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ABSTRACT In this study, glycerol, glycosyl, and tetraethyleneglycol monomethyl ether group substituted BODIPY compounds were successfully synthesized and characterized. The antioxidant, antidiabetic, antibiofilm, microbial cell viability, antimicrobial, antimicrobial photodynamic therapy, and DNA cleavage abilities of the BODIPY derivates were also tested. The highest antioxidant activity at 100 mg/L was 87.12% for BODIPY‐1, 85.61% for BODIPY‐2, and 92.78% for BODIPY‐3. The most effective inhibition of microbial cell viability was obtained for these BODIPY molecules at 100% at 50 and 100 mg/L. It was also observed that these BODIPY molecules exhibited excellent biofilm inhibition activities. The derivates were found to exhibit higher antibiofilm abilities against Staphylococcus aureus compared to Pseudomonas aeruginosa. When the antidiabetic effects of water‐soluble BODIPY compounds were examined, the highest effect was found to be 64.40% for BODIPY‐1 at 200 mg/L. [ABSTRACT FROM AUTHOR]

ABSTRACT Metal (Co3+, Ni2+, and Pt2+)–centered and noncentered ferrocene–Schiff bases were prepared. They have been structurally characterized by spectroscopic methods, elemental analysis, conductivity, and magnetic moment. Electron spin resonance was used for the characterization of the Co3+‐centered complex. All synthesized compounds showed antibacterial activity against all tested Gram‐positive and Gram‐negative strains, as well as potent antifungal effect against Candida species. Remarkably, these compounds demonstrated greater efficacy against Gram‐positive bacteria than Gram‐negative. Without an additional oxidant or reductant, the compounds exhibited DNA cleavage capability in the dark. The development of synthesized novel chemical compounds having DNA cleavage and antimicrobial activity plays a profound role in the advancement of medical therapeutics. [ABSTRACT FROM AUTHOR]

Three new Zn(II) Schiff base complexes, [ZnLCl2] (1), [ZnLBr2] (2) and [ZnL(SCN)2] (3), have been synthesized by adding Zn(II) salts of chloride and bromide to a compartmental ligand with NNO donor prepared through the simple condensation of pyridine‐2‐carboxaldehyde and a substituted aniline. The complexes have been identified using standard physicochemical methods, and their solid‐state structures have been determined through single‐crystal X‐ray analysis. The phosphatase‐like activity of all three complexes has been investigated using 4‐NPP as the model substrate in a DMSO/water medium. Complex 1 exhibits the highest level of phosphatase activity. The reactivity trend shows that complex 1 > complex 2 > complex 3. A plausible mechanism and the causes underlying the activity trend have been explored through DFT study, ESI‐mass spectra and 31P experiments. Complex 1 shows promising potential for use in gene‐targeted therapeutics due to its remarkable performance in DNA digestion. The excellent DNA cleavage result of complex 1 has led to the investigation of its molecular docking interaction with the liver cancer 2 receptor to study its anti‐liver cancer activity. Furthermore, in vitro anticancer activity of complex 1 also shows excellent antiproliferative activity against hepatocellular carcinoma. [ABSTRACT FROM AUTHOR]

One of the challenges for experimental structural biology in the 21st century is to see chemical reactions happen. Staphylococcus aureus (S. aureus) DNA gyrase is a type IIA topoisomerase that can create temporary double-stranded DNA breaks to regulate DNA topology. Drugs, such as gepotidacin, zoliflodacin and the quinolone moxifloxacin, can stabilize these normally transient DNA strand breaks and kill bacteria. Crystal structures of uncleaved DNA with a gepotidacin precursor (2.1 Å GSK2999423) or with doubly cleaved DNA and zoliflodacin (or with its progenitor QPT-1) have been solved in the same P61 space-group (a = b ≈ 93 Å, c ≈ 412 Å). This suggests that it may be possible to observe the two DNA cleavage steps (and two DNA-religation steps) in this P61 space-group. Here, a 2.58 Å anomalous manganese dataset in this crystal form is solved, and four previous crystal structures (1.98 Å, 2.1 Å, 2.5 Å and 2.65 Å) in this crystal form are re-refined to clarify crystal contacts. The structures clearly suggest a single moving metal mechanism—presented in an accompanying (second) paper. A previously published 2.98 Å structure of a yeast topoisomerase II, which has static disorder around a crystallographic twofold axis, was published as containing two metals at one active site. Re-refined coordinates of this 2.98 Å yeast structure are consistent with other type IIA topoisomerase structures in only having one metal ion at each of the two different active sites. [ABSTRACT FROM AUTHOR]

Chromium aluminum carbide (Cr2AlC) MAX phase and Cr2CTx (MXene-Cr) were synthesized by the pressureless sintering method and hydrothermal method, respectively. In addition to this, the free radical scavenging activities (FRSA) of MAX-Cr phase and MXene-Cr compounds were tested and compared with ascorbic acid and trolox as standard compounds. The obtained FRSA results of MAX-Cr phase and MXene-Cr were 42.82 and 59.64%, respectively, at 100 mg/L concentration. MXene-Cr showed a 66.90% inhibitory effect on α-amylase at 200 mg/L. The DNA nuclease activity of compounds was determined to be extremely satisfactory at 50, 100, and 200 mg/L concentrations. Moreover, the prepared MAX-Cr phase and MXene-Cr were investigated for antimicrobial activity against six bacterial and two fungal strains by the broth microdilution method. Compounds provided more significant inhibition against Gram-positive bacteria than Gram-negative bacteria and fungi. MAX-Cr phase and MXene-Cr almost completely inhibited microbial cell viability at a 25 mg/L concentration. Additionally, MXene-Cr showed 89.86% and 87.01% antibiofilm activity against S. aureus and P. aeruginosa, respectively, while the antibiofilm activity of the MAX-Cr phase was over 90%. [ABSTRACT FROM AUTHOR]

