DNA Cleavage Activity Research Articles

Exploring the antioxidant, antimicrobial, cytotoxic and biothermodynamic properties of novel morpholine derivative bioactive Mn(ii), Co(ii) and Ni(ii) complexes - combined experimental and theoretical measurements towards DNA/BSA/SARS-CoV-2 3CLPro.

A novel class of bioactive complexes (1-3) [MII(L)2(bpy)], where, L = 2-(4-morpholinobenzylideneamino)phenol, bpy = 2,2'-bipyridine, MII = Mn (1), Co (2) or Ni (3), were assigned to octahedral geometry based on analytical and spectral measurements. Gel electrophoresis showed that complex (2) demonstrated significant DNA cleavage activity compared to the other complexes under the action of oxidation agent (H2O2). The DNA binding constant properties measured by various techniques were in the following sequence: (2) > (3) > (1) > (HL), which suggests that the complexes might intercalate DNA, a possibility that is also supported by their biothermodynamic characteristics. The binding constant results for BSA from electronic absorption and fluorometric titrations demonstrate that complex (2) exhibits the highest binding effectiveness among them all, which means that all the compounds could interact with BSA through a static approach, additionally supported by FRET measurements. DFT and docking calculations were employed to realize the electronic structure, reactivity, and interaction capability of all substances with DNA, BSA, and the SARS-CoV-2 main protease. These binding energies fell within the ranges -7.7 to -8.5, -8.2 to -10.1 and -6.7 to -9.3 kcal mol-1, respectively. The higher reactivity of the complexes than the ligand is supported by FMO theory. The in vitro antibacterial, cytotoxicity, and radical scavenging characteristics revealed that complexes (2-3) have better biological efficacy than the others. The cytotoxicity and binding properties also show good correlation with the partition coefficient (log P), which is encouraging because all of the experimental findings are closely correlated with the theoretical measurements.

Flap Endonuclease 1 Endonucleolytically Processes RNA to Resolve R-Loops through DNA Base Excision Repair.

Flap endonuclease 1 (FEN1) is an essential enzyme that removes RNA primers and base lesions during DNA lagging strand maturation and long-patch base excision repair (BER). It plays a crucial role in maintaining genome stability and integrity. FEN1 is also implicated in RNA processing and biogenesis. A recent study from our group has shown that FEN1 is involved in trinucleotide repeat deletion by processing the RNA strand in R-loops through BER, further suggesting that the enzyme can modulate genome stability by facilitating the resolution of R-loops. However, it remains unknown how FEN1 can process RNA to resolve an R-loop. In this study, we examined the FEN1 cleavage activity on the RNA:DNA hybrid intermediates generated during DNA lagging strand processing and BER in R-loops. We found that both human and yeast FEN1 efficiently cleaved an RNA flap in the intermediates using its endonuclease activity. We further demonstrated that FEN1 was recruited to R-loops in normal human fibroblasts and senataxin-deficient (AOA2) fibroblasts, and its R-loop recruitment was significantly increased by oxidative DNA damage. We showed that FEN1 specifically employed its endonucleolytic cleavage activity to remove the RNA strand in an R-loop during BER. We found that FEN1 coordinated its DNA and RNA endonucleolytic cleavage activity with the 3'-5' exonuclease of APE1 to resolve the R-loop. Our results further suggest that FEN1 employed its unique tracking mechanism to endonucleolytically cleave the RNA strand in an R-loop by coordinating with other BER enzymes and cofactors during BER. Our study provides the first evidence that FEN1 endonucleolytic cleavage can result in the resolution of R-loops via the BER pathway, thereby maintaining genome integrity.

Miniature CRISPR-Cas12f1-Mediated Single-Nucleotide Microbial Genome Editing Using 3'-Truncated sgRNA.

The CRISPR-Cas system has been used as a convenient tool for genome editing because the nuclease that cuts the target DNA and the guide RNA that recognizes the target are separated into modules. Cas12f1, which has a smaller size than that of other Cas nucleases, is easily loaded into vectors and is emerging as a new genome editing tool. In this study, AsCas12f1 was used to negatively select only Escherichia coli cells obtained by oligonucleotide-directed genome editing. Although double-, triple-, and quadruple-base substitutions were accurately and efficiently performed in the genome, the performance of single-base editing was poor. To resolve this limitation, we serially truncated the 3'-end of sgRNAs and determined the maximal truncation required to maintain the target DNA cleavage activity of Cas12f1. Negative selection of single-nucleotide-edited cells was efficiently performed with the maximally 3'-truncated sgRNA-Cas12f1 complex in vivo. Moreover, Sanger sequencing showed that the accuracy of single-nucleotide substitution, insertion, and deletion in the microbial genome was improved. These results demonstrated that a truncated sgRNA approach could be widely used for accurate CRISPR-mediated genome editing.

