT-DNA with a binding constant of ~5.22 × 105 M−1. Several independent experiments like CD analysis, competitive displacement experiments and viscosity measurements revealed that rifampicin intercalates into the CT-DNA. Molecular docking studies corroborate this fact and depicted that this drug binds to the GC-rich region of DNA through multiple hydrogen bonding having the relative binding energy of −9.21 kcal mol−1. Besides, DNA binding ability, rifampicin causes the photo-cleavage of pUC19 DNA via singlet oxygen pathway. To the best of our knowledge, we report for the first time the DNA binding and DNA cleavage ability of rifampicin. This study provides a clue behind the execution of the anti-cancer activity of rifampicin. Overall, all these information can be used for further understanding the pharmacological effects of rifampicin.

Lateral gene transfer (LGT) promotes the proliferation of antibiotic resistance genes (ARGs). However, the lateral transfer efficiency of ARGs in the natural environment is little known. This study explored the lateral transfer of ARG-carrying plasmids to Escherichia coli (i.e., transformation) in the presence of metal oxide nanoparticles (MONPs). The presence of ZnO, Al2O3 and TiO2 nanoparticles (ZnONPs, Al2O3NPs and TiO2NPs, respectively) at concentrations of less than 50 mg L−1 inhibited the lateral transfer of pUC19 plasmid carrying the ampicillin resistance gene into E. coli DH5α in a decreasing degree of Al2O3NP > ZnONP > TiO2NP. The suppressed lateral ARG transfer was not due to the dissolution of metal ions from MONPs, but rather was caused by the binding of MONPs with the phosphate groups and bases of pUC19, as supported by microscopic, spectroscopic and computational evidence. The amount of plasmids bound to MONPs showed a significant negative correlation with the efficiency of ARG transfer, likely because plasmids and MONPs formed aggregates that were blocked out of E. coli cells, thus inhibiting the ARG lateral transfer. Our results provided new insight into the effect of MONPs on the lateral transfer of ARGs and improved our understanding of the ecological risks of plasmid-borne ARGs at the molecular and cellular level.

Three novel copper(ii) complexes, Cu(L1)2 (1), Cu(L2)2·2DMF (2), and Cu(L3)2·2DMF (3), were synthesized using three aroylhydrazone ligands, (E)-2-hydroxy-N'-(1-(pyrazin-2-yl)ethylidene)benzohydrazide (HL1), (E)-3-hydroxy-N'-(1-(pyrazin-2-yl)ethylidene)benzohydrazide (HL2) and (E)-4-hydroxy-N'-(1-(pyrazin-2-yl)ethylidene)benzohydrazide (HL3). The complexes were characterized by elemental analysis, infrared (IR), and Ultraviolet-visible light (UV-vis) spectroscopy. The X-ray crystal structures of the complexes all possess a distorted octahedral coordination geometry. Both an absorption spectral titration and a competitive binding assay (ethidium bromide, 4',6-diamidino-2-phenylindole (DAPI), and methyl green) revealed that complexes 2 and 3 bind readily to calf thymus DNA (ctDNA) through intercalative and minor groove binding modes. Complexes 2 and 3 also exhibited oxidative cleavage of supercoiled plasmid DNA (pUC19) in the presence of ascorbic acid as an activator. Cytotoxicity studies showed that complexes 2 and 3 possessed high cytotoxicities toward the HeLa human cervical cancer cell line, but weak toxicities toward the L929 normal mouse fibroblast cell line. We therefore have reason to believe that complexes 2 and 3 both show potential as promising anticancer candidate drugs.

