Nicked DNA Research Articles

Human MutLγ, the MLH1–MLH3 heterodimer, is an endonuclease that promotes DNA expansion

MutL proteins are ubiquitous and play important roles in DNA metabolism. MutLγ (MLH1-MLH3 heterodimer) is a poorly understood member of the eukaryotic family of MutL proteins that has been implicated in triplet repeat expansion, but its action in this deleterious process has remained unknown. In humans, triplet repeat expansion is the molecular basis for ∼40 neurological disorders. In addition to MutLγ, triplet repeat expansion involves the mismatch recognition factor MutSβ (MSH2-MSH3 heterodimer). We show here that human MutLγ is an endonuclease that nicks DNA. Strikingly, incision of covalently closed, relaxed loop-containing DNA by human MutLγ is promoted by MutSβ and targeted to the strand opposite the loop. The resulting strand break licenses downstream events that lead to a DNA expansion event in human cell extracts. Our data imply that the mammalian MutLγ is a unique endonuclease that can initiate triplet repeat DNA expansions.

Structure and mechanism of a Type III CRISPR defence DNA nuclease activated by cyclic oligoadenylate

The CRISPR system provides adaptive immunity against mobile genetic elements in prokaryotes. On binding invading RNA species, Type III CRISPR systems generate cyclic oligoadenylate (cOA) signalling molecules, potentiating a powerful immune response by activating downstream effector proteins, leading to viral clearance, cell dormancy or death. Here we describe the structure and mechanism of a cOA-activated CRISPR defence DNA endonuclease, CRISPR ancillary nuclease 1 (Can1). Can1 has a unique monomeric structure with two CRISPR associated Rossman fold (CARF) domains and two DNA nuclease-like domains. The crystal structure of the enzyme has been captured in the activated state, with a cyclic tetra-adenylate (cA4) molecule bound at the core of the protein. cA4 binding reorganises the structure to license a metal-dependent DNA nuclease activity specific for nicking of supercoiled DNA. DNA nicking by Can1 is predicted to slow down viral replication kinetics by leading to the collapse of DNA replication forks.

Dynamics of DNA nicking and unwinding by the RepC-PcrA complex.

The rolling-circle replication is the most common mechanism for the replication of small plasmids carrying antibiotic resistance genes in Gram-positive bacteria. It is initiated by the binding and nicking of double-stranded origin of replication by a replication initiator protein (Rep). Duplex unwinding is then performed by the PcrA helicase, whose processivity is critically promoted by its interaction with Rep. How Rep and PcrA proteins interact to nick and unwind the duplex is not fully understood. Here, we have used magnetic tweezers to monitor PcrA helicase unwinding and its relationship with the nicking activity of Staphylococcus aureus plasmid pT181 initiator RepC. Our results indicate that PcrA is a highly processive helicase prone to stochastic pausing, resulting in average translocation rates of 30 bp s−1, while a typical velocity of 50 bp s−1 is found in the absence of pausing. Single-strand DNA binding protein did not affect PcrA translocation velocity but slightly increased its processivity. Analysis of the degree of DNA supercoiling required for RepC nicking, and the time between RepC nicking and DNA unwinding, suggests that RepC and PcrA form a protein complex on the DNA binding site before nicking. A comprehensive model that rationalizes these findings is presented.

Extraction, characterization and biological properties of polysaccharide derived from green seaweed “Chaetomorpha linum” and its potential application in Tunisian beef sausages

A novel polysaccharide extracted from green algae Chaetomorpha linum (PS) was characterized, using infrared spectroscopy, HPLC-FID, gel filtration high-pressure chromatography, thin layer chromatography and spectrum visible UV. Natural antioxidant potential of PS was evaluated based on DPPH free radical, ferrous iron–chelating, β carotene bleaching inhibition activities and DNA nicking assay. Functional properties were estimated based on Water Holding Capacity (WHC), Oil Holding Capacity (OHC), emulsifying activity and foaming ability. Overall, data showed attractive chemical, functional and biological properties with an antioxidant capacity of PS in beef sausages during storage for 12 days. Indeed, as compared to standard formulation (with addition of vitamin C), samples formulated with PS presented lower values in terms of lipid oxidation (TBARS, MetMb), while preserving color properties compared with untreated samples. Furthermore, lower changes in pH, moisture, and the highest values of heme iron were obtained. Successful inhibition of microbial proliferation was proved. Endowed with high antioxidant and antimicrobial activities, PS could thus be used as a natural conservative in functional foods.