Green synthesis of iron oxide nanoparticles derived from water and methanol extract of Centaurea solstitialis leaves and tested for antimicrobial activity and dye decolorization capability

In this research, nanoparticles derived from water extract of Centaurea solstitialis leaves were used as green adsorbent in Fenton reaction for Reactive Red 180 (RR180) and Basic Red 18 (BR18) dyes removal. At optimum operating conditions, nanoparticles proved high performance in the tested dyes removal with more than 98% of removal elimination. The free-radical scavenging, DNA nuclease, biofilm inhibition capability, antimicrobial activity, microbial cell viability, and antimicrobial photodynamic therapy activities of the iron oxide nanoparticles (FeO-NPs) derived from water and methanol extract of plant were investigated. Each of the following analysis: SEM-EDX, XRD, and Zeta potential was implemented for the prepared NPs characterization and to describe their morphology, composition and its behavior in an aqueous solution, respectively. It was found that, the DPPH scavenging activities increased when the amount of nanoparticles increased. The highest radical scavenging activity achieved with FeO-NPs derived from water extract of plant as 97.41% at 200 mg/L. The new green synthesized FeO-NPs demonstrated good DNA cleavage activity. FeO-NPs showed good in vitro antimicrobial activities against human pathogens. The results showed that both synthesized FeO-NPs displayed 100% antimicrobial photodynamic therapy activity after LED irradiation. The water extract of FeO-NPs and methanol extract of FeO-NPs also showed a significant biofilm inhibition.

A bacterial Argonaute from Tepiditoga spiralis with the ability of RNA guided plasmid cleavage

The eukaryotic Argonaute proteins (eAgos) play an important role in the RNA interference pathway. The function and mechanism of prokaryotic Argonaute proteins (pAgos) in vivo are still unclear although the structure of pAgos and eAgos are highly homologous. Most of the reported pAgos have a preference for 5′P-gDNA, but MpAgo originated from bacteria Marinitoga piezophila preferentially uses 5′OH-gRNA to target DNA and RNA. To enrich our knowledge of this type of Argonaute proteins, here we report an Argonaute protein derived from Tepiditoga spiralis (TsAgo). Like MpAgo, TsAgo has a preference for 5′OH-gRNA. Meanwhile, TsAgo has DNA and RNA cleavage activity in presence of Mn2+ and Mg2+, and TsAgo has catalytic activity at 37–70 °C. In addition, TsAgo can tolerate mismatches in the 5′-end and 3′-tail regions of guides but is sensitive to mismatches in the 5′-seed and central regions of guides, especially the central region. Furthermore, the EMSA assay reveals that TsAgo exhibits a stronger binding affinity for 5′OH-gRNA than 5′P-gRNA which is consistent with its cleavage activity. Moreover, the structural modeling analysis demonstrates that like MpAgo, TsAgo has an ordered α5 at the C terminus of the PIWI domain which may hinder to binding of 5′ phosphate. Importantly, we find that TsAgo can target and cut plasmid DNA in vitro at 60 °C under the direction of RNA guides. These studies broaden our understanding of pAgos, and demonstrate that TsAgo can be regarded as an RNA-guided programmable nuclease for cleaving plasmids.

One-step synthesized biogenic nanoparticles using Linum usitatissimum: Application of sun-light photocatalytic, biological activity and electrochemical H2O2 sensor