Magnesium aminoclays were synthesized and used to transform non-competent Escherichia coli JM109 using the exogenous plasmid pUC19. The structure determined for the Mg aminoclays is analogous to 2:1 trioctahedral smectites such as talc, with an approximate composition R8Si8Mg6O16(OH)4, where R = CH2CH2NH2, morphologically arranged in layered sheets. Mg aminoclays were employed as a cationic vehicle that enabled the passage of plasmids across the cell envelope and led to genetic modification of the host. A stock solution of 10 mg/mL of Mg aminoclays was prepared, mixed with E. coli JM109 and pUC19 plasmid, and spread over Petri dishes containing lysogeny broth (LB), isopropyl ?-D-1-thiogalactopyranoside (IPTG), 5-bromo-4-chloro-3-indolyl-?-D-galactopyranoside (X-gal), ampicillin and various concentrations of agar (14%). The transformation efficiency obtained was higher for 1% and 2% agar even though transformation also occurred at agar concentrations of 3% and 4%. The optical density of E. coli JM109 and spreading time were also adjusted, favoring transformation when cells were used in their exponential growth phase (OD600 = 1.0) and spread for 90 s. Transformation was confirmed by the growth of blue colonies in LB/IPTG/X-gal/agar Petri dishes containing ampicillin, by regrowth of biomass in liquid media containing ampicillin and by agarose gel electrophoresis of the linearized pUC19 plasmid that followed plasmidic DNA extraction from 4 blue colonies. The maximum transformation efficiency achieved was 7.0 × 103 CFU/?g pUC19. This transformation approach proved to be suitable for a convenient, cost-effective, room-temperature, risk-free and rapid transformation of non-competent E. coli JM109., This study was supported by the Portuguese Foundation for Science and Technology (FCT) and the European Community fund FEDER, through Program COMPETE, under the scope of the Projects FCOMP-01- 0124-FEDER-007025 (PTDC/AMB/68393/2006), PEst-OE/EQB/LA0023/2013, UID/FIS/04650/2020, RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and the Project “BioEnv - Biotech nology and Bioengineering for a sustainable world”. The authors acknowledge the fellowship SFRH/BD/71661/2010 awarded to Gabriel Mendes under the scope of the MIT-Portugal Program. The authors also thank Paul Brown and Takuya Harada for the help in obtaining TEM images., info:eu-repo/semantics/publishedVersion

Degradation and deactivation kinetics of an antibiotic resistance gene (ARG) by ozone (O3) and free available chlorine (FAC) were investigated in phosphate-buffered solutions at pH 7 for O3 (in the presence of tert‑butanol), and pH 6.8 or 8.1 for FAC. We used a plasmid (pUC19)-encoded ampicillin resistance gene (ampR) in both extracellular (e-) and intracellular (i-) forms. The second-order rate constant (kO3) for degradation of 2686 base pair (bp) long e-pUC19 toward O3, which was determined by quantitative polymerase chain reaction assay, was calculated to be ~2 × 105 M−1s−1. The deactivation rate constants of e-pUC19 by O3 measured with various recipient E. coli strains were within a factor of 2 compared with the degradation rate constant for e-pUC19. The degradation/deactivation kinetics of i-pUC19 were similar to those of e-pUC19, indicating only a minor influence of cellular components on O3 reactivity toward i-pUC19. For FAC, the degradation and deactivation rates of e-pUC19 were decreased in the presence of tert‑butanol, implying involvement of direct FAC as well as some radical (e.g., •OH) reactions. The degradation rates of e-ampR segments by direct FAC reaction could be explained by a previously-reported two-step sequential reaction model, in which the rate constants increased linearly with e-ampR segment length. The deactivation rate constants of e-pUC19 during exposure to FAC were variable by a factor of up to 4.3 for the different recipient strains, revealing the role of DNA repair in the observed deactivation efficiencies. The degradation/deactivation of e-pUC19 were significantly faster at pH 6.8 than at pH 8.1 owing to pH-dependent FAC speciation variation, whereas i-pUC19 kinetics exhibited much smaller dependence on pH, demonstrating intracellular plasmid DNA reactions with FAC occurred at cytoplasmic pH (~7.5). Our results are useful for predicting and/or measuring the degradation/deactivation efficiency of plasmid-encoded ARGs by water treatment with ozonation and chlorination.