Full-length human cytomegalovirus terminase pUL89 adopts a two-domain structure specific for DNA packaging.

A key step in replication of human cytomegalovirus (HCMV) in the host cell is the generation and packaging of unit-length genomes into preformed capsids. The enzymes involved in this process are the terminases. The HCMV terminase complex consists of two terminase subunits, the ATPase pUL56 and the nuclease pUL89. A potential third component pUL51 has been proposed. Even though the terminase subunit pUL89 has been shown to be essential for DNA packaging and interaction with pUL56, it is not known how pUL89 mechanistically achieves sequence-specific DNA binding and nicking. To identify essential domains and invariant amino acids vis-a-vis nuclease activity and DNA binding, alanine substitutions of predicted motifs were analyzed. The analyses indicated that aspartate 463 is an invariant amino acid for the nuclease activity, while argine 544 is an invariant aa for DNA binding. Structural analysis of recombinant protein using electron microscopy in conjunction with single particle analysis revealed a curvilinear monomer with two distinct domains connected by a thinner hinge-like region that agrees well with the predicted structure. These results allow us to model how the terminase subunit pUL89’s structure may mediate its function.

Two-tiered enforcement of high-fidelity DNA ligation

DNA ligases catalyze the joining of DNA strands to complete DNA replication, recombination and repair transactions. To protect the integrity of the genome, DNA ligase 1 (LIG1) discriminates against DNA junctions harboring mutagenic 3′-DNA mismatches or oxidative DNA damage, but how such high-fidelity ligation is enforced is unknown. Here, X-ray structures and kinetic analyses of LIG1 complexes with undamaged and oxidatively damaged DNA unveil that LIG1 employs Mg2+-reinforced DNA binding to validate DNA base pairing during the adenylyl transfer and nick-sealing ligation reaction steps. Our results support a model whereby LIG1 fidelity is governed by a high-fidelity (HiFi) interface between LIG1, Mg2+, and the DNA substrate that tunes the enzyme to release pro-mutagenic DNA nicks. In a second tier of protection, LIG1 activity is surveilled by Aprataxin (APTX), which suppresses mutagenic and abortive ligation at sites of oxidative DNA damage.

Hybridization chain reaction (HCR) for amplifying nanopore signals

Circulating tumor DNA (ctDNA) in the blood is an important biomarker for noninvasive diagnosis, assessment, prediction and treatment of cancer. However, sensing performance of solid nanopore is limited by the fast kinetics of small DNA targets and unmatched dimensions. Here, we combines hybridization chain reaction (HCR) with nanopore detection to translate the presence of a small DNA target to characteristic nanopore signals of a long nicked DNA polymer. The amplification of nanopore signals obtained by HCR not only overcomes the functional limitation of solid nanopore, but also significantly elevates both selectivity and signal-to-noise ratio, which allows to detect ctDNA at a detection limit of 2.8 fM (S/N = 3) and the single-base resolution. Furthermore, the proposed method can apply in detection of ctDNA of KRAS G12DM in serum sample.

ATR, a DNA Damage Signaling Kinase, Is Involved in Aluminum Response in Barley.