This study aimed to synthesize Ag NPs as a green catalyst for photocatalytic activity and to examine their biological activities. It was determined that they have high activity in catalytic and biological activities. The green synthesis which is an environmentally friendly and inexpensive method was used to synthesize Ag-NPs using Linum usitatissimum as a reducing agent. Transmission electron microscopy (TEM), infrared to Fourier transform infrared (FTIR) spectroscopy, UV-Visible (UV–Vis) spectroscopy, and X-ray diffraction (XRD) were used to characterize the Ag NPs. In UV–Vis examination, Ag-NPs had intense peaks in the 435 nm region. The antibacterial activity of Ag NPs was investigated, and Ag NPs showed a high lethal effect against S. aureus, E. coli, B. subtilis, and MRSA. In addition, Ag NPs were tested for anticancer activity against the HT-29 colon cancer cell line, MDA-MB-231 breast cancer cell line, healthy cell line L929-Murine Fibroblast cell Lines, and MIA PaCa-2 human pancreatic cancer cell line at various concentrations (1–160 μg/mL) and showed a high anticancerogenic properties against MDA-MB-231 cells. Ag NPs showed the ability of DNA cleavage activity. Also, the antioxidant activity of Ag NPs against DPPH was found to be 80% approximately. Furthermore, the photocatalytic activity of Ag NPs against methylene blue (MB) was determined to be 67.13% at the 180th min. In addition, it was observed that biogenic Ag NPs have high electrocatalytic activity for hydrogen peroxide (H2O2) detection. In the sensor based on Ag NPs, linearity from 1 μM to 5 μM was observed with a detection limit (LOD) of 1.323 μM for H2O2. According to these results, we conclude that the biogenic Ag NPs synthesized using Linum usitatissimum extract can be developed as an efficient biological agent as an antibacterial and anticancer also can be used as a photocatalyst for industrial wastewater treatment to prevent wastewater pollution.

The role of filamentation in activation and DNA sequence specificity of the sequence-specific endonuclease SgrAI.

Filament formation by metabolic, biosynthetic, and other enzymes has recently come into focus as a mechanism to fine-tune enzyme activity in the cell. Filamentation is key to the function of SgrAI, a sequence-specific DNA endonuclease that has served as a model system to provide some of the deepest insights into the biophysical characteristics of filamentation and its functional consequences. Structure-function analyses reveal that, in the filamentous state, SgrAI stabilizes an activated enzyme conformation that leads to accelerated DNA cleavage activity and expanded DNA sequence specificity. The latter is thought to be mediated by sequence-specific DNA structure, protein-DNA interactions, and a disorder-to-order transition in the protein, which collectively affect the relative stabilities of the inactive, non-filamentous conformation and the active, filamentous conformation of SgrAI bound to DNA. Full global kinetic modeling of the DNA cleavage pathway reveals a slow, rate-limiting, second-order association rate constant for filament assembly, and simulations of in vivo activity predict that filamentation is superior to non-filamenting mechanisms in ensuring rapid activation and sequestration of SgrAI's DNA cleavage activity on phage DNA and away from the host chromosome. In vivo studies demonstrate the critical requirement for accelerated DNA cleavage by SgrAI in its biological role to safeguard the bacterial host. Collectively, these data have advanced our understanding of how filamentation can regulate enzyme structure and function, while the experimental strategies used for SgrAI can be applied to other enzymatic systems to identify novel functional roles for filamentation.

TOPOVIBL-REC114 interaction regulates meiotic DNA double-strand breaks

Meiosis requires the formation of programmed DNA double strand breaks (DSBs), essential for fertility and for generating genetic diversity. DSBs are induced by the catalytic activity of the TOPOVIL complex formed by SPO11 and TOPOVIBL. To ensure genomic integrity, DNA cleavage activity is tightly regulated, and several accessory factors (REC114, MEI4, IHO1, and MEI1) are needed for DSB formation in mice. How and when these proteins act is not understood. Here, we show that REC114 is a direct partner of TOPOVIBL, and identify their conserved interacting domains by structural analysis. We then analyse the role of this interaction by monitoring meiotic DSBs in female and male mice carrying point mutations in TOPOVIBL that decrease or disrupt its binding to REC114. In these mutants, DSB activity is strongly reduced genome-wide in oocytes, and only in sub-telomeric regions in spermatocytes. In addition, in mutant spermatocytes, DSB activity is delayed in autosomes. These results suggest that REC114 is a key member of the TOPOVIL catalytic complex, and that the REC114/TOPOVIBL interaction ensures the efficiency and timing of DSB activity.