Abstract Mesophilic Argonautes (Agos) from microbial resources have received significant attention due to their potential applications in genome editing and molecular diagnostics. This study characterizes a novel Ago from Pseudobutyrivibrio ruminis (PrAgo), which can cleave single-stranded DNA using guide DNA (gDNA). PrAgo, functioning as a multi-turnover enzyme, effectively cleaves DNA using 5′-phosphate gDNA, 14–30 nucleotides in length, in the presence of both Mn2+ and Mg2+ ions. PrAgo demonstrates DNA cleavage activity over a broad pH range (pH 4–12), with optimal activity at pH 11. As a mesophilic enzyme, PrAgo cleaves efficiently DNA at temperatures ranging from 25 to 65 °C, particularly at 65 °C. PrAgo does not show strong preferences for the 5′-nucleotide in gDNA. It shows high tolerance for single-base mismatches, except at positions 13 and 15 of gDNA. Continuous double-nucleotide mismatches at positions 10–16 of gDNA significantly reduce cleavage activity. Furthermore, PrAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at 65 °C. Additionally, molecular dynamic simulations suggest that interactions between the PAZ domain and different nucleic acids strongly influence cleavage efficiency. These findings expand our understanding of Protokaryotic Agos and their potential applications in biotechnology. Graphical Abstract

Vincetoxicum members offer important qualities in both conventional and modern medicine. In the present study, V. scandens from the northeast of Turkey (Trabzon city) was investigated for its phenolic compounds, antioxidant activities, DNA cleavage and binding effects. Phenolic compounds of the V. scandens methanol extract were analysed by high performance liquid chromatography (HPLC-DAD), while their total phenolic and total flavonoid contents were determined spectrophotometrically. The antioxidant potential of the extract was characterised using DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2 ́-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)), and FRAP (ferric reducing antioxidant power assay) tests. In addition, the DNA cleavage and binding features of the V. scandens extract were studied using pBR322 DNA and CTDNA, respectively. The results indicated that the V. scandens methanol extract contained a large amount of catechin, as well as a high total phenolic and total flavonoid content. On the other hand, the DPPH and ABTS antioxidant tests revealed that V. scandens did not exhibit high antioxidant activity. The methanol extract of the studied plant was also rich in catechin, p-coumaric acid and vanillic acid. While the V. scandens methanol extract exhibited weak antioxidant and DNA cleavage activity, it showed DNA binding activity at 50 μM, thus demonstrating the potential to be an intercalation agent in this concentration. This study provides the first report on the total phenolic content, total flavonoid content, DNA cleavage and DNA binding activity as well as antioxidant activity of V. scandens.

In an attempt to create models of phosphodiesterases, we previously investigated bis(guanidinium) naphthols. Such metal‐free anion receptors cleaved aryl phosphates and also plasmid DNA. Observed reaction rates, however, could not compete with those of highly reactive metal complexes. In the present study, we have replaced the guanidines by ethylene diamine side chains which accelerates the plasmid cleavage by compound 13 significantly (1 mM 13: t1/2=22 h). Further gains in reactivity are achieved by azo coupling of the naphthol unit. The electron accepting azo group decreases the pKa of the hydroxy group. It can also serve as a dye label and a handle for attaching DNA binding moieties. The resulting azo naphthol 17 not only nicks (1 mM 17: t1/2~1 h) but also linearizes pUC19 DNA. Although the high reactivity of 17 seems to result in part from aggregation, in the presence of EDTA azo naphthol 17 obeys first order kinetics (1 mM 17: t1/2=4.8 h), reacts four times faster than naphthol 13 and surpasses by far the former bis(guanidinium) naphthols 4 and 5. [ABSTRACT FROM AUTHOR]

Natural enediyne antibiotics undergo thermally induced cycloaromatization under physiological conditions to generate highly reactive diradicals that effectively abstract hydrogen atoms from biomacromolecules like DNA, leading to DNA cleavage and inducing programmed cell death in tumor cells, exhibiting potent cytotoxicity and broad‐spectrum antitumor properties. In order to further explore promising synthetic analogues of natural enediynes, a 3,6‐pyridazinedione moiety was fused at the ene‐position, and 10 new enediyne compounds with different atoms at the propargylic positions were synthesized. Differential scanning calorimetry (DSC) and electron paramagnetic resonance results indicated that these enediynes exhibited a rather low onset temperature and the capability to generate radical species at physiological temperatures. Density functional theory (DFT) calculations demonstrated that the pyridazinedione moiety facilitates cascade rearrangement processes, thereby endowing the key cycloaromatization with a low energy barrier. The enediynes with propargylic oxygen exhibit strong DNA cleavage capabilities, and all pyridazinedione‐fused enediynes showed IC50 values in the range of several tens of micromolars against HeLa cells. [ABSTRACT FROM AUTHOR]

Mesophilic Argonautes (Agos) from microbial resources have received significant attention due to their potential applications in genome editing and molecular diagnostics. This study characterizes a novel Ago from Pseudobutyrivibrio ruminis (PrAgo), which can cleave single-stranded DNA using guide DNA (gDNA). PrAgo, functioning as a multi-turnover enzyme, effectively cleaves DNA using 5′-phosphate gDNA, 14–30 nucleotides in length, in the presence of both Mn2+ and Mg2+ ions. PrAgo demonstrates DNA cleavage activity over a broad pH range (pH 4–12), with optimal activity at pH 11. As a mesophilic enzyme, PrAgo cleaves efficiently DNA at temperatures ranging from 25 to 65 °C, particularly at 65 °C. PrAgo does not show strong preferences for the 5′-nucleotide in gDNA. It shows high tolerance for single-base mismatches, except at positions 13 and 15 of gDNA. Continuous double-nucleotide mismatches at positions 10–16 of gDNA significantly reduce cleavage activity. Furthermore, PrAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at 65 °C. Additionally, molecular dynamic simulations suggest that interactions between the PAZ domain and different nucleic acids strongly influence cleavage efficiency. These findings expand our understanding of Protokaryotic Agos and their potential applications in biotechnology. [ABSTRACT FROM AUTHOR]