Background Halophiles offer an attractive source of genes conferring salt tolerance. Halobacillus trueperi SS1 strain of Lunsu, Himachal Pradesh, India, a strict halophile, was exploited to isolate and clone the genes for salt tolerance. The genomic library of BamH1 digest of H. trueperi SS1 was constructed in pUC19, and recombinants were screened for salt tolerance on an LB medium containing ampicillin (100 μg/ml) and NaCl (0 to 1.5 M). Results One recombinant clone named as salt-tolerant clone (STC) conferred salt tolerance to host Escherichia coli/DH5α, which showed growth in the LB medium supplemented with ampicillin and 1.2 M NaCl. Restriction digestion and PCR analysis revealed the presence of an insert of approximately 2000 bp in the STC. DNA sequencing of the 2-kb insert on both strands yielded a sequence of 2301 nucleotides. Protein BLAST analysis of 2301-bp sequence of H. trueperi SS1 present in STC showed 97% identity to multidrug transport ATP binding/permease protein of Halobacillus karajensis. The insert contained in STC was subcloned into pGEX4T2 vector, and the recombinant clone STC/pGEX4T2 conferred salt tolerance to the bacterial host E. coli. Conclusions The present study led to the isolation of salt tolerance gene encoding a putative multidrug transport ATP binding/permease protein from H. trueperi SS1. The salt tolerance gene can be subcloned for transferring salt tolerance traits into agricultural crop plants for cultivation in saline and coastal lands.

This study investigated the degradation and deactivation of an extracellular ampicillin resistance gene (ampR) encoded in plasmid pUC19 during exposure to UV254, •OH (generated by UV>290/H2O2), and combined exposure to UV254 and •OH (and/or SO4•−) using UV254/H2O2 and UV254/S2O82−. The degradation rates of ampR measured by quantitative polymerase chain reaction increased with increasing target amplicon length (192–851 bps). The rate constants for the degradation of pUC19 (2686 bps) were calculated as 0.26 cm2/mJ for UV254 and 1.5 × 1011 M−1s−1 for •OH, based on the degradation rates of ampR amplicons and assuming an equal sensitivity of DNA damage across the entire plasmid. DNA repair-proficient Escherichia coli (E. coli) AB1157 strain (wild-type) and its repair-deficient mutants including AB1886 (uvrA−), AB2463 (recA−), AB2480 (uvrA−, recA−), and DH5α (recA−, endA−) were applied as recipient cells in gene transformation assays. Results suggested that the elimination efficiency of transforming activity during UV254 and •OH exposure was dependent on the type of DNA repair genes in recipient E. coli strains. Losses of transforming activity were slower than the degradation of pUC19 by a factor of up to ∼5 (for E. coli DH5α), highlighting the importance of DNA repair in recipient cells. The degradation rates of ampR amplicons were much larger (by a factor of ∼4) in UV254/H2O2 and UV254/S2O82− than UV254 direct photolysis, indicating the significant contribution of •OH and SO4•− to the gene degradation. Not only UV254 and SO4•−, but also •OH contributed to the degradation of ampR during UV254/S2O82−, which was attributed to the conversion of SO4•− to •OH and a 10-fold larger reactivity of •OH towards ampR as compared to SO4•−. However, the enhanced gene degradation by radicals did not lead to a faster elimination of gene transforming activity during UV254/H2O2 and UV254/S2O82−, suggesting that UV254- and radical-induced DNA damage were not additive in their contributions to losses of gene transforming activity. Wastewater effluent organic matter (EfOM) accelerated the degradation of ampR during UV254 irradiation by means of reactive species production through indirect photolysis reactions, whereas EfOM mainly acted as a radical scavenger during UV254/H2O2 and UV254/S2O82− treatments.