Three novel Cas12a orthologs with robust DNA cleavage activity suitable for nucleic acid detection

Developing rapid and accurate pathogen detection methods is increasingly important, and CRISPR-Cas system can be optimized for this purpose. CRISPR-Cas12a is a single RNA-guided endonuclease system with the potential for nucleic acid detection. There is a broad diversity among Cas12a nucleases with robust detection capability. Herein, we characterised three Cas12a orthologs (ObCas12a, MbCas12a, and ScCas12a), including cis- and trans-cleavage activities, the identification of PAM, single-base resolution ability, and the application for nucleic acid detection. These Cas12a orthologs displayed robust cis- and trans-cleavage activities, and performed well in terms of specificity and sensitivity for nucleic acid detection. Furthermore, they have subtle differences in single-base resolution and recognised PAM sites in vitro. Therefore, these Cas12a nucleases are candidate proteins for CRISPR-based diagnostic methods. Addition of these enzymes to the nucleic acid detection toolbox will further expand the utility of this powerful technology.

Phosphorus-nitrogen compounds. Part 67. The reactions of Mono/bis (4-fluorobenzyl)spiro(N/N)cyclotriphosphazenes with sodium(3-amino-1-propanoxide): investigation of spectral properties, DNA-cleavage and antimicrobial activities

The starting reagents tetrachloro-mono- [1; R1=(CH2)3, R2=H] and tetrachloro-bis- [2; R1=(CH2)3, R2=FPhCH2- and 3; R1=(CH2)2, R2=FPhCH2-] (4-fluorobenzyl)monospiro(N/N)cyclotriphosphazenes, were obtained from the condensation reactions of hexachlorocyclotriphosphazatriene (N3P3Cl6; trimer; HCCP) with N-(4-fluorobenzyl)propane-1,3-diamine (L1), N,N'-bis(4-fluorobenzyl)propane-1,3-diamine (L2) and N,N'-bis(4-fluorobenzyl)ethane-1,2-diamine (L3), respectively. Substitution reactions of 1 and 2 with excess sodium(3-amino-1-propanoxide) separately yielded the only monospiro(N/O)amino-propanoxy-(4-fluorobenzyl)spiro(N/N) (1a) and monospiro-(N/O)aminopropanoxy-bis-(4-fluorobenzyl)spiro(N/N) (2a) cyclophosphazenes. Both monospiroaminopropanoxy (3a) and trans-bisspiroaminopropanoxy (3b) cyclotriphosphazenes were synthesized by the reactions of 3 with the excess sodium(3-amino-1-propanoxide). The structural features of all new products were identified using elemental analysis, FTIR, ESI-MS, 1H, 13C and 31P NMR data. DNA-cleavage activities of 1a-3a and 3b were tested with pBR322 plasmid DNA, and the four cyclotriphosphazenes caused changes in DNA intensity and mobility. It was found that compounds 2a and especially 3b were highly active against DNA. Furthermore, antimicrobial activities of the obtained cyclotriphosphazenes on some bacteria and yeast strains were evaluated and minimum inhibitory concentrations of the active compounds were calculated. According to these results, the most effective compound is 3b. It appears that this compound is even more active against yeasts than the standard antibiotic ketoconazole, even at very low concentrations (156 µM for C. albicans and 312.5 µM for C. tropicalis).

Computational, Experimental and Pharmacological Studies of Cu(II), Co(II) and Ni(II) Metal Complexes containing Azo Schiff base Ligand

The metal complexes containing Cu(II), Co(II) and Ni(II) metal ions were synthesised in the molar ratio of 1:2 [M:L] and azo Schiff base ligand as a primary ligand. The synthesized metal complexes were confirmed by the different analytical and spectroscopic techniques like UV-Visible, FT-IR, NMR, LC-MS and P-XRD techniques. The binding interaction of synthesized metal chelates with CT-DNA has been investigated by absorption spectra, viscosity measurements and thermal denaturation techniques. The mode of binding of the metal complexes with calf-thymus DNA has been explored. The metal complexes [Cu(II), Co(II) and Ni(II)] intercalated between the base pairs of the CT-DNA tightly with intrinsic binding constant (Kb) in Tris - HCl buffer containing 50 mMNaCl buffer. The DNA cleavage activities of the complexes were clearly assayed by using pUC-18 DNA which was monitored by the gel electrophoresis method. The antibacterial activity was carried out by a well-diffusion method against gram-positive bacteria S. aureus and gram-negative bacteria Shigella. Amylase inhibitory activity of pooled fractions was carried out according to the standard method with minor modification (antidiabetic activity). The in silico molecular docking was achieved with the DNA binding receptor PDB ID: 1E3Y taken from RCSB Protein Data Bank (www.rcsb.org/pdb). The receptor structre was prepared before use in docking study using protein preparation module of HEX 8.2 modelling package.