Ataxia Telangiectasia and Rad-3-related protein (ATR) is a DNA damage signaling kinase required for the monitoring of DNA integrity. Together with ATM and SOG1, it is a key player in the transcriptional regulation of DNA damage response (DDR) genes in plants. In this study, we describe the role of ATR in the DDR pathway in barley and the function of the HvATR gene in response to DNA damages induced by aluminum toxicity. Aluminum is the third most abundant element in the Earth’s crust. It becomes highly phytotoxic in acidic soils, which comprise more than 50% of arable lands worldwide. At low pH, Al is known to be a genotoxic agent causing DNA damage and cell cycle arrest. We present barley mutants, hvatr.g and hvatr.i, developed by TILLING strategy. The hvatr.g mutant carries a G6054A missense mutation in the ATR gene, leading to the substitution of a highly conserved amino acid in the protein (G1015S). The hvatr.g mutant showed the impaired DDR pathway. It accumulated DNA damages in the nuclei of root meristem cells when grown in control conditions. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis revealed that 60% of mutant nuclei possessed DNA nicks and breaks, whereas in the wild type only 2% of the nuclei were TUNEL-positive. The high frequency of DNA damages did not lead to the inhibition of the cell cycle progression, but the mutant showed an increased number of cells in the G2/M phase. In response to treatments with different Al doses, hvatr.g showed a high level of tolerance. The retention of root growth, which is the most evident symptom of Al toxicity, was not observed in the mutant, as it was in its parent variety. Furthermore, Al treatment increased the level of DNA damages, but did not affect the mitotic activity and the cell cycle profile in the hvatr.g mutant. A similar phenotype was observed for the hvatr.i mutant, carrying another missense mutation leading to G903E substitution in the HvATR protein. Our results demonstrate that the impaired mechanism of DNA damage response may lead to aluminum tolerance. They shed a new light on the role of the ATR-dependent DDR pathway in an agronomically important species.

Deletions associated with stabilization of the Top1 cleavage complex in yeast are products of the nonhomologous end-joining pathway.

Topoisomerase I (Top1) resolves supercoils by nicking one DNA strand and facilitating religation after torsional stress has been relieved. During its reaction cycle, Top1 forms a covalent cleavage complex (Top1cc) with the nicked DNA, and this intermediate can be converted into a toxic double-strand break (DSB) during DNA replication. We previously reported that Top1cc trapping in yeast increases DSB-independent, short deletions at tandemly repeated sequences. In the current study, we report a type of DSB-dependent mutation associated with Top1cc stabilization: large deletions (median size, ∼100 bp) with little or no homology at deletion junctions. Genetic analyses demonstrated that Top1cc-dependent large deletions are products of the nonhomologous end-joining (NHEJ) pathway and require Top1cc removal from DNA ends. Furthermore, these events accumulated in quiescent cells, suggesting that the causative DSBs may arise outside the context of replication. We propose a model in which the ends of different, Top1-associated DSBs are joined via NHEJ, which results in deletion of the intervening sequence. These findings have important implications for understanding the mutagenic effects of chemotherapeutic drugs that stabilize the Top1cc.

Three-way junction-induced isothermal amplification for nucleic acid detection

We, herein, describe a three-way junction (3WJ)-induced isothermal amplification (ThIsAmp) reaction for target nucleic acid detection. In this strategy, target nucleic acid induces the formation of 3WJ structure by associating a specially designed ThIsAmp template and ThIsAmp primer. Upon the formation of 3WJ structure, ThIsAmp primer is subjected to continuously repeated extension and nicking reaction by the combined activities of DNA polymerase and nicking endonuclease, consequently producing a large number of trigger strands. The trigger strands then initiate two separate but interconnected pathways by binding to either 3’ overhang of ThIsAmp template within the 3WJ structure or free ThIsAmp template. As a consequence, a large number of final double-stranded DNA products are produced under an isothermal condition, which can be monitored in real-time by detecting the fluorescence intensity resulting from SYBR Green I staining. Based on this principle, we successfully detected target DNA down to 78.1 aM with excellent specificity. The sophisticated design principle employed in this work would provide great insight for the development of self-operative isothermal amplifying system enabling target nucleic acid detection.

