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Avoiding biases in next generation sequencing (NGS) library preparation is crucial for obtaining reliable sequencing data. Recently, a new library preparation method has been introduced which has eliminated the need for the ligation step. This method, termed SMART (switching mechanism at the 5' end of the RNA transcript), is based on template switching reverse transcription. To date, there has been no systematic analysis of the additional biases introduced by this method. We analysed the genomic distribution of sequenced reads prepared from genomic DNA using the SMART methodology and found a strong bias toward long (≥12bp) poly dA/dT containing genomic loci. This bias is unique to the SMART-based library preparation and does not appear when libraries are prepared with conventional ligation based methods. Although this bias is obvious only when performing paired end sequencing, it affects single end sequenced samples as well. Our analysis demonstrates that sequenced reads originating from SMART-DNA libraries are heavily skewed toward genomic poly dA/dT tracts. This bias needs to be considered when deciding to use SMART based technology for library preparation.
The equilibrium binding of the cytotoxic plant alkaloid berberine to various DNAs and energetics of the interaction have been studied. At low ratios of bound alkaloid to base pair, the binding exhibited cooperativity to natural DNAs having almost equal proportions of AT and GC sequences. In contrast, the binding was non-cooperative to DNAs with predominantly high AT or GC sequences. Among the synthetic DNAs, cooperative binding was observed with poly(dA).poly(dT) and poly(dG).poly(dC) while non-cooperative binding was seen with poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC). Both cooperative and non-cooperative bindings were remarkably dependent on the salt concentration of the media. Linear plots of ln K(a) versus [Na(+)] for poly(dA).poly(dT) and poly(dA-dT).poly(dA-dT) showed the release of 0.56 and 0.75 sodium ions respectively per bound alkaloid. Isothermal titration calorimetry results revealed the binding to be exothermic and favoured by both enthalpy and entropy changes in all DNAs except the two AT polymers and AT rich DNA, where the same was predominantly entropy driven. Heat capacity values (DeltaCp(o)) of berberine binding to poly(dA).poly(dT), poly(dA-dT).poly(dA-dT), Clostridium perfringens and calf thymus DNA were -98, -140, -120 and -110 cal/mol K respectively. This study presents new insights into the binding dependent base pair heterogeneity in DNA conformation and the first complete thermodynamic profile of berberine binding to DNAs.
The base dependent binding of the cytotoxic alkaloid palmatine to four synthetic polynucleotides, poly(dA).poly(dT), poly(dA-dT).poly(dA-dT), poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) was examined by competition dialysis, spectrophotometric, spectrofluorimetric, thermal melting, circular dichroic, viscometric and isothermal titration calorimetric (ITC) studies. Binding of the alkaloid to various polynucleotides was dependent upon sequences of base pairs. Binding data obtained from absorbance measurements according to neighbour exclusion model indicated that the intrinsic binding constants decreased in the order poly(dA).poly(dT)>poly(dA-dT).poly(dA-dT)>poly(dG-dC).poly(dG-dC)>poly(dG).poly(dC). This affinity was also revealed by the competition dialysis, increase of steady state fluorescence intensity, increase in fluorescence quantum yield, stabilization against thermal denaturation and perturbations in circular dichroic spectrum. Among the polynucleotides, poly(dA).poly(dT) showed positive cooperativity at binding values lower than r=0.05. Viscosity studies revealed that in the strong binding region, the increase of contour length of DNA depended strongly on the sequence of base pairs being higher for AT polymers and induction of unwinding-rewinding process of covalently closed superhelical DNA. Isothermal titration calorimetric data showed a single entropy driven binding event in the AT homo polymer while that with the hetero polymer involved two binding modes, an entropy driven strong binding followed by an enthalpy driven weak binding. These results unequivocally established that the alkaloid palmatine binds strongly to AT homo and hetero polymers by mechanism of intercalation.