Copper(II) Complexes of a New Hydrazones with Aliphatic Groups: Synthesis, Characterisation and Nuclease Activity

Two new copper(II) complexes of bidentate arylhydrazone ligands have prepared and studied. Elemental analysis, SEM-EDS, thermal gravimetric analysis (TGA), powder X-ray diffraction (XRD), and a number of spectroscopic techniques were used to determine the structures of the compounds (FTIR, UV-Vis, 1H and 13C-NMR). Using agarose gel electrophoresis, the DNA cleavage activities of the produced copper complexes were investigated in the absence and presence of hydrogen peroxide. It was also investigated the influence of compound dose on the DNA cleavage process. The results show that in the presence of an oxidant agent, all of the complexes break pBR322 DNA. At the concentration of 10 µM, [Cu(L1)2] converted the supercoil DNA into two forms, while [Cu(L1)2] formed only the nicked form. The mechanistic analyses show that the active oxidative species for DNA breakage is a species generated by hydrogen peroxide and the copper(II) metal. The compounds also cleaveged pBR322 DNA in the absence of H2O2, marginally.

Synthesis and Characterization of a Polydioxanone-Coated Dipeptide-Functionalized Magnetic γ-Fe2O3 Nanoparticles-Modified PES Membrane and Its Biological Applications

This work is focused on the synthesis of leucyl-glycine-functionalized γ-Fe2O3 magnetic nanoparticles coated by polydioxanone (γ-Fe2O3-CA-Leu-Gly-PDX) as a polyethersulphone (PES) membrane for biotechnological applications. The physicochemical characteristics were investigated by FT-IR, SEM, XRD, a vibrating sample magnetometer (VSM), and ICP-OES. The present investigation also centered on the several biological activities of γ-Fe2O3-CA-Leu-Gly-PDX. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and metal chelating activity was studied for evaluation of its antioxidant activity potential. It exhibited 100% DPPH radical scavenging and 93.33% metal chelating activity. With applicability to antimicrobial photodynamic therapy, DNA cleavage and antimicrobial activity, the cell viability of γ-Fe2O3-CA-Leu-Gly-PDX was investigated in detail. The γ-Fe2O3-CA-Leu-Gly-PDX demonstrated the significant biofilm inhibition activity as being 81.54% and 86.34% for P. aeruginosa and S. aureus, respectively. Moreover, a novel polyethersulphone nanocomposite membrane incorporated with γ-Fe2O3-CA-Leu-Gly-PDX was prepared. The performance of the γ-Fe2O3-CA-Leu-Gly-PDX-blended polyethersulphone (PES) membrane was investigated by measuring the antifouling and E. coli rejection. The nanocomposite membranes demonstrated remarkable antifouling properties in contrast with the pristine PES when BSA (bovine serum albumin) and E. coli were filtrated. A complete rejection was obtained by the composite membrane. After an application of the membrane study, the modified polyethersulphone (PES) membrane blended with γ-Fe2O3-CA-Leu-Gly-PDX removed 100% of the E. coli.

Calcineurin dephosphorylates topoisomerase IIβ and regulates the formation of neuronal-activity-induced DNA breaks

Neuronal activity induces topoisomerase IIβ (Top2B) to generate DNA double-strand breaks (DSBs) within the promoters of neuronal early response genes (ERGs) and facilitate their transcription, and yet, the mechanisms that control Top2B-mediated DSB formation are unknown. Here, we report that stimulus-dependent calcium influx through NMDA receptors activates the phosphatase calcineurin to dephosphorylate Top2B at residues S1509 and S1511, which stimulates its DNA cleavage activity and induces it to form DSBs. Exposing mice to a fear conditioning paradigm also triggers Top2B dephosphorylation at S1509 and S1511 in the hippocampus, indicating that calcineurin also regulates Top2B-mediated DSB formation following physiological neuronal activity. Furthermore, calcineurin-Top2B interactions following neuronal activity and sites that incur activity-induced DSBs are preferentially localized at the nuclear periphery in neurons. Together, these results reveal how radial gene positioning and the compartmentalization of activity-dependent signaling govern the position and timing of activity-induced DSBs and regulate gene expression patterns in neurons.