Transformation, the uptake of DNA directly from the environment, is a major driver of gene flow in microbial populations. In bacteria, DNA uptake requires a nuclease that processes dsDNA to ssDNA, which is subsequently transferred into the cell and incorporated into the genome. However, the process of DNA uptake in archaea is still unknown. Previously, we cataloged genes essential to natural transformation in Methanococcus maripaludis, but few homologs of bacterial transformation‐associated genes were identified. Here, we characterize one gene, MMJJ_16440 (named here as ecnA), to be an extracellular nuclease. We show that EcnA is Ca2+‐activated, present on the cell surface, and essential for transformation. While EcnA can degrade several forms of DNA, the highest activity was observed with ssDNA as a substrate. Activity was also observed with circular dsDNA, suggesting that EcnA is an endonuclease. This is the first biochemical characterization of a transformation‐associated protein in a member of the archaeal domain and suggests that both archaeal and bacterial transformation initiate in an analogous fashion. [ABSTRACT FROM AUTHOR]

Herein, we report a new strategy for the design of antibiotic agents based on the electrostatic interaction and hydrogen bonding, highlighting the significance of hydrogen bonding and the increased recognition sites in facilitating the interaction with bacterial cell membranes and DNA. A series of quaternary ammonium functionalized urea-based anion receptors were studied. While the monodentate mono-urea M1, bisurea M2, and trisurea M3 failed to break through the cell membrane barrier and thus could not kill bacteria, the extended bidentate dimers D1–D3 presented gradually increased membrane penetrating capabilities, DNA conformation perturbation abilities, and broad-spectrum antibacterial activities against E. coli, P. aeruginosa, S. aureus, E. faecalis, and S. epidermidis. [ABSTRACT FROM AUTHOR]

CRISPR-interference (CRISPRi), a highly effective method for silencing genes in mammalian cells, employs an enzymatically dead form of Cas9 (dCas9) complexed with one or more guide RNAs (gRNAs) with 20 nucleotides (nt) of complementarity to transcription initiation sites of target genes. Such gRNA/dCas9 complexes bind to DNA, impeding transcription of the targeted locus. Here, we present an alternative gene-suppression strategy using active Cas9 complexed with truncated gRNAs (tgRNAs). Cas9/tgRNA complexes bind to specific target sites without triggering DNA cleavage. When targeted near transcriptional start sites, these short 14-15 nts tgRNAs efficiently repress expression of several target genes throughout somatic tissues in Drosophila melanogaster without generating any detectable target site mutations. tgRNAs also can activate target gene expression when complexed with a Cas9-VPR fusion protein or modulate enhancer activity, and can be incorporated into a gene-drive, wherein a traditional gRNA sustains drive while a tgRNA inhibits target gene expression.

In the present work, a novel coordination polymer {[Cu(phen)(Tmdipy)(NO3)H2O](NO3) 2.5(H2O)}n (phen = 1,10-phenanthroline, Tmdipy = 4,4′- Trimethylenedipyridine), has been prepared and structurally determined by elemental analysis, IR, EPR, UV–visible and single-crystal X-ray crystallography. The complex crystallized in triclinic space group P -1, and the copper(II) shows a distorted octahedral geometry, with Jahn-Teller distortion occurring in the (4 + 2) CuN4O2 coordination sphere. The crystal structure evidences that the CP1 is formed of dinuclear [Cu(phen)(Tmdipy)(NO3)(H2O)]2 units weakly connected by nitrate anions giving rise to 1D polymeric chain structure. EPR measurement confirms about the distorted octahedral geometry of the complex. By using UV–Vis absorption, fluorescence spectroscopy, and cyclic voltammetric techniques, the binding studies of copper(II) complex with calf thymus DNA (CT-DNA) were investigated. Additionally, the gel electrophoresis method was used to examine the cleavage of pBR322 DNA by copper(II) complex and exhibited potent cytotoxic effects against human cell line (HepG2). Finally, magnetic properties, molecular docking studies and photoluminescence characteristics have been evaluated.

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Comprehensive Summary: Currently, CRISPR/Cas9 technology has found widespread applications across various domains. However, the utility of CRISPR/Cas9 is encumbered by issues pertaining to its reliability and safety, primarily stemming from the uncontrolled activity of the system. Therefore, the design and development of CRISPR/Cas9 systems with controllable activity is of paramount importance. Biotin, characterized by its small molecular weight, and streptavidin, distinguished by its substantial spatial steric hindrance, can be harnessed as an ideal OFF switch (termed a "bioactivity brake") due to their interaction characteristics. In this work, we present a strategy that employs the streptavidin‐biotin interaction as a "brake system" for CRISPR/Cas9, effectively allowing for the shutdown of the enzymatic activity of CRISPR/Cas9. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: Currently, CRISPR/Cas9 technology has found widespread applications across various domains. However, the utility of CRISPR/Cas9 is encumbered by issues pertaining to its reliability and safety, primarily stemming from the uncontrolled activity of the system. Therefore, the design and development of CRISPR/Cas9 systems with controllable activity is of paramount importance. Biotin, characterized by its small molecular weight, and streptavidin, distinguished by its substantial spatial steric hindrance, can be harnessed as an ideal OFF switch (termed a "bioactivity brake") due to their interaction characteristics. In this work, we present a strategy that employs the streptavidin‐biotin interaction as a "brake system" for CRISPR/Cas9, effectively allowing for the shutdown of the enzymatic activity of CRISPR/Cas9. [ABSTRACT FROM AUTHOR]

The present study focused on the antioxidant, cytotoxic, antimicrobial, and DNA-protective effects of Usnea lapponica, Usnea intermedia, and Usnea florida, which are distributed in Turkey. The methanol extracts of these three lichen species were subjected to high-performance liquid chromatography (HPLC) analysis to quantify the amount of usnic acid and the highest amount was found in U. lapponica (3345.9±92.18 mg/gextract). Furthermore, the total phenolic and flavonoid contents were analyzed. Antioxidant capacity was evaluated by different chemical assays, together with antimicrobial, cytotoxic, and DNA-protective properties. The results of the antioxidant assays showed that U. lapponica exhibited the highest DPPH radical scavenging activity, whereas the extract that scavenged ABTS radicals more potently was identified as U. intermedia. The disc diffusion method was used to investigate the antimicrobial activity, and the strongest effect was observed in U. intermedia methanol extract with a zone value of 13 mm on Micrococcus luteus. In cytotoxicity experiments, U. florida extract (250 µg/mL; 36.03% viability) exhibited a high antiproliferative effect on A549 cells and U. intermedia (62.5 µg/mL; 23.65% viability) on MDA-MB-231 cells. DNA-protective effects were investigated using pBR322 plasmid DNA, and all the studied species were found to have DNA-protective effects. These results suggest that Usnea species may be potential candidates for the development of new phytopharmaceuticals and functional ingredients. [ABSTRACT FROM AUTHOR]

