Endonuclease Activity Assay Research Articles

SgRNA design and in vitro nucleolytic analysis of the Cas9‐RNP complex for transgene‐free genome editing of the eIF4E1 gene from Capsicum an‐ nuum L.

Chili (Capsicum annuum L.) is a highly valued vegetable, renowned for its unique taste and aroma. However, chili production faces challenges in meeting the high demand due to infections caused by pathogens such as ChiVMV (potyvirus). Previous studies have suggested that chili eIF4E1 plays a crucial role in potyvirus gene transcription. Therefore, this study explores the potential of CRISPR‐Cas9‐based genome editing to enhance chili resistance by introducing premature stop codons or truncated proteins. Two sgRNAs were designed, targeting the first and second intron of the eIF4E1 gene. The production of Cas9 protein was assessed with varying IPTG concentrations in Escherichia coli BL21(DE3), carrying 4xNLS‐pMJ915v2‐sfGFP plasmid with a TEV protease cut‐site at the N terminal. The findings indicate that the optimal IPTG concentration is 500 µM. Purification using an IMAC column confirmed the presence of Cas9 in the initial 2 mL of the eluted fractions, as indicated by numerous background proteins. Nevertheless, successful formation of Cas9‐RNP complexes was achieved for both sgRNAs. The nucleolytic activity of Tag‐Cas9 (carrying the MBP‐tag) and Cas9 was confirmed through in vitro endonuclease activity assays. The next step involved transfecting chili protoplasts with these RNP complexes to edit the chili eIF4E1 gene.

Unraveling the anti-influenza effect of flavonoids: Experimental validation of luteolin and its congeners as potent influenza endonuclease inhibitors.

The biological effects of flavonoids on mammal cells are diverse, ranging from scavenging free radicals and anti-cancer activity to anti-influenza activity. Despite appreciable effort to understand the anti-influenza activity of flavonoids, there is no clear consensus about their precise mode-of-action at a cellular level. Here, we report the development and validation of a screening assay based on AlphaScreen technology and illustrate its application for determination of the inhibitory potency of a large set of polyols against PA N-terminal domain (PA-Nter) of influenza RNA-dependent RNA polymerase featuring endonuclease activity. The most potent inhibitors we identified were luteolin with an IC50 of 72±2nM and its 8-C-glucoside orientin with an IC50 of 43±2nM. Submicromolar inhibitors were also evaluated by an invitro endonuclease activity assay using single-stranded DNA, and the results were in full agreement with data from the competitive AlphaScreen assay. Using X-ray crystallography, we analyzed structures of the PA-Nter in complex with luteolin at 2.0Å resolution and quambalarine B at 2.5Å resolution, which clearly revealed the binding pose of these polyols coordinated to two manganese ions in the endonuclease active site. Using two distinct assays along with the structural work, we have presumably identified and characterized the molecular mode-of-action of flavonoids in influenza-infected cells.

Alkyne-DNA-Functionalized Alloyed Au/Ag Nanospheres for Ratiometric Surface-Enhanced Raman Scattering Imaging Assay of Endonuclease Activity in Live Cells.

A novel ratiometric surface-enhanced Raman scattering (SERS) nanosensor has been developed to probe the activity of endonuclease under in vitro and in living cells conditions. The optimized alloyed Au/Ag nanoparticles (NPs) were synthesized as the SERS substrate, which combined the superior properties of both pure Au and pure Ag nanoparticles: they exhibit excellent plasmonic property with high chemical stability and low cytotoxicity. They were then employed for quantitative detection of endonuclease through functionalization with single-stranded DNA (ssDNA) carrying 3-[4-(phenylethynyl)benzylthio]propanoic acid (PEB) as endonuclease-responsive SERS signaling molecule and 4-thiophenylacetylene (TPA) as the internal standard SERS signaling molecule. In the presence of endonuclease, the ssDNA was cleaved, releasing PEB molecules from the particle surface and decreasing the SERS signal at 2215 cm-1 from PEB. Since the SERS signal at 1983 cm-1 from alkynyl TPA remained the same, quantitative detection of endonuclease was achieved, based on the ratiometric peak intensity of I1983/ I2215, with a detection limit as low as 0.056 unit/mL. A highly biocompatible and antijamming ratiometric SERS sensor was established by combining the alloyed Au/AgNPs with two unique alkynes molecules with Raman signals in the cellular silent region. The ratiometric sensor was successfully employed to detect intracellular endonuclease activity as well as endonuclease in living cells for the first time.