In-vitro antibacterial activity of Ni(II), Cu(II), and Zn(II) complexes incorporating new azo-azomethine ligand possessing excellent antioxidant, anti-inflammatory activity and protective effect of free radicals against plasmid DNA

Azo Schiff base ligand 2-hydroxy-3-methoxy-5-(tolyldiazenyl)benzaldehyde oxime (HL1) and 2-hydroxy-3-methoxy-5-(methoxyphenyl)benzaldehyde oxime (HL2) were prepared along with their transition metal complexes of Ni(II), Cu(II), and Zn(II). Ligands and their metal complexes were characterized by several analysis techniques. In- vitro antibacterial, antioxidant and anti-inflammatory activities of synthesized ligands and their metal complexes have been studied. Biological study showed that amongst all the synthesized compounds, Cu(II) complexes possessed excellent antibacterial activity than standard antibiotic Chloramphenicol. Ligands (HL1) and (HL2) showed excellent antioxidant as well as anti-inflammatory activity. Both the ligands were tested for their protective effect of free radicals against plasmid DNA and it was found that both the ligands showed good DNA nicking activity.

Selecting spermatozoa with intact chromatin may reduce embryo aneuploidy

We tested a novel approach for treating couples with complete and persistent embryo aneuploidy. Using a microfluidic device, we selected spermatozoa with the highest progressive motility and genomic integrity, capable of generating euploid embryos. From October 2016 to April 2019, 13 consenting couples with male partners with high sperm chromatin fragmentation (SCF) in their ejaculate and a history of embryo aneuploidy and/or recurring implantation failure underwent a new ICSI cycle in which semen specimens were processed by microfluidics sperm selection (MFSS) and density gradient centrifugation (DGC). Consenting men had their ejaculates screened by standard semen analysis according to WHO 2010 criteria. Specimens were processed by DGC and MFSS. SCF was measured by TUNEL utilizing a commercial kit (In Situ Cell Death Detection Kit, Roche). At least 500 spermatozoa were counted under fluorescent microscopy, with an established threshold of 15%. Fertilization and clinical pregnancy rates were assessed and compared between the two preparation methods. Preimplantation genetic testing for aneuploidy (PGT-A) was performed on the resulting embryos. Embryo implantation and pregnancies after replacement of thawed euploid blastocysts were recorded. A total of 13 men with an average age of 41.5±10 years had the following average semen parameters: concentration of 40.5±44 x106/mL, 26±19 motility, and 2.2±1% morphology. After DGC and MFSS, the sperm concentration was 27±41 and 4.4±7 x106/mL, with 47.7±43% and 97.1±4% motility, respectively (P<0.0001). The average SCF decreased from 29% in the raw samples to 20% following DGC (P=NS), and dropped to 2.2% after MFSS processing (P<0.0001). These couples (female partner, 39±6 years) underwent 15 ICSI cycles with DGC-selected spermatozoa and achieved a fertilization rate of 71.3% (97/136), which generated 64.5% (31/48) morphologically good-quality embryos; of these, 12.5% (6/48) were determined by PGT-A to be euploid. Two of these euploid embryos were transferred and did not yield a pregnancy. In a subsequent ICSI cycle with MFSS processing, a fertilization rate of 73.2% (104/142) resulted in 61.8% (34/55) good-quality embryos. Of these, 36.3% (20/55) were identified as euploid and were cryopreserved (P<0.05). Seven couples received a thawed single euploid blastocyst and all 7 became pregnant (P<0.0001), resulting in the delivery of two healthy babies, with 71.4% (5/7) still ongoing. Dysfunction of the male genital tract increases both single-strand (ss) and double-strand (ds) DNA nicks and breaks, resulting in spermatozoa that impair embryonic development. Because dsDNA breaks in the male gamete can be responsible for embryo aneuploidy, the use of MFSS processing to select spermatozoa with the highest motility and genomic integrity may enhance the chances of obtaining a euploid conceptus for transfer.