Metallic nanoparticles have attracted great attention in catalytic, medical diagnosis, and treatment research in recent years. The formation of palladium nanoparticles using rosemary (Rosmarinus officinalis L.) extract was carried out using the green synthesis method. The plant was extracted using 70% ethanol by microwave techniques. The novelty of this study is the investigation of the biological activities of green synthesis of Pd nanoparticles, such as DNA cleavage activity, antimicrobial activity, DPPH scavenging activity, and its electro-catalytic performance in alcohol oxidation. Additionally, photocatalytic activities were also evaluated. The characterization of synthesized palladium nanoparticles (Pd NPs) was performed by UV-spectrometry, XRD, FTIR, and TEM. According to TEM results, Pd nanoparticles were observed to have a spherical shape and an average particle size of 4.91 nm. The Pd NPs showed the photodegradation of MB solution by up to 79.9% at 120 min. The newly synthesized plant-mediated green synthesized Pd NPs showed the max and the min antimicrobial activity at 16 µg/mL and 256 µg/mL against L. pneumophila and C. albicans, respectively. The current density ratio of 48.22 mA/cm2 obtained in the study indicates that the obtained materials may be of interest in different applications. According to the results obtained, a direct relationship of extract use is observed in the synthesis of Pd nanoparticles and is a good way to reduce and stabilize metal salts. It has been determined that green Pd NPs have potential for use in energy production from alcohol oxidation and in medical applications. [ABSTRACT FROM AUTHOR]

Non‐peripherally, glycerol terminal groups substituted copper (II) phthalocyanine were non‐covalently (electrostatic and/or π–π interaction) attached to carbon (CQD) and carbon‐boron quantum dots (CBQD) to form QDs‐Pc nanoconjugate systems. Synthesized novel phthalocyanine compounds and QDs‐Pc conjugate systems were characterized using different spectroscopic techniques. Various biological assessments were applied to newly synthesized compounds. Conjugates 4 and 5 had a maximal free radical scavenging activity of 71.3% and 68.1% at a 100 mg/L concentration. Compounds exhibited high antidiabetic activities at 200 mg/L. Also, compounds showed significant DNA nuclease activity at all tested concentrations. The most efficient MIC value was obtained as 4 mg/L against Enterococcus hirae and Enterococcus feacalis. This MIC value was further decreased after photodynamic therapy, and it was observed that the antimicrobial effects of the compounds increased. Inhibition of microbial cell viability was obtained as 100% for all compounds. In addition, compounds exerted perfect biofilm inhibitory effects. [ABSTRACT FROM AUTHOR]

Allosteric signaling within multidomain proteins is a driver of communication between spatially distant functional sites. Understanding the mechanism of allosteric coupling in large multidomain proteins is the most promising route to achieving spatial and temporal control of the system. The recent explosion of CRISPR-Cas9 applications in molecular biology and medicine has created a need to understand how the atomic level protein dynamics of Cas9, which are the driving force of its allosteric crosstalk, influence its biophysical characteristics. In this study, we used a synergistic approach of nuclear magnetic resonance (NMR) and computation to pinpoint an allosteric hotspot in the HNH domain of the thermostable GeoCas9. We show that mutation of K597 to alanine disrupts a salt-bridge network, which in turn alters the structure, the timescale of allosteric motions, and the thermostability of the GeoHNH domain. This homologous lysine-to-alanine mutation in the extensively studied mesophilic S. pyogenes Cas9 similarly alters the dynamics of the SpHNH domain. We have previously demonstrated that the alteration of allostery via mutations is a source for the specificity enhancement of SpCas9 (eSpCas9). Hence, this may also be true in GeoCas9.

The RNA programmed non-specific (trans) nuclease activity of CRISPR-Cas Type V and VI systems has opened a new era in the field of nucleic acid-based detection. Here, we report on the enhancement of trans-cleavage activity of Cas12a enzymes using hairpin DNA sequences as FRET-based reporters. We discover faster rate of trans-cleavage activity of Cas12a due to its improved affinity (Km) for hairpin DNA structures, and provide mechanistic insights of our findings through Molecular Dynamics simulations. Using hairpin DNA probes we significantly enhance FRET-based signal transduction compared to the widely used linear single stranded DNA reporters. Our signal transduction enables faster detection of clinically relevant double stranded DNA targets with improved sensitivity and specificity either in the presence or in the absence of an upstream pre-amplification step.

The clustered regularly interspaced short palindromic repeat (CRISPR) genome-editing revolution established the beginning of a new era in life sciences. Here, we review the role of state-of-the-art computations in the CRISPR-Cas9 revolution, from the early refinement of cryo-EM data to enhanced simulations of large-scale conformational transitions. Molecular simulations reported a mechanism for RNA binding and the formation of a catalytically competent Cas9 enzyme, in agreement with subsequent structural studies. Inspired by single-molecule experiments, molecular dynamics offered a rationale for the onset of off-target effects, while graph theory unveiled the allosteric regulation. Finally, the use of a mixed quantum-classical approach established the catalytic mechanism of DNA cleavage. Overall, molecular simulations have been instrumental in understanding the dynamics and mechanism of CRISPR-Cas9, contributing to understanding function, catalysis, allostery, and specificity.