There is a lack of shuttle vectors to be needed for investigations into the genetics of Porphyromonas gingivalis and related species. To better understand the prevalence of candidates for such tools, we have examined multiple strains of black-pigmented anaerobes (clinical and laboratory isolates) for plasmids. As no plasmids were found in P. gingivalis strains, we have used the pYH420 plasmid, derived from P. asaccharolytica, as backbone to construct a shuttle vector in combination with pUC19 from Escherichia coli. Nucleotide sequence determination of the pYH420 plasmid revealed that it contained a gene with similarity to rep from plasmid pTS1 (isolated from Treponema denticola) as well as a homolog of mobA, a member of a gene family found on mobilizable genetic elements found in the genus Bacteroides. We constructed the pG106 and pG108 shuttle vectors using parts of the pUC19 and pYH420 vectors. This resulted in a vector with a multiple cloning site (MCS) in the lacZ gene enabling us to perform blue–white colony selection. The pG106 and pG108 shuttle vectors are electro-transformable into E. coli, P. gingivalis and B. thetaiotaomicron, where they are stable. We demonstrated that these vectors were suitable in these species for applications of molecular cloning including complementation and gene expression studies. Using the pG108 vector, we complement the hcpR mutant strain of P. gingivalis and rescued its [Formula: see text]-sensitive phenotype. We also performed a gene expression study using the P-glow BS2 fluorescent reporter gene and the ahpC promoter in B. thetaiotaomicron.

Herein we present the synthesis of two ligands containing two di(2-picolyl)amine (DPA) units linked by either a 1,1′-(pyridine-2,6-diyl)bis(3-ethylurea) (L1) or a 1,1′-(1,3-phenylene)bis(3-ethylurea) (L2) spacer. The corresponding binuclear CuII and ZnII complexes were prepared and isolated. The X-ray structures of the L1 ligand and the [Cu2L1Cl2]2+ complex evidence an unusual cis/trans conformation of one of the urea groups stabilized by an intramolecular hydrogen bond with the nitrogen atom of the pyridyl spacer. The CuII complexes form rather strong ternary complexes with phosphorylated anions. The [Cu2L1]4+ complex presents a rather high affinity for pyrophosphate (logK11 = 8.19 at pH 7, 25 °C), while [Cu2L2]4+ stands out because of its strong binding to AMP2− (logK11 = 9.3 at pH 7, 25 °C). The interaction of the CuII complexes with deoxyribonucleic acid from calf thymus (ct-DNA) was monitored using circular dichroism (CD) and luminescence spectroscopies. These studies revealed a quite strong interaction of the complexes with ct-DNA (Kb = (6.4 ± 0.7) × 103 for [Cu2L1]4+ and Kb = (6.3 ± 1.0) × 103 for [Cu2L2]4+). Competition experiments carried out in the presence of methyl green and BAPPA (N1,N3-Bis(4-amidinophenyl)propane-1,3-diamine) as major and minor groove competitors, respectively, confirm that the interaction of both complexes with DNA takes place through the minor groove, in agreement with docking studies. The [Cu2L2]4+ complex is quite efficient in promoting the cleavage of the double-stranded pUC19 plasmid DNA, by favoring the conversion of the supercoiled form to the nicked form following a hydrolytic mechanism.

The sequences of two commonly used pIJ101-based Streptomyces-E. coli shuttle vectors, pEM4 and pUWL201, were determined using next-generation sequencing and mapped. pEM4 was found to be 8.3 kbp long, containing genes encoding β-lactamase, thiostrepton resistance, the lacZ□ fragment, an E. coli pUC19 origin of replication, and the Streptomyces pIJ101 origin of replication. pUWL201 was found to be 6.78 kbp long, containing genes encoding β-lactamase, thiostrepton resistance, the lacZ□ fragment, an E. coli pUC19 origin of replication, and the Streptomyces pIJ101 origin of replication. Interestingly, the sequences for both pEM4 and pUWL201 exceed their previously reported size by 1.1 kbp and 0.4 kbp, respectively. This report updates the literature with the corrected sequences for these two popular vectors, ensuring their compatibility with modern synthetic biology cloning methodologies.

Electrons and •OH-radicals have been generated by using low-energy laser pulses of 6 ns duration (1064 nm wavelength) to create plasma in a suspension of plasmid DNA (pUC19) in water. Upon thermali...