Integrative toxicogenomic analysis for elucidating molecular interference on DNA integrity and repair system with underlying signaling networks in response to low-level lead acetate in rat liver model

Lead (Pb) is widely used in consumer products as an alloy and pigment despite its well-known serious toxic effects. Pb absorbed through the gastrointestinal and respiratory system flows through the bloodstream and accumulates in the brain, bone and liver. Especially, the liver is well known as an important organ for detoxification, and a number of prior studies have confirmed the alteration of diverse biological mechanisms caused by long-term Pb exposure in the liver. In this study, we investigated the genotoxic potential of Pb on nuclear DNA and repair capacity using comet and AP endonuclease activity assay, respectively in rat liver tissue at long-term and low-level exposure. Significant extent of DNA break damages and the impairment of enzymatic repair activities were observed in the Pb exposed group, in comparison to the control group. In addition, using integrative toxicogenomic approach we comprehensively analyzed the Pb-induced gene alteration in genome-wide scale and DNA excision repair-focused biological networks using microarray and pathway analysis, respectively. Based on integrative approach, we suggest that genotoxic effects of low-level Pb exposure are, in part, involved in the enhancement of DNA strand break and impairment of repair activity, with complex molecular signaling pathways. Our study provides perspective on novel biomarker responsible for Pb-induced hepatotoxicity at long-term and low-dose exposure, which would be useful for further development of gene-targeted therapeutic strategies and/or natural chemo-preventive agents for effective reduction of the Pb toxicity in both occupational and environmental perspectives. The exact mechanism of genotoxicity in liver exposed to low-level Pb will be further warranted.

Structure-based virtual screening toward the discovery of novel inhibitors of the DNA repair activity of the human apurinic/apyrimidinic endonuclease 1.

The DNA repair activity of human apurinic/apyrimidinic endonuclease 1 (APE1) has been recognized as a promising target for the development of small-molecule inhibitors to be used in combination with anticancer agents. In an attempt to identify novel inhibitors of APE1, we present a structure-based virtual screening (SBVS) study based on molecular docking analysis of the compounds of NCI database using the GOLD 5.1.0 (Genetic Optimization for Ligand Docking) suite of programs. Compounds selected in this screening were tested with a fluorescence-based APE1 endonuclease activity assay. Two compounds (37 and 41) were able to inhibit the multifunctional enzyme APE1 in the micromolar range, while compound 22 showed inhibitory effects at nanomolar concentrations. These results were confirmed by a plasmid DNA nicking assay. In addition, the potential APE1 inhibitors did not affect the cell viability of non-tumor MCF10A cells. Overall, compounds 22, 37, and 41 appear to be important scaffolds for the design of novel APE1 inhibitors and this study highlights the relevance of in silico-based approaches as valuable tools in drug discovery.

One facile fluorescence strategy for sensitive detection of endonuclease activity using DNA-templated copper nanoclusters as signal indicators

Assay of endonuclease activity plays an important role in biologic and medicinal study. Herein, a simple and facile strategy was developed for detecting endonuclease activity. In this strategy, poly(AT-TA) double-stranded DNA containing restriction cutting site for EcoRI endonuclease was rationally designed and acted as the substrate of EcoRI and the template for copper nanoclusters (CuNCs). In the presence of EcoRI, the double-stranded DNA was cleaved, and then the introduction of Cu2+ and ascorbate cannot form the fluorescent CuNCs due to the lack of the double-stranded DNA templates. The endonuclease actvity was quantified by monitoring the change in fluorescence intensity. Under the optimized conditions, the fluorescence change was linear with EcoRI concentration in the range from 0.002U/μL to 0.1U/μL with the detection limit of 0.00087U/μL. This method was easy and convenient to operate in homogeneous solution, and the whole assay process can be completed in a single tube.