The recently identified novel Holliday junction-resolving enzyme, termed Hjc_15-6, activity investigation results imply DNA cleavage by Hjc_15-6 in a manner that potentially enhances the molecular self-assembly that may be exploited for creating DNA-networks and nanostructures. The study also demonstrates Pwo DNA polymerase acting in combination with Hjc_15-6 capability to produce large amounts of DNA that transforms into large DNA-network structures even without DNA template and primers. Furthermore, it is demonstrated that Hjc_15-6 prefers Holliday junction oligonucleotides as compared to Y-shaped oligonucleotides as well as efficiently cleaves typical branched products from isothermal DNA amplification of both linear and circular DNA templates amplified by phi29-like DNA polymerase. The assembly of large DNA network structures was observed in real time, by transmission electron microscopy, on negative stained grids that were freshly prepared, and also on the same grids after incubation for 4 days under constant cooling. Hence, Hjc_15-6 is a promising molecular tool for efficient production of various DNA origamis that may be implemented for a wide range of applications such as within medical biomaterials, catalytic materials, molecular devices and biosensors. [ABSTRACT FROM AUTHOR]

In the present study, phenolic compounds, antioxidant activities, DNA cleavage and binding effects of Vincetoxicum tmoleum (Vt) collected from west of Anatolia (Manisa city) were investigated. Antioxidant potentials of the extracts were characterized with their total phenolic and flavonoid contents, DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), and FRAP (ferric reducing antioxidant power assay) tests. Besides, the DNA cleavage and binding features of V. tmoleum extract were studied using pBR322 DNA and CT-DNA, respectively. Phenolic compounds of the extract were analyzed by high performance liquid chromatography (HPLC-DAD). As a result, V. tmoleum methanol extract was found to have the high total phenolic and total flavonoid content and antioxidant effect. Strong positive correlations were also found between DPPH and TFC (r = 0.995; p < 0.01), and TPC (r = 0.989;p < 0.01), ABTS (r = 0.994;p < 0.01) and FRAP(r=0,995; p < 0.01). Methanol extract of V. tmoleum had large amounts of p-coumaric acid, ferulic acid and protocatechuic acid. While V. tmoleum samples showed weak DNA cleavage activity, they showed DNA binding activity at 50 µM concentration, that is, the potential to be an intercalation agent in this concentration. [ABSTRACT FROM AUTHOR]

Search for new antimicrobial agents is of great significance due to the issue of antimicrobial resistance, which nowadays has become more important than many diseases. The aim of this study was to evaluate the toxicity and biological effects of a dextran-graft-polyacrylamide (D-PAA) polymer-nanocarrier with/without silver or gold nanoparticles (AgNPs/D-PAA and AuNPs/D-PAA, respectively) to analyze their potential to replace or supplement conventional antibiotic therapy. The toxicity of nanocomplexes against eukaryotic cells was assessed on primary dermal fibroblasts using scratch, micronucleus and proliferation assays. DPPH (2,2-diphenyl-1-picrylhydrazylradical) assay was used to evaluate the antioxidant capacity of D-PAA, AgNPs/D-PAA and AuNPs/D-PAA. DNA cleavage, antimicrobial and biofilm inhibition effects of nanocomplexes were investigated. Nanocomplexes were found to be of moderate toxicity against fibroblasts with no genotoxicity observed. AgNPs/D-PAA reduced motility and proliferation at lower concentrations compared with the other studied nanomaterials. AgNPs/D-PAA and AuNPs/D-PAA showed radical scavenging capacities in a dose-dependent manner. The antimicrobial activity of AgNPs/D-PAA against various bacteria was found to be much higher compared to D-PAA and AuNPs/D-PAA, especially against E. hirae, E. faecalis and S. aureus, respectively. D-PAA, AgNPs/D-PAA and AuNPs/D-PAA showed DNA-cleaving and biofilm inhibitory activity, while AgNPs/D-PAA displayed the highest anti-biofilm activity. AgNPs/D-PAA and AuNPs/D-PAA were characterized by good antimicrobial activity. According to the findings of the study, AgNPs/D-PAA and AuNPs/D-PAA can be evaluated as alternatives for the preparation of new antimicrobial agents, the fight against biofilms, sterilization and disinfection processes. Our findings confirm the versatility of nanosystems based on dextran–polyacrylamide polymers and indicate that AgNPs/D-PAA and AuNPs/D-PAA can be evaluated as alternatives for the preparation of novel antimicrobial agents. [ABSTRACT FROM AUTHOR]

Iodine-induced cleavage at phosphorothioate DNA (PT-DNA) is characterized by extremely high sensitivity (∼1 phosphorothioate link per 106 nucleotides), which has been used for detecting and sequencing PT-DNA in bacteria. Despite its foreseeable potential for wide applications, the cleavage mechanism at the PT-modified site has not been well established, and it remains unknown as to whether or not cleavage of the bridging P-O occurs at every PT-modified site. In this work, we conducted accurate ωB97X-D calculations and high-performance liquid chromatography-mass spectrometry to investigate the process of PT-DNA cleavage at the atomic and molecular levels. We have found that iodine chemoselectively binds to the sulfur atom of the phosphorothioate link via a strong halogen-chalcogen interaction (a type of halogen bond, with binding affinity as high as 14.9 kcal/mol) and thus triggers P-O bond cleavage via phosphotriester-like hydrolysis. Additionally, aside from cleavage of the bridging P-O bond, the downstream hydrolyses lead to unwanted P-S/P-O conversions and a loss of the phosphorothioate handle. The mechanism we outline helps to explain specific selectivity at the PT-modified site but also predicts the dynamic stoichiometry of P-S and P-O bond breaking. For instance, Tris is involved in the cascade derivation of S-iodo-phosphorothioate to S-amino-phosphorothioate, suppressing the S-iodo-phosphorothioate hydrolysis to a phosphate diester. However, hydrolysis of one-third of the Tris-O-grafting phosphotriester results in unwanted P-S/P-O conversions. Our study suggests that bacterial DNA phosphorothioation may more frequently occur than previous bioinformatic estimations have predicted from iodine-induced deep sequencing data.