The vaccinia virus E3 protein is an important intracellular modulator of innate immunity that can be split into distinct halves. The C terminus contains a well defined dsRNA-binding domain, whereas the N terminus contains a Z-DNA-binding domain, and both domains are required for virulence. In this study, we investigated whether the E3 Z-DNA-binding domain functions by sequestering cytoplasmic dsDNA thereby preventing the induction of type I interferon (IFN). In line with this hypothesis, expression of E3 ablated both IFN-beta expression and NF-kappaB activity in response to the dsDNA, poly(dA-dT). However, surprisingly, the ability of E3 to block poly(dA-dT) signalling was independent of the N terminus, whereas the dsRNA-binding domain was essential, suggesting that the Z-DNA-binding domain does not bind immunostimulatory dsDNA. This was confirmed by the failure of E3 to co-precipitate with biotinylated dsDNA, whereas the recruitment of several cytoplasmic DNA-binding proteins could be detected. Recently, AT-rich dsDNA was reported to be transcribed into 5'-triphosphate poly(A-U) RNA by RNA polymerase III, which then activates retinoic acid-inducible gene I (RIG-I). Consistent with this, RNA from poly(dA-dT) transfected cells induced IFN-beta and expression of the E3 dsRNA-binding domain was sufficient to ablate this response. Given the well documented function of the E3 dsRNA-binding domain we propose that E3 blocks signalling in response to poly(dA-dT) by binding to transcribed poly(A-U) RNA preventing RIG-I activation. This report describes a DNA virus-encoded inhibitor of the RNA polymerase III-dsDNA-sensing pathway and extends our knowledge of E3 as a modulator of innate immunity.
Sequence-dependent configuration changes and condensation of double-stranded poly(dG-dC).(dG-dC) (GC-DNA) and ds poly(dA-dT).(dA-dT) (AT-DNA) were observed by atomic force microscopy in the presence of Ni(II). Less condensing agent was required to generate configuration changes in GC-DNA as compared to AT-DNA. In the presence of Ni(II) cations, GC-DNA adopted a Z-type conformation and underwent a stepwise condensation, starting with partial intramolecular folding, followed by intermolecular condensation of two to several molecules and ending with the formation of toroids, rods, and jumbles. GC-DNA condensates were unusual in that the most highly condensed regions were surrounded by loops of ds GC-DNA. In contrast, AT-DNA retained its B-type conformation and displayed only minor condensation even at high Ni(II) concentrations. The Ni(II)-dependent differences in condensation between GC-DNA and AT-DNA are predicted by an extension of the electrostatic zipper motif proposed by Kornyshev and Leikin, in which we account for shorter than Debye screening length surface separations between the DNA molecules and for the Ni(II)-induced conformation change of GC-DNA to Z-DNA.
The DNA polymerase accessory factor proliferating cell nuclear antigen (PCNA) has been caught in interaction with an ever increasing number of proteins. To characterize the sites and functions of some of these interactions, we constructed four mutants of human PCNA and analysed them in a variety of assays. By targeting loops on the surface of the PCNA trimer and changing three or four residues at a time to alanine, we found that a region including part of the domain-connecting loop of PCNA and loops on one face of the trimer, close to the C-termini, is involved in binding to all of the following proteins: DNA polymerase delta, replication factor C, the flap endonuclease Fen1, the cyclin dependent kinase inhibitor p21 and DNA ligase I. An inhibition of DNA ligation caused by the interaction of PCNA with DNA ligase I was found, and we show that DNA ligase I and Fen1 can inhibit DNA synthesis by DNA polymerase delta/PCNA. We demonstrate that PCNA must be located below a 5' flap on a forked template to stimulate Fen1 activity, and considering the interacting region on PCNA for Fen1, this suggests an orientation for PCNA during DNA replication with the C-termini facing forwards, in the direction of DNA synthesis.