TR-04 * NANOPARTICLE siRNA DELIVERY VEHICLES INHIBIT DNA REPAIR AND SENSITIZE PEDIATRIC BRAIN TUMOR CELLS TO RADIATION THERAPY

Radiotherapy (RT) is an integral component of the treatment for medulloblastoma (MB) and the only effective adjuvant therapy for ependymoma (EP), two of the most common pediatric brain tumors. However, survival is frequently accompanied by one or more radiation-induced adverse developmental and psychosocial sequelae. Therefore, strategies that enhance the tumoricidal action of RT while sparing adjacent normal brain are expected to reduce the amount of radiation required to generate a therapeutic effect in these tumors. The multifunctional DNA repair protein Ape1/Ref-1 has been implicated in conferring radiation resistance in pediatric brain tumors. However, inhibiting Ape1 activity in the clinic has been hindered by the lack of safe and effective drugs and siRNA delivery vehicles. Here, we used our nanoparticle delivery vehicles to deliver siRNA against Ape1 (siApe1) and improve cell kill after RT. Nanoparticles were loaded with siApe1, or siGFP as a control, and used to treat UW228 (MB) and Res196 (EP) cells. Ape1 expression and activity were measured using PCR, Western blot, and an abasic endonuclease activity assay. DNA repair and clonogenic survival were assessed after exposure to 137Cs-γ-rays. We found that nanoparticle-mediated siApe1 delivery reduced Ape1 expression and activity by greater than 80%, which was accompanied by a greater abundance of DNA damage. This diminished the shoulder of resistance in survival curves decreasing survival to ∼50% at 1 Gy. Sensitization was specific to abasic site generating treatments as response to paclitaxel was not affected. Therefore, nanoparticle-mediated inhibition of Ape1 may help enhance the therapeutic effect of RT in pediatric brain tumor patients and reduce treatment-induced morbidity.

A new label-free and turn-on strategy for endonuclease detection using a DNA–silver nanocluster probe

Endonuclease plays a vital role in a variety of biological processes and the assay of endonuclease activity and inhibitors is of high importance in the fields ranging from biotechnology to pharmacology. Howerer, traditional techniques usually suffer from time intensive, laborious, and cost-expensive. This work aims to develop a facile and sensitive method for endonuclease activity assay by making use of the fluorescence enhancement effect when DNA–silver nanoclusters (DNA–Ag NCs) are in proximity to guanine-rich DNA sequences. The system mainly consists of block DNA (B-DNA), G-DNA and Ag-DNA. B-DNA serves as the substrate of the endonuclease (S1 nuclease as the model enzyme). G-DNA, which is predesigned entirely complementary to B strand, contains a guanine-rich overhang sequence and hybridization part at the 5′-end. Ag–DNA involves a sequence for Ag NCs synthesis and a sequence complementary to the hybridization part of the G-DNA. In the “off” state, B-DNA plays the role as a blocker that inhibit the proximity between Ag NCs and guanine-rich DNA sequences, resulting in a low fluorescence readout. However, if S1 nuclease is introduced into the system, B-DNA was cleaved into mono- or short-oligonucleotides fragments, which could not hybridize with G-DNA. As a result, the subsequent addition of DNA–Ag NCs could bring guanine-rich DNA sequences close to the Ag NCs, accompanied by a significant fluorescence enhancement. Therefore, endonuclease activity could be successfully quantified by monitoring the variation in fluorescence intensity. In addition, this approach can also be applied for inhibitor screening of endonuclease. This label-free and turn-on fluorescent assays employing the mechanism proposed here for the detection of nuclease and inhibitors turn out to be sensitive, selective, and convenient.

In vitro enzymology of Cas9.

Cas9 is a bacterial RNA-guided endonuclease that uses base pairing to recognize and cleave target DNAs with complementarity to the guide RNA. The programmable sequence specificity of Cas9 has been harnessed for genome editing and gene expression control in many organisms. Here, we describe protocols for the heterologous expression and purification of recombinant Cas9 protein and for in vitro transcription of guide RNAs. We describe in vitro reconstitution of the Cas9-guide RNA ribonucleoprotein complex and its use in endonuclease activity assays. The methods outlined here enable mechanistic characterization of the RNA-guided DNA cleavage activity of Cas9 and may assist in further development of the enzyme for genetic engineering applications.