Six triruthenium complexes (1–6) ([Ru3O(CH3COO)6(L)3]PF6, L = quinazoline (qui) (complex 1), 5-nitroisoquinoline (5-nitroiq) (complex 2), 5-bromoisoquinoline (5-briq) (complex 3), isoquinoline (iq) (complex 4), 5-aminoisoquinoline (5-amiq) (complex 5), and 5,6,7,8-tetrahydroisoquinoline (thiq) (complex 6)) have been studied regarding their anti-cancer activity (B16F10 and A549 cells) and their interactions with fish sperm DNA (fs-DNA). The crystallographic data of complex 4 show the typical triangular geometry, where the three isoquinoline ligands display different degrees of co-planarity with the [Ru3O] unit. In the range of 2 to 200 μM, these complexes make B16F10 cells less viable. The overall cytotoxicity order is complex 4 > complex 6 > complex 2 > complex 5 > complex 1. The two first complexes have IC50 of 10 and 50 μM, respectively, being less toxic to L929 non-tumoral cells. The complexes can displace ethidium bromide (EB) from DNA, but the intercalation constant values (Kapp ~105 M−1) are low due to the smaller size of the azanaphtalene ligands as compared to classic intercalating molecules. Complex 5 provides the highest constants in both the EB displacement (Kapp = 12.5 × 105 M−1) and direct spectrophotometric titration with fs-DNA (Kb = 6.3 × 104 M−1) assays, possibly because its amino group can assist interaction with DNA through hydrogen bonds. Hypochromism of complexes IC band was observed (T = 310 K), suggesting hydrophobic contributions. Low values of Kapp and circular dichroism spectra for DNA-compound 6 mixtures, suggest semi-intercalative and major groove binding interaction modes. Compared to cisplatin, incubation of complexes 1–6 with plasmid pUC19 shows no DNA cleavage. Results for the interaction with DNA do not correlate with cytotoxicity, which suggests that DNA is not the pharmacological target of this class of compounds.

Tert-butylquinone (TBQ) and its alkylamino and aralkylamino derivatives are of high interest as a potential antitumor agent. Therefore, it was necessary to investigate if the compounds exert undesirable activities such as interaction with DNA molecule which could result in negative side effects in the case of their use in the diseases treatment. The major aim of this study was to investigate genotoxic potential of TBQ and selected derivatives in an acellular model by using plasmid DNA, in the prokaryotic model by the SOS/umuC assay in Salmonella typhimurium TA1535/pSK1002 and in eukaryotic models by using comet assay in human fetal lung cell line (MRC-5) and human liver cancer cell line (HepG2). Results indicated that in the acellular model TBQ and its derivatives do not interact with plasmid pUC19. In the prokaryotic model, only TBQ exerted weak genotoxic potential and only at highly cytotoxic concentrations. In eukaryotic models, genotoxic potential was detected mainly at the highest concentrations of the tested substances but the effect was lower in both cell lines in comparison with benzo[a]pyrene and etoposide which were used as positive controls. Weak genotoxic potential of tested compounds recommends them as good candidates for further testing in development of new antitumor agents. © 2018, © 2018 Informa UK Limited, trading as Taylor & Francis Group.

Square planar mononuclear platinum(II) complexes having general formula [Pt(Ln)Cl2], (where, Ln = L1-4) were synthesized with neutral bidentate heterocyclic 1,3,5-trisubstituted bipyrazole based ligands. The synthesized compounds were characterized by physicochemical method such as TGA, molar conductance, micro-elemental analysis and magnetic moment, and spectroscopic method such as, FT-IR, UV-vis, 1H NMR, 13C NMR and mass spectrometry. Biological applications of the compounds were carried out using in vitro brine shrimp lethality bioassay, in vitro antimicrobial study against five different pathogens, and cellular level cytotoxicity against Schizosaccharomyces pombe (S. Pombe) cells. Pt(II) complexes were tested for DNA interaction activities using electronic absorption titration, viscosity measurements study, fluorescence quenching technique and molecular docking assay. Binding constants (Kb) of ligands and complexes were observed in the range of 0.23-1.07 × 105 M-1 and 0.51-3.13 × 105 M-1, respectively. Pt(II) complexes (I-IV) display an excellent binding tendency to biomolecule (DNA) and possess comparatively high binding constant (Kb) values than the ligands. The DNA binding study indicate partial intercalative mode of binding in complex-DNA. The gel electrophoresis activity was carried out to examine DNA nuclease property of pUC19 plasmid DNA.