A new series of trimethoprim (TMP) analogs containing amide bonds (1-6) have been synthesized. Molecular docking, as well as dihydrofolate reductase (DHFR) inhibition assay were used to confirm their affinity to bind dihydrofolate reductase enzyme. Data from the ethidium displacement test showed their DNA-binding capacity. Tests confirming the possibility of DNA binding in a minor groove as well as determination of the association constants were performed using calf thymus DNA, T4 coliphage DNA, poly (dA-dT)2 and poly (dG-dC)2. Additionally, the mechanism of action of the new compounds was studied. In conclusion, some of our new analogs inhibited DHFR activity more strongly than TMP did, which confirms, that the addition of amide bonds into the analogs of TMP increases their affinity towards DHFR.
Caspase-mediated cleavage of PARP1 is a surrogate marker for apoptosis. However, the biological significance of PARP1 cleavage during apoptosis is still unclear. Here, using unbiased protein affinity purification, we show that truncated PARP1 (tPARP1) recognizes the RNA polymerase III (Pol III) complex in the cytosol. tPARP1 mono-ADP-ribosylates RNA Pol III in vitro and mediates ADP-ribosylation of RNA Pol III during poly(dA-dT)-stimulated apoptosis in cells. tPARP1-mediated activation of RNA Pol III facilitates IFN-β production and apoptosis. In contrast, suppression of PARP1 or expressing the non-cleavable form of PARP1 impairs these molecular events. Taken together, these studies reveal a novel biological role of tPARP1 during cytosolic DNA-induced apoptosis.
Eighteen previously undescribed trimethoprim (TMP) analogs containing amide bonds (1-18) were synthesized and compared with TMP, methotrexate (MTX), and netropsin (NT). These compounds were designed as potential minor groove binding agents (MGBAs) and inhibitors of human dihydrofolate reductase (hDHFR). The all-new derivatives were obtained via solid phase synthesis using 4-nitrophenyl Wang resin. Data from the ethidium displacement test confirmed their DNA-binding capacity. Compounds 13-14 (49.89% and 43.85%) and 17-18 (41.68% and 42.99%) showed a higher binding affinity to pBR322 plasmid than NT. The possibility of binding in a minor groove as well as determination of association constants were performed using calf thymus DNA, T4 coliphage DNA, poly (dA-dT)2, and poly (dG-dC)2. With the exception of compounds 9 (IC50 = 56.05 µM) and 11 (IC50 = 55.32 µM), all of the compounds showed better inhibitory properties against hDHFR than standard, which confirms that the addition of the amide bond into the TMP structures increases affinity towards hDHFR. Derivatives 2, 6, 13, 14, and 16 were found to be the most potent hDHFR inhibitors. This molecular modelling study shows that they interact strongly with a catalytically important residue Glu-30.
RORalpha4 is a nuclear receptor activating the transcription of genes that are important for a variety of physiological processes like muscle differentiation, lipid and bone metabolism, cerebellar development, and inflammation. Furthermore, it plays an essential role in maintaining circadian rhythmicity of the core clock in the suprachiasmatic nuclei (SCN). Here, we describe the successful overexpression and purification of human full-length RORalpha4 in Escherichia coli using a T7 expression system. The expressed protein formed inclusion bodies which were solubilized in the presence of 6M guanidinium-HCl and renatured by gradual removal of guanidinium-HCl and addition of l-arginine. The refolded protein was purified by nickel affinity chromatography due to an N-terminal polyhistidine tag which can be cleaved with thrombin subsequently. This method permitted us to obtain up to 20mg of pure and native RORalpha4 protein per liter of E. coli culture. The DNA binding activity of the refolded protein was demonstrated by electrophoretic mobility shift assay (EMSA) using an oligonucleotide comprising the ROR-response element (RORE) motif (A/G)GGTCA. In addition, we developed a new monoclonal antibody to human RORalpha in mice with high sensitivity and specificity.