Probing Sequence and Topological Specificity in the Binding of Tetra(Methylpyridyl)Porphines to DNAs

The binding of a family of tetra(methylpyridyl)porphines to relaxed and supercoiled circular phiX197RF DNA has been probed using restriction endonuclease activity assays employing nine restriction enzymes (selected for different cleavage sequences and different flanking sequences), mung bean nuclease assays and E.coli topoisomerase assays. The restriction enzyme assays indicate that placement of the methyl substituent alters the sequence and topological specificity of binding. For example, the the restriction enzyme Mlu I (cleavage site ACGCGT), the ortho isomer yields partial inhibition of cleavage of supercoiled and relaxed DNA, the meta isomer yields inhibition of the relaxed DNA and no effect with supercoiled DNA, and the para isomer gives enhanced cleavage of supercoiled and relaxed DNA. With Dra I (TTTAAA), the ortho isomer again gives partial inhibition cleavage of supercoiled and relaxed DNA, the meta isomer gives inhibition of cleavage of relaxed AND supercoiled DNA, and the para isomer has no effect on cleavage of either form. Variations in flanking sequences affect the selectivity in binding. Mung bean digestion assays indicate that all three isomers alter the conformation of the supercoiled DNA, with the ortho isomer giving the greatest distortion. Topoisomerase studies suggest that these molecules unwind the DNA. Molecular modeling suggests that the placement of the methyl substituent on the pyridyl rings affects the planarity of the molecules, which may alter the binding to DNA and lead to the observations above.

Qualitative analysis of sequence specific binding of flavones to DNA using restriction endonuclease activity assays

Flavones, found in nature as secondary plant metabolites, have shown efficacy as anti-cancer agents. We have examined the binding of two flavones, 5,7-dihydroxy-3,6,8-trimethoxy-2-phenyl-4H-chromen-4-one (5,7-dihydroxy-3,6,8-trimethoxy flavone; FlavA) and 3,5-dihydroxy-6,7,8-trimethoxy-2-phenyl-4H-chromen-4-one (3,5-dihydroxy-6,7,8-trimethoxy flavone; FlavB), to phiX174 RF DNA using restriction enzyme activity assays employing the restriction enzymes Alw44, AvaII, BssHII, DraI, MluI, NarI, NciI, NruI, PstI, and XhoI. These enzymes possess differing target and flanking sequences allowing for observation of sequence specificity analysis. Using restriction enzymes that cleave once with a mixture of supercoiled and relaxed DNA substrates provides for observation of topological effects on binding. FlavA and FlavB show differing sequence specificities in their respective binding to phiX. For example, with relaxed DNA, FlavA shows inhibition of cleavage with DraI (reaction site (5') TTTAAA) but not BssHII ((5') GCGCGC) while FlavB shows the opposite results. Evidence for tolological specificity is also observed, Molecular modeling and conformational analysis of the flavones suggests that the phenyl ring of FlavB is coplanar with the flavonoid ring while the phenyl ring of FlavA is at an angle relative to the flavonoid ring. This may account for aspects of the observed sequence and topological specificities in the effects on restriction enzyme activity.

G-quadruplex-Based Fluorescent Assay of S1 Nuclease Activity and K+

Endonuclease plays an important role in many biological processes, and an assay of endonuclease activity is of great significance. However, traditional methods for the assay of endonuclease activity have undesirable limitations, such as high cost, DNA-consuming and laboriousness. In the present work, a G-quadruplex-based, fluorescent assay of endonuclease activity has been developed with protoporphyrin IX (PPIX) as a signal reporter. S1 nuclease, a single strand DNA (ssDNA)-specific endonuclease, is employed as model system. In the "on" state, G-quadruplex DNA can greatly enhance the fluorescence of PPIX. However, if S1 nuclease could cleave G-quadruplex DNA into small fragments, there would be no formation of G-quadruplexes, accompanied by low emission response of PPIX. This fluorescent discrimination before or after digestion by nuclease can be used to monitor the activity of S1 nuclease. This assay is simple in design and offers a convenient protocol for homogeneous, rapid and high-throughput detection. In addition, the proposed strategy avoids complicated covalent modifications or chemical labeling, and thus offers advantages of simplicity and cost efficiency. More importantly, K(+) is found to well inhibit the activity of S1 nuclease when using certain G-quadruplex DNA as substrate, and thus this system is further used for turn-on detection of K(+). S1 nuclease is critical in the detection of K(+) since it helps to reduce the background signal.