A compact protein with a size of

A series of mononuclear Cu(II) complexes were synthesized using a potential tetradentate hydrazone ligand obtained from the reaction between phenylhydrazine groups and 2,5-thiophenedicarboxaldehyde. The structures of ligands and complexes were elucidated through various spectroscopic techniques, confirming their composition. All complexes were found to adopt four-coordinated geometries, indicating the formation of stable structures. Spectroscopic analysis revealed that the hydrazone ligand coordinated with the Cu(II) metal ions as a dibasic tetradentate ligand by utilizing the phenolic oxygen and azomethine nitrogen atoms. The binding affinity of the complexes with calf thymus DNA (CT-DNA) was investigated using absorption and viscosity measurements, demonstrating their interaction through the intercalation mode. The binding studies showed that the Cu(II) complexes exhibited varying degrees of binding affinity, with Cu(L4) demonstrating the highest affinity, followed by Cu(L1), Cu(L2), and Cu(L3). Moreover, the DNA fragmentation properties of the Cu(II) complexes were evaluated, suggesting their potential utilization in pharmaceutical applications. The obtained results highlight the significance of these novel complexes in the field of medicinal chemistry.

In this study, 4‐(2‐(4‐methylthiazol‐5‐yl) ethoxy) phthalonitrile (3) and zinc phthalocyanine complex (4) were synthesized and characterized. UV–vis and fluorescence spectra, aggregation, and fluorescence parameters of the zinc phthalocyanine compound were investigated. As biological properties, DPPH radical capture, antidiabetic, DNA cutting, antimicrobial, photodynamic antimicrobial, anti‐biofilm activities, and microbial cell viability parameters of the compounds were determined. The highest antioxidant activity was found as 62.80% at 100 mg/L concentration with compound 4, and compound 4 also showed the best antidiabetic activity as 65.17% at 400 mg/L concentration. In DNA cutting activity, it was determined that 3 and 4 cut DNA at all concentrations. For compounds 3–4, the minimum inhibitory concentration (MIC) values were found to be 32 and 16 mg/L for Enterococcus hirae and Enterococcus feacalis, respectively. The strongest MIC value of photodynamic antimicrobial activity was achieved as 4 mg/L for E. feacalis with 4. It was observed that both compounds inhibited the microbial viability activity of Escherichia coli by 100% at concentrations of 100 mg/L. Compound 4 inhibited biofilms of Pseudomonas aureginosa and Staphylococcus aureus as 92.48% and 98.56% at 50 mg/L concentration, respectively. [ABSTRACT FROM AUTHOR]

In this research, Vitis vinifera extract was used as a reducing molecule for the biogenic synthesis of platinum nanoparticles (Vv-Pt NPs). Vv-Pt NPs were extensively characterized by various methods. It has been determined that Pt NPs were spherical shaped and 1.513 ± 0.35 nm in size with TEM analysis. The biological activity of synthesized Pt NPs using Vitis vinifera extract was investigated such as antimicrobial, antioxidant, and DNA cleavage studies. Pt NPs exhibited the highest 2,2-Diphenyl-1-picrylhydrazil (DPPH) radical scavenging activity at 200 mg/L. The synthesized Pt NPs were observed to have significant chemical nuclease activity. Pt NPs were found to have significant antibacterial activity against Gram (−) and Gram (+) bacteria, and the highest antimicrobial activity was observed against E. coli. Then, hydrogen production was investigated due to the catalytic effects of Vv-Pt NPs on the hydrolysis of sodium borohydride (NaBH4). Turnover of frequency (TOF), enthalpy (∆H), entropy (∆S), and activation energy (Ea) values were determined as 1217.20 h−1, 37.70 kJ/mol, −153.66 kJ/mol K, and 40.24 kJ/mol, respectively. It has been observed that synthesized Vv-Pt NPs will be used as nano-drugs and will guide sustainable, renewable, and clean energy studies. [ABSTRACT FROM AUTHOR]

RNA has received enormous attention as a biomolecule in the fields of medicine, public health, and biotechnology. Over the last decade, various types of RNA molecules have been exploited as valuable drug targets by different classes of pharmaceutical agents in combating infections and diseases. In this work, we have investigated the binding of Cisplatin to RNA within double stranded RNA : DNA hybrid structures. UV melting and Isothermal Titration Calorimetry studies reveal that Cisplatin binding leads to destabilization of hybrid duplex likely due to Cisplatin‐RNA adduct formation. Gel electrophoretic analysis using an in vitro CRISPR‐Cas9 model system shows that Cisplatin binding to guide RNA leads to a concentration‐dependent reduction in target DNA cleavage. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: The control of protein functions with light is valuable for spatiotemporal probing of biological systems. Current small‐molecule photo‐ modulation methods include the light‐induced uncaging of inhibitors and chromophore‐assisted light inactivation with reactive oxygen species (ROS). However, the constant target protein expression results in inadequate photo‐modulation efficiency, particularly for less potent inhibitors and chromophores. Herein, we report a novel bifunctional small‐molecule ligands strategy to photo‐modulate gene‐editing enzymes CRISPR/Cas9. A coumarin‐derived small‐molecule ligand Bhc‐BRD0539 is developed to uncage the active inhibitor upon light irradiation and to generate ROS in the Cas9 proximity for the dual inhibition of Cas9 activity. Our results highlight the synergistic photo‐modulation with bifunctional small‐molecule ligands, which offers a valuable addition to current CRISPR/Cas9 photo‐modulation technologies and may extend to other protein classes. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: The control of protein functions with light is valuable for spatiotemporal probing of biological systems. Current small‐molecule photo‐ modulation methods include the light‐induced uncaging of inhibitors and chromophore‐assisted light inactivation with reactive oxygen species (ROS). However, the constant target protein expression results in inadequate photo‐modulation efficiency, particularly for less potent inhibitors and chromophores. Herein, we report a novel bifunctional small‐molecule ligands strategy to photo‐modulate gene‐editing enzymes CRISPR/Cas9. A coumarin‐derived small‐molecule ligand Bhc‐BRD0539 is developed to uncage the active inhibitor upon light irradiation and to generate ROS in the Cas9 proximity for the dual inhibition of Cas9 activity. Our results highlight the synergistic photo‐modulation with bifunctional small‐molecule ligands, which offers a valuable addition to current CRISPR/Cas9 photo‐modulation technologies and may extend to other protein classes. [ABSTRACT FROM AUTHOR]