Here we use single-molecule imaging to determine coarse-grained intrinsic energy landscapes for nucleosome deposition on model DNA substrates. Our results reveal distributions that are correlated with recent in silico predictions, reinforcing the hypothesis that DNA contains some intrinsic positioning information. We also show that cis-regulatory sequences in human DNA coincide with peaks in the intrinsic landscape, whereas valleys correspond to nonregulatory regions, and we present evidence arguing that nucleosome deposition in vertebrates is influenced by factors that are not accounted for by current theory. Finally, we demonstrate that intrinsic landscapes of nucleosomes containing the centromere-specific variant CenH3 are correlated with patterns observed for canonical nucleosomes, arguing that CenH3 does not alter sequence preferences of centromeric nucleosomes. However, the nonhistone protein Scm3 alters the intrinsic landscape of CenH3-containing nucleosomes, enabling them to overcome the otherwise exclusionary effects of poly(dA-dT) tracts, which are enriched in centromeric DNA.
Dicationic diamidines have been well established as potent antiparasitic agents with proven activity against tropical diseases like trypanosomiasis and malaria. This work presents the synthesis of new mono and diflexible triaryl amidines (6a-c, 13a,b and 17), their aza analogues (23 and 27) and respective methoxyamidine prodrugs (5, 7, 12a,b, 22 and 26). All diamidines were assessed in vitro against Trypanosoma brucei rhodesiense (T. b. r.) and Plasmodium falciparum (P. f.) where they displayed potent to moderate activities at the nanomolar level with IC50s = 11-378 nM for T. b. r. and 4-323 nM against P. f.. In vivo efficacy testing against T. b. r. STIB900 has shown the monoflexible diamidine 6c as the most potent derivative in this study eliciting 4/4 cures of infected mice for a treatment period of >60 days upon a 4 × 5 mg/kg dose i. p. treatment. Moreover, thermal melting analysis measurement ΔTm for this series of diamidines/poly (dA-dT) complexes fell between 0.5 and 19 °C with 6c showing the highest binding to the DNA minor groove. Finally, a 50 ns molecular dynamics study of an AT-rich DNA dodecamer with compound 6c revealed a strong binding complex supported by vdW and electrostatic interactions.
Keratinocytes are non-professional immune cells contributing actively to innate immune responses partially by reacting to a wide range of molecular patterns by activating pattern recognition receptors. Cytosolic nucleotide fragments as pathogen- or self-derived trigger factors are activating inflammasomes and inducing anti-viral signal transduction pathways as well as inducing expression of inflammatory cytokines. We aimed to compare the induced inflammatory reactions in three keratinocyte cell types-normal human epidermal keratinocytes, the HaCaT cell line and the HPV-KER cell line-upon exposure to the synthetic RNA and DNA analogues poly(I:C) and poly(dA:dT) to reveal the underlying signaling events. Both agents induced the expression of interleukin-6 and tumor necrosis factor α in all cell types; however, notable kinetic and expression level differences were found. Western blot analysis revealed rapid activation of the nuclear factor κB (NF-κB), mitogen activated protein kinase and signal transducers of activator of transcription (STAT) signal transduction pathways in keratinocytes upon poly(I:C) treatment, while poly(dA:dT) induced slower activation. Inhibition of NF-κB, p38, STAT-1 and STAT-3 signaling resulted in decreased cytokine expression, whereas inhibition of mitogen-activated protein kinase kinase 1/2 (MEK1/2) signaling showed a negative feedback role in both poly(I:C)- and poly(dA:dT)-induced cytokine expression. Based on our in vitro results nucleotide fragments are able to induce inflammatory reactions in keratinocytes, but with different rate and kinetics of cytokine expression, explained by faster activation of signaling routes by poly(I:C) than poly(dA:dT).