Structural and Biochemical Basis for Development of Influenza Virus Inhibitors Targeting the PA Endonuclease

Emerging influenza viruses are a serious threat to human health because of their pandemic potential. A promising target for the development of novel anti-influenza therapeutics is the PA protein, whose endonuclease activity is essential for viral replication. Translation of viral mRNAs by the host ribosome requires mRNA capping for recognition and binding, and the necessary mRNA caps are cleaved or “snatched” from host pre-mRNAs by the PA endonuclease. The structure-based development of inhibitors that target PA endonuclease is now possible with the recent crystal structure of the PA catalytic domain. In this study, we sought to understand the molecular mechanism of inhibition by several compounds that are known or predicted to block endonuclease-dependent polymerase activity. Using an in vitro endonuclease activity assay, we show that these compounds block the enzymatic activity of the isolated PA endonuclease domain. Using X-ray crystallography, we show how these inhibitors coordinate the two-metal endonuclease active site and engage the active site residues. Two structures also reveal an induced-fit mode of inhibitor binding. The structures allow a molecular understanding of the structure-activity relationship of several known influenza inhibitors and the mechanism of drug resistance by a PA mutation. Taken together, our data reveal new strategies for structure-based design and optimization of PA endonuclease inhibitors.

Post-ischemic administration of peptide with apurinic/apyrimidinic endonuclease activity inhibits induction of cell death after focal cerebral ischemia/reperfusion in mice

Previous scientific research has elucidated the correlation between changes in levels of the DNA base excision repair protein, apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1), and ischemic neuronal DNA damage. However, to date, no studies have addressed the question of whether treatment involving this protein's repair function may prevent ischemic neuron death in vivo. Therefore, we aimed to investigate whether treatment with APE peptide is sufficient to prevent neuron death after ischemia/reperfusion (I/R) in mice. Mice were subjected to intraluminal suture occlusion of the middle cerebral artery for 1 h followed by reperfusion. Post-ischemic treatment with the peptide containing only the APE repair functional domain was introduced intracerebroventricularly. Endonuclease activity assay and immunohistochemistry were performed. Assays of apurinic/apyrimidinic (AP) sites, single-strand DNA breaks, caspase-3 activity, and cell death were examined and quantified. We found that post-ischemic administration of the APE peptide up to 4 h after reperfusion significantly inhibited the induction of cell death and subsequent infarct volume, measured 24 h after I/R.

Real-time monitoring of restriction endonuclease activity using molecular beacon

Restriction endonucleases are one of the most important enzymes in molecular biology. These enzymes are essential in recombinant technology, genotyping, mapping, and sequencing of large strands of DNA [1,2]. Because of the biological importance of restriction endonucleases and their wide use, restriction endonuclease activity assays are essential. Traditional methods for assaying activity of restriction endonucleases, such as gel electrophoresis, Wlter binding, high-performance liquid chromatography (HPLC), and enzyme-linked immunosorbent assay (ELISA) [3–6], have been used. All of these methods, however, are discontinuous, time-consuming, and laborious, and they usually require isotope labeling. Several spectroscopic methods using Xuorescence measurements have been developed [7–12]. These assays are continuous. However, up to now all of them have been designed only for doubly labeled DNA substrates with very large Xuorophores that may interfere with recognition and reactivity with certain restriction endonucleases. Furthermore, the potential for achieving high sensitivity through Xuorescent resonance energy transfer (FRET) has not been fully realized. Therefore, it is necessary to develop a sensitive, convenient, and non-isotope-labeled method for assaying restriction endonuclease activity. Since they were Wrst reported in 1996 [13], molecular beacons (MBs) have become a class of molecules widely

Identification of an Essential Cleavage Site in ColE7 Required for Import and Killing of Cells