Selenium nanoparticles (SeNPs) have gained wide importance in the scientific community and have emerged as an optimistic therapeutic carrier agent for targeted drug delivery. In the present study, the effectiveness of nano selenium conjugated with Morin (Ba-SeNp-Mo) produced from endophytic bacteria Bacillus endophyticus reported in our earlier research was tested against various Gram-positive, Gram-negative bacterial pathogens and fungal pathogens that showed good zone of inhibition against all selected pathogens. Antioxidant activities of these NPs were studied by 1, 1-diphenyl-2- picrylhydrazyl (DPPH), 2,2′-Azino-bis-3-ethylbenzothiozoline-6-sulfonic acid (ABTS), hydrogen peroxide (H2O2), superoxide (O2−), and nitric oxide (NO) radical scavenging assays that exhibited dose-dependent free radical scavenging activity with IC50 values 6.92 ± 1.0, 16.85 ± 1.39, 31.60 ± 1.36, 18.87 ± 1.46, and 6.95 ± 1.27 μg/mL. The efficiency of DNA cleavage and thrombolytic activity of Ba-SeNp-Mo were also studied. The antiproliferative effect of Ba-SeNp-Mo was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in COLON-26 cell lines that resulted in IC50 value of 63.11 μg/mL. Further increased intracellular reactive oxygen species (ROS) levels up to 2.03 and significant early, late and necrotic cells were also observed in AO/EtBr assay. CASPASE 3 expression was upregulated to 1.22 (40 μg/mL) and 1.85 (80 μg/mL) fold. Thus, the current investigation suggested that the Ba-SeNp-Mo has offered remarkable pharmacological activity. [ABSTRACT FROM AUTHOR]

The recent thrust in research has projected the type II clustered regularly interspaced short palindromic repeats and associated protein 9 (CRISPR-Cas9) system as an avant-garde plant genome editing tool. It facilitates the induction of site-specific double-stranded DNA cleavage by the RNA-guided DNA endonuclease (RGEN), Cas9. Elimination, addition, or alteration of sections in DNA sequence besides the creation of a knockout genotype (CRISPRko) is aided by the CRISPR-Cas9 system in its wild form (wtCas9). The inactivation of the nuclease domain generates a dead Cas9 (dCas9), which is capable of targeting genomic DNA without scissoring it. The dCas9 system can be engineered by fusing it with different effectors to facilitate transcriptional activation (CRISPRa) and transcriptional interference (CRISPRi). CRISPR-Cas thus holds tremendous prospects as a genome-manipulating stratagem for a wide gamut of crops. In this article, we present a brief on the fundamentals and the general workflow of the CRISPR-Cas system followed by an overview of the prospects of bioinformatics in propelling CRISPR-Cas research with a special thrust on the available databases and algorithms/web-accessible applications that have aided in increasing the usage and efficiency of editing. The article also provides an update on the current regulatory landscape in different countries on the CRISPR-Cas edited plants to emphasize the far-reaching impact of the genomic editing technology. [ABSTRACT FROM AUTHOR]

CRISPR-CasΦ, a small RNA-guided enzyme found uniquely in bacteriophages, achieves programmable DNA cutting as well as genome editing. To investigate how the hypercompact enzyme recognizes and cleaves double-stranded DNA, we determined cryo-EM structures of CasΦ (Cas12j) in pre- and post-DNA-binding states. The structures reveal a streamlined protein architecture that tightly encircles the CRISPR RNA and DNA target to capture, unwind and cleave DNA. Comparison of the pre- and post-DNA-binding states reveals how the protein rearranges for DNA cleavage upon target recognition. On the basis of these structures, we created and tested mutant forms of CasΦ that cut DNA up to 20-fold faster relative to wild type, showing how this system may be naturally attenuated to improve the fidelity of DNA interference. The structural and mechanistic insights into how CasΦ binds and cleaves DNA should allow for protein engineering for both in vitro diagnostics and genome editing.

Agents that cause double-strand breaks (DSBs) of DNA via radical formation have been demonstrated to be effective in treating cancer because DSBs result in cellular apoptosis. Light-responsive agents for the treatment of cancer have been of interest for decades because they afford the ability to spatially control chemical reactions limiting the effects by controlling the area of illumination. Alkylcobalamins, which are structurally related to Vitamin B12 (B12), produce radicals with very high quantum yields when illuminated with green light (approximately 530 nm). Cancerous cells uptake alkylcobalamins to a greater extent than healthy cells because these rapidly dividing cells have an increased demand for B12. Tethering two cobalamins with a propyl group results in a complex that causes true DNA DSBs in a light-mediated manner.

Restriction endonucleases are a component of restriction–modification systems, where the main biological function is to protect bacterial cells from incoming foreign DNA molecules. There are four main types of restriction enzymes (types I, II, III, and IV), which differ in protein composition, cofactor requirements, and mode of action. The most studied are representatives of type II, which specifically recognize DNA sequences of 4–8 bp and catalyze DNA cleavage within these sequences or not far from them. The exceptional precision of type II enzymes has made them indispensable for DNA manipulations. Although hundreds of DNA restriction enzymes are currently known, there is still a need for enzymes that recognize new DNA targets. For this reason, the discovery of new natural restriction endonucleases and rational design of their properties (to obtain enzymes with high specificity for a unique nucleotide sequence at a restriction site and without nonspecific activity) will expand the list of enzymes for use in biotechnology and genetic engineering. This review briefly touches upon the main types of restriction endonucleases, their classification, nomenclature, and typical properties, and it concisely describes approaches to the construction of enzymes with altered properties. [ABSTRACT FROM AUTHOR]

Activation-induced cytidine deaminase (AID)-dependent DNA cleavage is the initial event of antibody gene-diversification processes such as class switch recombination (CSR) and somatic hypermutation (SHM). We previously reported the requirement of an AID-dependent decrease of topoisomerase 1 (Top1) for efficient DNA cleavage, but the underlying molecular mechanism has remained elusive. This study focuses on HuR / ELAVL1 , a protein that binds to AU-rich elements in RNA. HuR -knockout (KO) CH12 cells derived from murine B lymphoma cells were found to have lower CSR and hypermutation efficiencies due to decreased AID-dependent DNA cleavage levels. The HuR -KO CH12 cells do not show impairment in cell cycles and Myc expression, which have been reported in HuR-reduced spleen B cells. Furthermore, drugs that scavenge reactive oxygen species (ROS) do not rescue the lower CSR in HuR -KO CH12 cells, meaning that ROS or decreased c-Myc protein amount is not the reason for the deficiencies of CSR and hypermutation in HuR -KO CH12 cells. We show that HuR binds to Top1 mRNA and that complete deletion of HuR abolishes AID-dependent repression of Top1 protein synthesis in CH12 cells. Additionally, reduction of CSR to IgG3 in HuR -KO cells is rescued by knockdown of Top1, indicating that elimination of the AID-dependent Top1 decrease is the cause of the inefficiency of DNA cleavage, CSR and hypermutation in HuR -KO cells. These results show that HuR is required for initiation of antibody diversification and acquired immunity through the regulation of AID-dependent DNA cleavage by repressing Top1 protein synthesis. [ABSTRACT FROM AUTHOR]