Accumulation of potentially mutagenic abasic sites lesions in DNA are often considered as stress induced biomarker for radiation exposure and carcinogenesis. A simple and cost-effective fluorescence based method for detection of abasic sites in dsDNA is demonstrated. DNA was used as a template for copper nanoclusters (Cu NCs) formation. Presence of abasic sites in DNA hinder the formation of Cu NCs resulting in increased presence of Cu(II) in solution. These free copper ions were traced quantitatively by fluorescence quenching of carbon dots (CDs) in solution that report the presence of abasic sites in those DNA samples. Apart from fluorescence properties, binding of Cu NCs are markedly different for normal and abasic sites containing DNA. To demonstrate the inclusiveness of Cu NCs and CD based biosensing of abasic sites, oligomeric DNA, plasmid DNA in linear and condensed form and DNA extracted from onion and HeLa cells were tested and respond sensitively to the presence of abasic sites in nanomolar range of those DNA that is visually noticeable with UV light. The detection strategy works very well for clustered abasic sites DNA damage also, where multiple abasic sites lesions are present in close proximity in DNA and easily detectable by this method through visual inspection under UV light.

To better understand the elimination of transforming activity of antibiotic resistance genes (ARGs), this study investigated the deactivation of transforming activity of an ARG (in Escherichia coli as a host) and ARG degradation (according to quantitative PCR [qPCR] with different amplicon sizes) during UV (254 nm) and UV/H2O2 treatments of plasmid pUC19 containing an ampicillin resistance gene (ampR). The required UV fluence for each log10 reduction of the transforming activity during UV treatment was ∼37 mJ cm−2 for both extra- and intra-cellular pUC19 (the latter within E. coli). The resulting fluence-based rate constant (k) of ∼6.2 × 10−2 cm2 mJ−1 was comparable to the k determined previously for transforming activity loss of plasmids using host cells capable of DNA repair, but much lower (∼10-fold) than that for DNA repair-deficient cells. The k value for pUC19 transforming activity loss was similarly much lower than the k calculated for cyclobutane-pyrimidine dimer (CPD) formation in the entire plasmid. These results indicate the significant role of CPD repair in the host cells. The degradation rate constants (k) of ampR measured by qPCR increased with increasing target amplicon size (192–851 bp) and were close to the k calculated for the CPD formation in the given amplicons. Further analysis of the degradation kinetics of plasmid-encoded genes from this study and from the literature revealed that qPCR detected most UV-induced DNA damage. In the extracellular plasmid, DNA damage mechanisms other than CPD formation (e.g., base oxidation) were detectable by qPCR and gel electrophoresis, especially during UV/H2O2 treatment. Nevertheless, the enhanced DNA damage for the extracellular plasmids did not result in faster elimination of the transforming activity. Our results indicate that calculated CPD formation rates and qPCR analyses are useful for predicting and/or measuring the rate of DNA damage and predicting the efficiency of transforming activity elimination for plasmid-encoded ARGs during UV-based water disinfection and oxidation processes.

The DNA damage in the presence of dipyrone (used as its sodium salt, NaDip) and some transition metal ions in an air saturated ([O2] ≈ 0.25 mM) non-buffered solution at T = (25.0 ± 0.5)°C was investigated by agarose gel electrophoresis. As metal ions Cu2+, Fe3+, Ni2+ and Mn3+ were selected and evaluated in the present study because of the important role they play in a biological system. pUC19 plasmid DNA damage-induced by NaDip (80–600 μM) was observed in the presence of 100 μM Cu2+. The damage was proportional to the NaDip concentration provided that the order of addition of reagents (pUC19 plasmid DNA + Cu2+ + NaDip) is obeyed. Addition in the reaction medium of ligands for Cu2+ and Cu+, respectively EDTA and neocuproine, promoted total inhibition or reduction of the pUC19 plasmid DNA damage suggesting the involvement of the Cu2+/Cu+ cycle. Besides, the decrease in the pUC19 plasmid DNA damage after addition of catalase (1.0 × 10−4 mg μL−1) in the same reaction medium indicates that H2O2 is also involved in the damage process. In NaDip concentration range (80–600 μM), and under same the experimental conditions, it was not possible to conclude whether there was pUC19 plasmid DNA damage caused by 10 μM Fe3+. No damage was observed in the presence of Mn3+ or Ni2+. Although the technique used in this study is sensitive to detect the pUC19 plasmid DNA damage it was not possible to identify in which DNA base this damage occurs. Further studies with other techniques should be made to unambiguously identify the oxidative intermediates that are responsible for the DNA damage. As far as we know, this is the first study dealing with the pUC19 plasmid DNA damage-induced by NaDip in presence of copper, iron, nickel and manganese ions.