Proteome of HU-Lacking E. coli Studied by Means of 2D Gel Electrophoresis

Histone-like protein HU is a dimeric nucleoid-associated protein (NAP). HU is the most conserved NAP. It binds nonspecifically to duplex DNA with a preference for targeting nicked and bent DNA. HU limits the architecture of the bacterial nucleoid and its deletion is lethal for Bacillus subtilis and Mycoplasma genitalium which do not contain other NAPs. E. coli lacking HU is viable but has numerous growth defects. The effects of the HU protein on gene expression is known from microarray analysis and HU regulons were identified. In HU-deficient E. coli, absence of this DNA architectural protein causes a disorder in gene regulation; on the other hand, E. coli growth under standard conditions is almost unaltered in the absence of HU. To understand how the bacterium confronts the chromosomal disorder, we performed proteome analysis to compare protein abundances in cells containing the HU protein or not. Comparison of the proteomic profile of wild-type and HU-deficient E. coli shows how the altered gene expression influences the protein content. We show that proteome profile changes are very similar to the gene expression profile changes in HU-deficient E. coli. Several exceptions show that proteome studies are very important.

Gd-EOB-DTPA MRI Imaging and Apoptosis Research in Orthotopic Rats Model of Hepatocellular Carcinoma after Stereotactic Radiotherapy

To investigate the characteristics of Gd-EOB-DTPA MRI imaging tumor cell apoptosis and the correlation between them in orthotopic rats model of hepatocellular carcinoma after stereotactic radiotherapy. Forty healthy male Sprague-Dawley rats were randomly divided into model group (n=40) and control group (n=20). When the tumor diameter was ≥1.0cm, the rats in the experimental group underwent single stereotactic radiotherapy with a dose of 15Gy. 40 rats after radiotherapy were randomly divided into 4 groups, 10 in each group. The control group was randomly divided into 4 groups of 5 each, and the above group was added. MR rats were routinely scanned and DWI scans were performed on the 1st, 7th, 14th, and 21st day after radiotherapy. The region of interest (ROI) ADC value and signal intensity (SI) of the tumor and normal life after radiotherapy were measured, and the ratio of ADC to SI (tumor/liver) was calculated. After the end of the scan, the animals were sacrificed, and the liver cell tumor specimens were subjected to DNA nick end labeling (TUNEL) to determine the apoptosis of the tumor cells, and the apoptotic index (apoptotic cells/tumor cells) was calculated. Two independent sample t-tests were used to compare the apoptotic index and ADC ratio between the radiotherapy group and the control group. Comparison of apoptotic index and ADC ratio between the radiotherapy groups was analyzed by one-way analysis of variance; the correlation between the two was performed using the Pearson test. The ADC and SI ratios were statistically significant at 7 days, 14 days and 21 days after radiotherapy (P<0.01=, there was no significant difference at 1 day after radiotherapy (P>0.05). 1 day, 7 days, 14 days, The apoptotic index of the 21-day radiotherapy group was significantly different from that of the control group (P<0.05=. The ADC and SI ratios were positively correlated with the apoptotic index (AI) (P<0.05). The imaging characteristics of Gd-EOB-DTPA MRI can reflect the dynamic changes of apoptosis at different time points after stereotactic radiotherapy in rat liver cancer.

Chromatin-bound oxidized α-synuclein causes strand breaks in neuronal genomes in in vitro models of Parkinson's Disease