Colicin E7 (ColE7), a nuclease toxin released from Escherichia coli, kills susceptible bacteria under environmental stress. Nuclease colicins are processed during translocation with only the cytotoxic nuclease domains traversing the inner membrane to cleave tRNA, rRNA, or DNA in the cytoplasm of target cells. In this study, we show that the E. coli periplasmic extract cleaves ColE7 between Lys(446) and Arg(447) in the presence or absence of its inhibitor Im7 protein. Several residues near cleavage sites were mutated, but only mutants of Arg(447) completely lost in vivo cell-killing activity. Both the full-length and the nuclease domain of Arg(447) mutants retained their nuclease activities, indicating that failure to kill cells was not a consequence of damage to the endonuclease activity of the enzyme. Moreover, the R447E ColE7 mutant was not cleaved at its 447 site by periplasmic extracts or transported into the cytoplasm of target cells. Collectively, these results suggest that ColE7 is cleaved at Arg(447) during translocation and that cleavage is an essential step for ColE7 import into the cytoplasm of target cells and its cell-killing activity. Conserved basic residues aligned with Arg(447) have also been found in other nuclease colicins, implying that the processing at this position may be common to other colicins during translocation.

Ceramide synthase is essential for endonuclease-mediated death of renal tubular epithelial cells induced by hypoxia-reoxygenation.

Ceramide is known to play a role in the cell signaling pathway involved in apoptosis. Most studies suggest that enhanced ceramide generation is the result of hydrolysis of sphingomyelin by sphingomyelinases. However, the role of ceramide synthase in enhanced ceramide generation has not been previously examined in hypoxia-reoxygenation injury. In the present study, we demonstrated that 60-min hypoxia of rat renal tubular epithelial NRK-52E cells in a gas chamber with 95% N2-5% CO2 with glucose deprivation resulted in a significant increase in ceramide generation. The ceramide level further increased after reoxygenation for 60 min. Exposure of cells to hypoxia-reoxygenation resulted in a significant increase in ceramide synthase activity without any significant change in acid or neutral sphingomyelinase. The hypoxia-reoxygenation of NRK-52E cells was also associated with the release of endonuclease G (EndoG) from mitochondria to cytoplasm measured by Western blot analysis and endonuclease activity assay. It further led to the fragmentation of DNA and cell death. A specific inhibitor of ceramide synthase, fumonisin B1 (50 microM), suppressed hypoxia-reoxygenation-induced ceramide generation and provided protection against hypoxia-reoxygenation-induced EndoG release, DNA fragmentation, and cell death. Taken together, our data suggest that hypoxia-reoxygenation results in an activation of ceramide synthase rather than sphingomyelinase and that ceramide synthase-dependent ceramide generation is a key modulator of EndoG-mediated cytotoxicity in hypoxia-reoxygenation injury to renal tubular epithelial cells.

Enhancement of assays of activities of endonucleases on defined substrates by poisson and non-poisson combinatoric analysis

Assay of endonuclease activity, as performed in most laboratories, depends upon change in form of small, defined substrate molecules, with a secondary computation required to obtain a determination of enzyme activity. We now explore the assumptions inherent in these computations and provide a series of equations that permit more accurate determinations of enzyme activity from assays of this type. These equations allow information to be obtained not only from substrate fractions left uncleaved by the endonuclease, upon which conventional systems rely, but also from products cleaved by the enzyme. Some information yielded by these equations is unobtainable using conventional methods of analysis.

A rapid immunological assay of endonuclease activity

Denatured T4 DNA mixed with specific antibody forms a complex which is retained by a glass fiber filter. The retention of this complex is dependent on the size of the DNA: single-stranded fragments with a molecular weight greater than 10 6 are retained, whereas fragments with a molecular weight of less than 2 · 10 4 are not retained. The effects of pH, temperature, NaCl concentration, antiserum concentration, and DNA concentration on the formation and retention of antibody-DNA complexes have been studied. This technique can be used as an endonuclease assay and will detect 1 ng pancreatic deoxyribonuclease. It is 100 times more sensitive than an acid-solubility assay. This endonuclease assay was used to monitor the purification of Mustelis canis liver endonuclease.