We have developed a new method for estimating the localization of DNA damage such as apurinic/apyrimidinic sites (APs) on DNA using fluorescence anisotropy. This method is aimed at characterizing clustered DNA damage produced by DNA-damaging agents such as ionizing radiation and genotoxic chemicals. A fluorescent probe with an aminooxy group (AlexaFluor488) was used to label APs. We prepared a pUC19 plasmid with APs by heating under acidic conditions as a model for damaged DNA, and subsequently labeled the APs. We found that the observed fluorescence anisotropy (robs) decreases as averaged AP density (λAP: number of APs per base pair) increases due to homo-FRET, and that the APs were randomly distributed. We applied this method to three DNA-damaging agents, 60Co γ-rays, methyl methanesulfonate (MMS), and neocarzinostatin (NCS). We found that robs–λAP relationships differed significantly between MMS and NCS. At low AP density (λAP

DNA transformation that delivers plasmid DNAs into bacterial cells is fundamental in genetic manipulation to engineer and study bacteria. Developed transformation methods to date are optimized to specific bacterial species for high efficiency. Thus, there is always a demand for simple and species-independent transformation methods. We herein describe the development of a chemico-physical transformation method that combines a rubidium chloride (RbCl)-based chemical method and sepiolite-based physical method, and report its use for the simple and efficient delivery of DNA into various bacterial species. Using this method, the best transformation efficiency for Escherichia coli DH5α was 4.3×106CFU/μg of pUC19 plasmid, which is higher than or comparable to the reported transformation efficiencies to date. This method also allowed the introduction of plasmid DNAs into Bacillus subtilis (5.7×103CFU/μg of pSEVA3b67Rb), Bacillus megaterium (2.5×103CFU/μg of pSPAsp-hp), Lactococcus lactis subsp. lactis (1.0×102CFU/μg of pTRKH3-ermGFP), and Lactococcus lactis subsp. cremoris (2.2×102CFU/μg of pMSP3535VA). Remarkably, even when the conventional chemical and physical methods failed to generate transformed cells in Bacillus sp. and Enterococcus faecalis, E. malodoratus and E. mundtii, our combined method showed a significant transformation efficiency (2.4×104, 4.5×102, 2×101, and 0.5×101CFU/μg of plasmid DNA). Based on our results, we anticipate that our simple and efficient transformation method should prove usefulness for introducing DNA into various bacterial species without complicated optimization of parameters affecting DNA entry into the cell.

This paper describes the synthesis of a trinuclear Cu(II) complex ( 4 ) containing a central 1,4,5,8,9,12-hexaazatriphenylene-hexacarboxylate (hat) core ( 3 ). Low, micromolar concentrations of the negatively charged parent ligand 3 and the neutral trinuclear complex 4 were found to photocleave negatively charged pUC19 plasmid DNA with high efficiency at neutral pH (350 nm, 50 min, 22 °C). The interactions of complex 4 with double-helical DNA were studied in detail. Scavenger and colorimetric assays pointed to the formation of Cu(I), superoxide anion radicals, hydrogen peroxide, and hydroxyl radicals during photocleavage reactions. UV–visible absorption, circular dichroism, DNA thermal denaturation, and fluorescence data suggested that the Cu(II) complex contacts double-stranded DNA in an external fashion. The persistent association of ligand 3 and complex 4 with Na(I) and/or other cations in aqueous solution might facilitate electrostatic DNA interactions.