Alpha-synuclein (α-Syn) overexpression and misfolding/aggregation in degenerating dopaminergic neurons have long been implicated in Parkinson's disease (PD). The neurotoxicity of α-Syn is enhanced by iron (Fe) and other pro-oxidant metals, leading to generation of reactive oxygen species in PD brain. Although α-Syn is predominantly localized in presynaptic nerve terminals, a small fraction exists in neuronal nuclei. However, the functional and/or pathological role of nuclear α-Syn is unclear. Following up on our earlier report that α-Syn directly binds DNA in vitro, here we confirm the nuclear localization and chromatin association of α-Syn in neurons using proximity ligation and chromatin immunoprecipitation analysis. Moderate (∼2-fold) increase in α-Syn expression in neural lineage progenitor cells (NPC) derived from induced pluripotent human stem cells (iPSCs) or differentiated SHSY-5Y cells caused DNA strand breaks in the nuclear genome, which was further enhanced synergistically by Fe salts. Furthermore, α-Syn required nuclear localization for inducing genome damage as revealed by the effect of nucleus versus cytosol-specific mutants. Enhanced DNA damage by oxidized and misfolded/oligomeric α-Syn suggests that DNA nicking activity is mediated by the chemical nuclease activity of an oxidized peptide segment in the misfolded α-Syn. Consistent with this finding, a marked increase in Fe-dependent DNA breaks was observed in NPCs from a PD patient-derived iPSC line harboring triplication of the SNCA gene. Finally, α-Syn combined with Fe significantly promoted neuronal cell death. Together, these findings provide a novel molecular insight into the direct role of α-Syn in inducing neuronal genome damage, which could possibly contribute to neurodegeneration in PD.

The Aggregation Behavior and Antioxidative Activity of Amphiphilic Surfactants Based on Quinuclidin‐3‐ol

AbstractThe self‐aggregation and thermodynamic properties of three cationic quaternary ammonium surfactants were investigated. The physicochemical properties of compounds containing quinuclidin‐3‐ol with even number of carbon atoms (10, 12, and 14) in the hydrophobic tail were measured by conductivity, dynamic light scattering (DLS), and Zeta‐potential measurements. DLS and Zeta‐potential measurements show a similar size distribution for all surfactants with excellent uniformity, and Zeta‐potential increases significantly with increase in the size of hydrocarbon tail. The critical micelle concentration (CMC) and the degree of micelle ionization (β) were determined using conductivity measurements. The CMC values of surfactants were found to be between 3.4 and 23.8 × 10−3 M. The standard Gibbs free energy () was derived from conductivity measurements and suggests that surfactants containing longer chains spontaneously form micelles. The antioxidative properties of these cationic surfactants were evaluated using the 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) and oxygen radical absorbance capacity (ORAC) assays. Among the tested samples, N‐tetradecyl‐3‐hydroxyquinuclidinium bromide (QOH‐C14) exhibited the highest antioxidative potential (388.30 nmol (TE) equivalents mL−1), which was further investigated by the DNA nicking assay.

Xanthine Oxidase Inhibitors Screening, Antioxidation, and DNA Protection Properties of Geranium wilfordii Maxim

Geranium wilfordii Maxim is a commonly used traditional medicine and as decoction pieces in drinks for treating acute and chronic rheumatalgia. Earlier research showed it could inhibit xanthine oxidase and had a strong antioxidative activity. In this study, four compounds namely, corilagin, geraniin, ellagic acid, and myricetrin were screened and identified as xanthine oxidase inhibitors and antioxidants using ultrafiltration combined liquid chromatography—mass spectrometry (LC—MS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) spiking combined LC—MS methods. Ellagic acid showed the greatest inhibition with IC50 of 0.018 ± 0.001 mM better than that of allopurinol. The kinetic parameters exhibited the mode of xanthine oxidase inhibitions by corilagin, geraniin, and myricetrin were the competitive types, whereas the ellagic acid was found as an uncompetitive inhibitor. In vitro DNA nicking assay showed these four compounds protected DNA from reactive oxygen species. Correlation analysis and partial least squares analysis showed there was a moderate correlation between ellagic acid and DPPH˙ scavenging activity. Our findings revealed four compounds in G. wilfordii Maxim contributed satisfied radicals scavenging, xanthine oxidase inhibition, and DNA damage protective activities. More comprehensive research on pharmaceutical applications and phytochemical profile of G. wilfordii Maxim could be further studied.