The dysregulation of NLRP3 inflammasome plays a critical role in pathogenesis of various human inflammatory diseases, thus NLRP3 inflammasome activation must be tightly controlled at multiple levels. However, the underlying mechanism regulating NLRP3 inflammasome activation remains unclear. Herein, the effects of Tripartite motif-containing protein 65 (TRIM65) on NLRP3 inflammasome activation and the underlying molecular mechanism were investigated in vitro and in vivo. Inhibition or deletion of Trim65 could significantly strengthen agonist induced NLRP3 inflammasome activation in THP-1 cells and BMDMs, indicated by increased caspase-1 activation and interleukin-1β secretion. However, TRIM65 had no effect on poly (dA: dT)-induced AIM2 inflammasome activation or flagellin-induced IPAF inflammasome activation. Mechanistically, immunoprecipitation assays demonstrated that TRIM65 binds to NACHT domain of NLRP3, promotes lys48- and lys63- linked ubiquitination of NLRP3 and restrains the NEK7-NLRP3 interaction, thereby inhibiting NLRP3 inflammasome assembly, caspase-1 activation, and IL-1β secretion. In vivo, three models of inflammatory diseases were used to confirm the suppression role of TRIM65 in NLRP3 inflammasome activation. TRIM65-deficient mice had a higher production of IL-1β induced by lipopolysaccharide in sera, and more IL-1β secretion and neutrophil migration in the ascites, and more severity of joint swelling and associated IL-1β production induced by monosodium urate, suggesting that TRIM65 deficiency was susceptible to inflammation. Therefore, the data elucidate a TRIM65-dependent negative regulation mechanism of NLRP3 inflammasome activation and provide potential therapeutic strategies for the treatment of NLRP3 inflammasome-related diseases.
Inflammasomes are cytosolic complexes that mature and secrete the inflammatory cytokines interleukin 1β (IL-1β) and IL-18 and induce pyroptosis. The NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome detects many pathogen- and danger-associated molecular patterns, and reactive oxygen species (ROS)/reactive nitrogen species (RNS) have been implicated in its activation. The phenazine pyocyanin (PCN) is a virulence factor of Pseudomonas aeruginosa and generates superoxide in cells. Here we report that PCN inhibits IL-1β and IL-18 release and pyroptosis upon NLRP3 inflammasome activation in macrophages by preventing speck formation and Caspase-1 maturation. Of note, PCN did not regulate the AIM2 (absent in melanoma 2) or NLRC4 inflammasomes or tumor necrosis factor (TNF) secretion. Imaging of the fluorescent glutathione redox potential sensor Grx1-roGFP2 indicated that PCN provokes cytosolic and nuclear but not mitochondrial redox changes. PCN-induced intracellular ROS/RNS inhibited the NLRP3 inflammasome posttranslationally, and hydrogen peroxide or peroxynitrite alone were sufficient to block its activation. We propose that cytosolic ROS/RNS inhibit the NLRP3 inflammasome and that PCN's anti-inflammatory activity may help P. aeruginosa evade immune recognition.
Ischemia-reperfusion (IR) injury induces activation of several inflammasomes that widely affect neuroinflammation and, subsequently, neuronal viability. The absent in melanoma 2 (AIM2) inflammasome is highly expressed in neurons after traumatic injury. This study was performed to investigate whether the AIM2 molecule acts as an initiator to trigger AIM2 inflammasome activation and regulate neuronal pyroptosis in a mouse IR model. The early motor dysfunction that occurred within the first 8 h post-IR injury was closely associated with a massive increase in dsDNA in serum and cerebrospinal fluid (CSF) at the same observed timepoints. However, the subsequent persistent dysfunction was consistent with the continuously increasing protein levels of apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), cleaved caspase-1 and IL-1β with time. Upregulated AIM2 immunoreactivity was primarily visualized in neurons. The si-AIM2 treatment in vivo preserved part of motor function, accompanied by decreased protein levels of AIM2, ASC, cleaved caspase-1 and IL-1β. In vitro, the direct interactions between the AIM2 molecule and caspase-1 were demonstrated by immunofluorescence staining and coimmunoprecipitation. In this context, both si-AIM2 and Ac-YVAD-CMK treatments effectively maintained neuronal viability, as demonstrated by the decreased percentage of cells with pyroptosis and release of lactate dehydrogenase (LDH), accompanied by weak immunoreactivity and a decreased number of AIM2-caspase-1 positive neurons. By contrast, poly(dA-dT) treatment exacerbated neuronal pyroptosis by reversing the above-mentioned effects. However, no significant differences were observed after si-Con treatment. These results suggest AIM2 molecule played an important role in initiating AIM2 inflammasome activation through IR-induced release of ectopic dsDNA.