A highly negatively charged binuclear ZrIV-substituted Keggin polyoxometalate [{α-PW11O39Zr(μ-OH)(H2O)}2]8- (ZrK 2 : 2) has been shown to promote the hydrolytic cleavage of phosphoester bonds in the supercoiled plasmid pUC19 DNA under physiological pH and temperature, giving relaxed and linear forms of pUC19 as hydrolysis products. The interaction between ZrK 2 : 2 and DNA was experimentally proven by circular dichroism (CD) spectroscopy and 31P diffusion ordered NMR spectroscopy. crosscheck: This document is CrossCheck deposited related_data: Supplementary Information identifier: T. N. Parac-Vogt (ORCID) copyright_licence: The Royal Society of Chemistry has an exclusive publication licence for this journal history: Received 24 October 2016; Accepted 9 December 2016; Accepted Manuscript published 9 December 2016; Advance Article published 16 December 2016; Version of Record published 3 January 2017 ispartof: Chemical Communications vol:53 issue:3 pages:617-620 ispartof: location:England status: published

Plasmid DNA is commonly used as a simpler substitute for a cell in studies of early effects of ionizing radiation because it allows to determine yields of primary DNA lesions. Experimental studies often employ plasmids of different lengths, in different concentrations in the aqueous solution. Influence of these parameters on the heavy-ion induced yields of primary DNA damage has been studied, using plasmids pUC19 (2686 bp), pBR322 (4361 bp) and pKLAC2 (9107 bp) in 10 and 50 ng/μl concentration. Results demonstrate the impact of plasmid length, while no significant difference was observed between the two concentrations. The uncertainty of the results is discussed.

Type IB DNA topoisomerases are enzymes to change the topological state of DNA molecules and are essential in studying replication, transcription, and recombination of nucleic acids in vitro. DNA topoisomerase IB from Vaccinia virus (vTopIB) is a 32 kDa, type I eukaryotic topoisomerase, which relaxed positively and negatively supercoiled DNAs without Mg2+ and ATP. Although vTopIB has been effectively produced in E. coli expression system, no studies remain available to explore an alternative platform to express recombinant vTopIB (rvTopIB) in a higher eukaryote, where the one can expect post-translational modifications that affect the activity of rvTopIB. Here in this study, rvTopIB with N-terminal tags was constructed and expressed in a silkworm–baculovirus expression vector system (silkworm–BEVS). We developed a simple two consecutive chromatography purification to obtain highly pure rvTopIB. The final yield of rvTopIB obtained from a baculovirus-infected silkworm larva was 83.25 μg. We also evaluated the activity and function of rvTopIB by the DNA relaxation activity assays using a negatively supercoiled pUC19 plasmid DNA as a substrate. With carefully assessing optimized conditions for the reaction buffer, we found that divalent ions, Mg2+, Mn2+, Ca2+, as well as ATP stimulate the DNA relaxation activity by rvTopIB. The functional and active form of rvTopIB, together with the yields of the protein we obtained, suggests that silkworm–BEVS would be a potential alternative platform to produce eukaryotic topoisomerases on an industrial scale.

Quantitative description of the interaction between doxorubicin (DOX), a broadly used anticancer drug, and DNA is the key to understand the action mechanism and side effects of its clinical use. However, the reported equilibrium constants of DOX–DNA interaction obtained using a range of different analytical methods vary even by several orders of magnitude. Herein, we propose a novel application of a single-molecule technique – fluorescence correlation spectroscopy (FCS) – to probe the interaction between DOX and two types of DNA (pUC19 and calf thymus DNA), taking advantage of intrinsic self-fluorescence of DOX. We provide an analytical formula for autocorrelation analysis to determine the equilibrium constant of DOX–DNA complex-formation, where binding of multiple DOX molecules to a DNA chain is included in the reaction–diffusion model. Our FCS-based method not only quantitatively revealed the values of equilibrium constant, but also implied that the stability of DOX–DNA complex is related to the types of base pair rather than the length or structure of the DNA. This work opens a promising pathway toward quantitative determination of molecular interactions in complex systems such as living cells or organisms at single-molecule level.