A bacterial Argonaute with efficient DNA and RNA cleavage activity guided by small DNA and RNA

Argonaute proteins are widespread in prokaryotes and eukaryotes with diversified catalytic activities. Here,we describe an Argonaute from Marinitoga hydrogenitolerans (MhAgo) with all eight cleavage activities. Utilization of all four types of guides and efficient cleavage of single-stranded DNA (ssDNA) and RNA targets are revealed. The preference for the 5'-terminus nucleotides of 5'P guides, but no obvious preferences for that in 5'OH guides, is further uncovered. Moreover, the cleavage efficiency is heavily impaired by mismatches in the central and 3'-supplementary regions of guides, and the affinity between guides or guides/target duplex and MhAgo is proved as one of the factors affecting cleavage efficiency. Structural and mutational analyses imply some unknown distinctive structural features behind the cleavage activity of MhAgo. Meanwhile, 5'OH-guide RNA (gRNA)-mediated plasmid cleavage activity is unveiled. Conclusively, MhAgo is versatile, and its biochemical characteristics improve our understanding of pAgos and the pAgo-based techniques.

Synthesis, structural, spectral and molecular docking of Ni(II) and Pd(II) complexes with isatin moiety and their DNA cleavage activity

New complexes of Ni(II) and Pd(II) with Schiff base ligand derived from 5-nitro isatin with 4-phenyl-3-thiosemicarbazide have been synthesized in absolute ethanol. The isolated solid compounds have been characterized by elemental analyses and spectral (FT-IR, 1 H NMR and UV-Vis.) measurements. The Schiff base ligand has been further characterized by single-crystal XRD. It has been found that the Schiff base ligand behaves as a tridentate ligand forming chelates with 1:2 (metal:ligand) in the case of Ni-complex and 1:1 (metal:ligand) in the case of Pd-complex. Octahedral-geometry has been suggested for the Ni(II) chelate while square-planar geometry has been suggested for the Pd(II) chelate. The supramolecular structure of the thiosemicarbazide is controlled by large number of short intermolecular contacts such as O…H, H…H, C…C, S…C, S…H, S…N, H…C, N…O and C…O interactions where the most dominant contacts are H…H, H…C, C…C, O…H and S…H interactions. The optimized molecular geometry calculated at B3LYP/6-31G(d,p) level agreed very well with the reported X-ray structure. The two bands observed at 434 and 250 nm in the UV-Vis. spectrum of the compound in DMSO as solvent have been calculated using TD-DFT method at 430.9 nm (f = 0.1178) and 257.2 nm (f = 0.387), respectively. These bands have been assigned to HOMO→LUMO (96%) and HOMO→LUMO+2 (90%) excitations which belong to n-π* and π-π* transitions, respectively. All the synthesized compounds have been tested against supercoiled pBR322 DNA. These compounds have been found to promote the cleavage of plasmid DNA in the presence of H 2 O 2 . The molecular docking of the ligand and its metal complexes refer to groove binding toward the DNA with high docking score of Ni(II) complex rather than the corresponding ligand and Pd(II) complex.

Allosteric activation of CRISPR-Cas12a requires the concerted movement of the bridge helix and helix 1 of the RuvC II domain.

Nucleases derived from the prokaryotic defense system CRISPR-Cas are frequently re-purposed for gene editing and molecular diagnostics. Hence, an in-depth understanding of the molecular mechanisms of these enzymes is of crucial importance. We focused on Cas12a from Francisella novicida (FnCas12a) and investigated the functional role of helix 1, a structural element that together with the bridge helix (BH) connects the recognition and the nuclease lobes of FnCas12a. Helix 1 is structurally connected to the lid domain that opens upon DNA target loading thereby activating the active site of FnCas12a. We probed the structural states of FnCas12a variants altered in helix 1 and/or the bridge helix using single-molecule FRET measurements and assayed the pre-crRNA processing, cis- and trans-DNA cleavage activity. We show that helix 1 and not the bridge helix is the predominant structural element that confers conformational stability of FnCas12a. Even small perturbations in helix 1 lead to a decrease in DNA cleavage activity while the structural integrity is not affected. Our data, therefore, implicate that the concerted remodeling of helix 1 and the bridge helix upon DNA binding is structurally linked to the opening of the lid and therefore involved in the allosteric activation of the active site.