Novel bacterial topoisomerase inhibitors (NBTIs) are an emerging class of antibacterials that target gyrase and topoisomerase IV. A hallmark of NBTIs is their ability to induce gyrase/topoisomerase IV-mediated single-stranded DNA breaks and suppress the generation of double-stranded breaks. However, a previous study reported that some dioxane-linked amide NBTIs induced double-stranded DNA breaks mediated by Staphylococcus aureus gyrase. To further explore the ability of this NBTI subclass to increase double-stranded DNA breaks, we examined the effects of OSUAB-185 on DNA cleavage mediated by Neisseria gonorrhoeae gyrase and topoisomerase IV. OSUAB-185 induced single-stranded and suppressed double-stranded DNA breaks mediated by N. gonorrhoeae gyrase. However, the compound stabilized both single- and double-stranded DNA breaks mediated by topoisomerase IV. The induction of double-stranded breaks does not appear to correlate with the binding of a second OSUAB-185 molecule and extends to fluoroquinolone-resistant N. gonorrhoeae topoisomerase IV, as well as type II enzymes from other bacteria and humans. The double-stranded DNA cleavage activity of OSUAB-185 and other dioxane-linked NBTIs represents a paradigm shift in a hallmark characteristic of NBTIs and suggests that some members of this subclass may have alternative binding motifs in the cleavage complex. [ABSTRACT FROM AUTHOR]

It is known that heavy metal containing nanomaterials can easily prevent the formation of microbial cultures. The emergence of new generation epidemic diseases in the last 2 years has increased the importance of both personal and environmental hygiene. For this reason, in addition to preventing the spread of diseases, studies on alternative disinfectant substances are also carried out. In this study, the antibacterial activity of nanoflower and nanocube, which are easily synthesized and nanoparticle species containing iron, were compared. The antioxidant abilities of new synthesized NF@FeO(OH) and NC@α-Fe2O3 were tested by DPPH scavenging activity assay. The highest DPPH inhibition was achieved with NC@α-Fe2O3 as 71.30% at 200 mg/L. NF@FeO(OH) and NC@α-Fe2O3 demonstrated excellent DNA cleavage ability. The antimicrobial capabilities of NF@FeO(OH) and NC@α-Fe2O3 were analyzed with micro dilution procedure. In 500 mg/L, the antimicrobial activity was 100%. In addition to these, the biofilm inhibition of NF@FeO(OH) and NC@α-Fe2O3 were investigated against S. aureus and P. aeruginosa and it was found that they showed significant antibiofilm inhibition. It is suggested that additional studies can be continued to be developed and used as an antibacterial according to the results of the nanoparticles after various toxicological test systems. [ABSTRACT FROM AUTHOR]

Under physiological conditions, natural enediynes antibiotics generate free radicals through a cycloaromatization reaction. The resulting highly reactive radicals can abstract hydrogen atoms on the phosphate backbone of DNA, leading to high cytotoxicity. On the other hand, anthraquinone‐fused compounds such as daunomycin and adriamycin can insert into DNA through planar aromatic ring structures, resulting in strong DNA damage. To this end, a series of naphthoquinone‐based enediynes were designed and synthesized by adjusting the type of terminal alkyne to modulate their water solubility and radical generation property. Theoretical calculations suggest that the naphthoquinone‐based enediyne generates radicals through a rearrangement (1,3‐proton transfer) followed by Myers‐Saito cyclization processes, with a much lower activation free energy barrier than Bergman cyclization (24.2 kcal/mol vs 39.8 kcal/mol). Differential scanning calorimetry and electron paramagnetic resonance results showed that naphthoquinone‐based enediynes undergo cycloaromatization at relatively low temperatures, resulting in the generation of highly reactive radical species. The enediynes with optimal water solubility displayed strong DNA cleavage ability at micromolar level and high cytotoxicity towards Hela cells, providing a half‐inhibitory concentration of 8.19 μM. [ABSTRACT FROM AUTHOR]

CRISPR-Cas12a is a genome-editing system, recently also harnessed for nucleic acid detection, which is promising for the diagnosis of the SARS-CoV-2 coronavirus through the DETECTR technology. Here, a collective ensemble of multimicrosecond molecular dynamics characterizes the key dynamic determinants allowing nucleic acid processing in CRISPR-Cas12a. We show that DNA binding induces a switch in the conformational dynamics of Cas12a, which results in the activation of the peripheral REC2 and Nuc domains to enable cleavage of nucleic acids. The simulations reveal that large-amplitude motions of the Nuc domain could favor the conformational activation of the system toward DNA cleavages. In this process, the REC lobe plays a critical role. Accordingly, the joint dynamics of REC and Nuc shows the tendency to prime the conformational transition of the DNA target strand toward the catalytic site. Most notably, the highly coupled dynamics of the REC2 region and Nuc domain suggests that REC2 could act as a regulator of the Nuc function, similar to what was observed previously for the HNH domain in the CRISPR-associated nuclease Cas9. These mutual domain dynamics could be critical for the nonspecific binding of DNA and thereby for the underlying mechanistic functioning of the DETECTR technology. Considering that REC is a key determinant in the system's specificity, our findings provide a rational basis for future biophysical studies aimed at characterizing its function in CRISPR-Cas12a. Overall, our outcomes advance our mechanistic understanding of CRISPR-Cas12a and provide grounds for novel engineering efforts to improve genome editing and viral detection.