The DNA interactions of PT-BIS(ACRAMTU) ([Pt(en)(ACRAMTU)2](NO3)4; ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea, en = ethylenediamine), a bifunctional platinum-acridine conjugate, have been studied in native and synthetic double-stranded DNAs and model duplexes using various biophysical techniques. These include ethidium-DNA fluorescence quenching and thermal melting experiments, circular dichroism (CD) spectroscopy and plasmid unwinding assays. In addition, the binding mode was studied in a short octamer by NMR spectroscopy in conjunction with molecular modeling. In alternating copolymers, PT-BIS(ACRAMTU) shows a distinct preference for poly(dA-dT)2, which is approximately 3-fold higher than that of ACRAMTU. In the ligand-oligomer complex, d(GCTATAGC)2.PT-BIS(ACRAMTU) (complex I*), PT-BIS(ACRAMTU) increases the thermal stability of the B-form host duplex by DeltaT(m) > 30 K (CD and UV melting experiments). The agent unwinds pSP73 plasmid DNA by 44(+/-2) degrees per bound molecule, indicating bisintercalative binding. A 2-D NMR study unequivocally demonstrates that PT-BIS(ACRAMTU)'s chromophores deeply bisintercalate into the 5'-TA/TA base pair steps in I*, while the platinum linker lies in the minor groove. An AMBER model reflecting the NMR results shows that bracketing of the central AT base pairs in a classical nearest neighbor excluded fashion is feasible. PT-BIS(ACRAMTU) inhibits DNA hydrolysis by BstZ17 I at the enzyme's restriction site, GTA downward arrowTAC. Possible consequences for other relevant DNA-protein interactions, such as those involved in TATA-box-mediated transcription initiation and the utility of the platinum-intercalator technology for the design of sequence-specific agents are discussed.
The cytosolic DNA sensing pathway mediates innate immune defense against infection by many DNA viruses; however, viruses have evolved multiple strategies to evade the host immune response. Duck enteritis virus (DEV) causes an acute and contagious disease with high mortality in waterfowl. The mechanisms employed by DEV to block the DNA sensing pathway are not well understood. Here, we sought to investigate the role of DEV US3, a serine/threonine protein kinase, in the inhibition of DNA sensing. We found that ectopic expression of DEV US3 significantly inhibited the production of IFN-β and expression of interferon-stimulated genes induced by interferon-stimulatory DNA and poly(dA-dT). US3 also inhibited viral DNA-triggered IFN-β activation and promoted DEV replication in duck embryo fibroblasts, while knockdown of US3 during DEV infection enhances the IFN-β response and suppresses viral replication. US3 inhibited the DNA-sensing signaling pathway by targeting interferon regulatory factor 7 (IRF7), and the kinase activity of US3 was indispensable for its inhibitory function. Furthermore, we found that US3 interacts with the activation domain of IRF7, phosphorylating IRF7, blocking its dimerization and nuclear translocation, and finally leading to the inhibition of IFN-β production. These findings expand our knowledge on DNA sensing in ducks and reveal a novel mechanism whereby DEV evades host antiviral immunity. IMPORTANCE Duck enteritis virus (DEV) is a duck alphaherpesvirus that causes an acute and contagious disease with high mortality, resulting in substantial economic losses in the commercial waterfowl industry. The evasion of DNA-sensing pathway-mediated antiviral innate immunity is essential for the persistent infection and replication for many DNA viruses. However, the strategies used by DEV to block the DNA-sensing pathway are not well understood. In this study, DEV US3 protein kinase was demonstrated to inhibit the DNA-sensing signaling via binding to the activation domain of interferon regulatory factor 7 (IRF7), which induced the hyperphosphorylation of IRF7 and abolished IRF7 dimerization and nuclear translocation. Our findings provide insights into how duck herpesviral kinase counteracts host antiviral innate immunity to ensure viral replication and spread.
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