Melinjo (Gnetum Gnemon) Seed Protein Activity Against pBSKS DNA Cleavage and Its Cytotoxicity in T47D and 4T1 Cells

The plant protein of melinjo (Gnetum gnemon) in seeds is known to have antioxidant properties. Apart from being an antioxidant, melinjo seed protein also has the potential to be developed as an anticancer compound. One of the potential anticancer compounds has been screened based on supercoiled DNA cleavage activity (pBSKS). The purpose of this study was to determine the activity of the protein fraction isolated with DEAE and BUTYL matrices against supercoiled DNA cleavage of pBSKS. Supercoiled DNA cleavage test activity of pBSKS was determined using the electrophoresis method and visualized with a UV transilluminator at a wavelength of 312 nm. The cytotoxic activity of melinjo seeds was tested through the MTT assay method and was read on an ELISA reader with a wavelength of 550 and 595 nm seen from the IC50 value. The protein fraction of melinjo seeds isolated with DEAE and BUTYL matrices had concentrations of 470.1 and 81.02 g/mL, respectively. The visualization results showed that the DEAE and BUTYL protein fractions resulted in depletion of supercoiled DNA bands of pBSKS. The concentration of active protein in the melinjo seeds fractionated using the DEAE-650M matrix was higher than that of the BUTYL-650M matrix. In this study, after several cytotoxic tests were carried out, various results were obtained. The activity of protein isolates from melinjo seeds fractionated using DEAE-650M and BUTYL-650M against 4T1 and T47D cells could be categorized as non-toxic because the IC50 value was > 1000 g/mL and the average percentage of viable cells was more than 50%, except for the seed protein fraction. Melinjo fractionated using DEAE-650M against T47D cells which had an IC50 value of 127.62 g/mL could be categorized as quite active and cytotoxic.
 Keywords: Gnetum gnemon L., protein isolation, ribosome-inactivating proteins (RIPs), DNA cleavage, cytotoxic, T47D, 4T1

Biological Activity and Optical Sensor Properties of Green Synthesis Polymer

AbstractIn this study, 4‐amino‐3‐hydroxy‐1‐naphthalene sulfonic acid (Monomer) was converted to its enzyme‐catalyzed oxidative polymerization (Poly‐enz) in dioxane solvent by horseradish peroxidase enzyme (HRP) in the presence of hydrogen peroxide as oxidant. For the analysis of the monomer and its synthesized Poly‐enz, 1H‐NMR, 13C‐NMR, FT‐IR, TGA, and SEM measurements were determined. In addition, the optical ion sensor properties of Poly‐enz against metals such as Ag+, Cd2+, Co2+, Hg2+, Ni2+, Pb2+, Zn2+, Cu2+ were examined with UV‐Vis spectrophotometer. It was observed that Cu metal showed sensory properties. DNA cleavage and DNA binding activities of monomer and Poly‐enz were done using by agarose gel electrophoresis and UV‐Vis methods, respectively. It was seen that the Poly‐enz interacted with DNA electrostatically. This enzymatic polymer was found to cleave pBR322 plasmid DNA both hydrolytically and oxidatively as well. The antioxidant activities of the compounds were examined by three different methods (DPPH, ABTS, FRAP) and it was found that the Poly‐enz showed high antioxidant activity compared to its monomer. Antimicrobial activities were studied against various bacterial and yeast cultures by the minimum inhibition concentration (MIC) method. It was observed that the antimicrobial activities of the enzymatic polymer in different microorganisms increased depending on the concentration.

CHEF induced fluorometric sensing of Al3+ and picric acid with bioimaging in human peripheral blood mononuclear cell

Herein we report a quinoline modified oxacalixarene architecture (HQOC) that shows chelation enhanced turn-on fluorescence response toward Al3+. The presence of hard carbonyl oxygen and quinoline nitrogen plays an important role in switching-on the system through coordinating with hard Al3+ ions. The photophysical of the probe HQOC toward aluminium were thoroughly investigated using various techniques including UV–vis spectroscopy, fluorescence and NMR spectroscopy. The sequential detection of Al3+ and picric acid is immensely significant in evaluating its presence in the human ecosystem. HQOC allowed the specific recognition and quantification of Al3+ and picric acid with the detection limits of 24.60 nM and 34.28 nM. The Density Functional Theory (DFT) calculations further provide insight into the mechanistic aspects and electronic transitions involved during sensing. Additionally, we extended the work toward several bio-analytical applications. The cytotoxicity of the synthesized complex was screened against Peripheral Blood Mononuclear Cells (PBMC) by MTT assay and subsequently cell imaging, DNA cleavage activity was also explored for amenable therapeutic needs.