Plasmid DNA nicking- a Novel Activity of Soybean Trypsin Inhibitor and Bovine Aprotinin

Protease inhibitors, such as trypsin inhibitor, serum alpha-1 antitrypsin, or liver aprotinin, are a class of proteins that competitively bind and block the catalytic activity of proteolytic enzymes with wide ranging biological functions. A significant number of protease inhibitors have also been shown to possess antimicrobial activity, presumed to contribute in defense against pathogenic microorganisms as plants with higher levels of protease inhibitors tend to exhibit increased resistance towards pathogens. Two proposed mechanisms for the antimicrobial activity are combating microbial proteases that play roles in disease development and disruption of microbial cell wall & membrane necessary for survival. Here we show for the first time a novel activity of soybean trypsin inhibitor and bovine aprotinin that they nick supercoiled, circular plasmid DNA. A number of experiments conducted to demonstrate the observed DNA nicking activity is inherent, rather than a co-purified, contaminating nuclease. The nicking of the plasmid results in markedly reduced efficiencies in transformation of E. coli and transfection of HEK293T cells. Thus, this work reveals yet a new mechanism for the antimicrobial activity by protease inhibitors.

Dx.doi.org/10.18081/2333-5106/016-7/253-264

DNA topoisomerases are ubiquitous, enzymes needed to control topological problems encountered during DNA replication, transcription, recombination and maintenance of genomic stability. They proved to be valuable targets for cancer chemotherapy. The seeds alkaloids extract of Rue plant (Peganum harmala L. Zygophyllaceae) were fractioned by high performance liquid chromatography as a suitable technique, HPLC waters company, gradient, Nova-Pak, C18*, 4µm, 3.9x150mm, mobile phase ACN-water pH10.7. Thirty-four separate solution depending on the retention time of eluents. The aim of this study is to investigate the trace compounds relatively and used as inhibitors for the topoisomerase II enzyme, the study the poison and suppressor model inhibitions and the synergy and antagonism activity of fractions. The visicinone, harmine, visicine and harmaline, were (2.86, 0.75, 13.14, and 16.62 mg/100mg of plant powder. The harmine ˃vicicinone˃ harmaline˃ vicicine as enzyme inhibitors. The unknown trace compounds gave the best inhibition relatively, the alkaloid fractions gave higher inhibitory units of fraction effective (FE). Results of seeds alkaloid fractions showed that the FE in state of fraction mixture was 36% inhibitory units, while summation of FE of each fraction alone gave higher level than their mixture, where sum of FE of them was 1229% inhibitory units. The suppressor inhibition model according to highest percent of the remain supercoiled DNA plasmid than production of linear and open nicked circular with high value in F8 (84%), the poisons inhibition model activity according to the highest percent of linear and nicked open circular DNA plasmid with high level in F31 (88%). There are a trace compounds within some fraction but that the effectiveness of inhibitory relatively higher than known compounds (harmine, vicisinone, harmaline, and vicisine), this can be purified and used as somewhat better inhibitors. There is antagonism activity among seed alkaloids fractions. The all fractions showed a poison and a suppressor model inhibition action in varies percentage, the suppressor inhibition appear in high value in F8 (84%), while the poisons inhibition model gave high level in F31 (88%).

A Universal Assay for Making DNA, RNA, and RNA-DNA Hybrid Configurations for Single-Molecule Manipulation in Two or Three Steps without Ligation.

Despite having a great variety of topologies, most DNA, RNA, and RNA-DNA hybrid (RDH) configurations for single-molecule manipulation are composed of several single-stranded (ss) DNA and ssRNA strands, with functional labels at the two ends for surface tethering. On this basis, we developed a simple, robust, and universal amplification-annealing (AA) assay for making all these configurations in two or three steps without inefficient digestion and ligation reactions. As examples, we made ssDNA, short ssDNA with double-stranded (ds) DNA handles, dsDNA with ssDNA handles, replication-fork shaped DNA/RDH/RNA, DNA holiday junction, three-site multiple-labeled and nicked DNA, torsion-constrained RNA/RDH, and short ssRNA with RDH handles. In addition to single-molecule manipulation techniques including optical tweezers, magnetic tweezers, and atomic force microscopy, these configurations can be applied in other surface-tethering techniques as well.