Efficient DNA Cleavage Research Articles

Machine learning based predictive analysis of DNA cleavage induced by diverse nanomaterials

DNA cleavage by nanomaterials has the potential to be utilized as an innovative tool for gene editing. Numerous nanomaterials exhibiting DNA cleavage properties have been identified and cataloged. Yet, the exploitation of property data through data-driven machine-learning approaches remains unexplored. A database was developed, compiling thirty distinctive characteristics, encompassing physical and chemical properties, as well as experimental conditions of nanomaterials that have demonstrated DNA cleavage capability such as in articles published over the past two decades. The DNA cleavage effect and efficiency of nanomaterials were predicted using machine learning algorithms such as support vector machines, deep neural networks, and random forest, and a classification accuracy of 0.93 for the cleavage effect was achieved. Moreover, the potential of utilizing larger datasets to enhance the predictive capacity of models was discussed. The findings indicate the feasibility of predicting nanomaterial properties based on experimental data. Evaluating the performance and effectiveness of the machine learning models trained using the existing data can furnish valuable insights for future materials research endeavors, especially for the design of DNA cleavage with specific sites.

Eco-benevolent synthesis of ZnO-NPs and ZnO-MFs from Inula oculus-christi L. (Asteraceae) with effective antioxidant, antimicrobial, DNA cleavage, and decolorization efficiencies.

As a result of the changes occurring globally in recent years, millions of people are facing challenging and even life-threatening diseases such as cancer and the COVID-19 pandemic, among others. This phenomenon has spurred researchers towards developing and implementing innovative and environmentally friendly scientific methods, merging disciplines with significant technological potential, such as nanotechnology with medicinal plants. Therefore, the focus of this research is to synthesize zinc nanoparticles (ZnO-NPs) and microflowers (ZnO-MFs) using extracts of the medicinal plant I. oculus christi prepared in n-hexane and methanol as new bioreduction and capping agents through a simple and environmentally friendly chemical approach. Optical, thermal, and morphological structural analyses of ZnO-NPs and ZnO-MFs were conducted using Ultraviolet-Visible (UV-Vis) spectroscopy, Fourier Transform Infrared (FT-IR) spectroscopy, Thermogravimetric Analysis (TGA), and Field Emission Scanning Electron Microscopy (FE-SEM). Metabolic profiles of extracts from different plant parts were analyzed using Gas Chromatography-Mass Spectrometry (GC-MS) and supported by visualization of contents through Principal Component Analysis (PCA), hierarchical cluster analysis heatmaps, and Pearson correlation graphs. Interestingly, ZnO-NPs and ZnO-MFs exhibited strong antioxidant properties and demonstrated particularly potent antimicrobial activity against Micrococcus luteus NRRL B-4375, Escherichia coli ATCC 25922, and Candida albicans ATCC 10231 strains compared to standard antibiotics. Furthermore, ZnO-NPs and ZnO-MFs showed excellent plasmid DNA-cleavage activity of pBR322 with increasing doses. The photocatalytic performance of the synthesized ZnO-NPs and ZnO-MFs was evaluated for methylene blue (MB), congo red (CR), and safranin-O(SO) dyes, demonstrating remarkable color removal efficiency. Overall, the results provide a promising avenue for the green synthesis of ZnO-NPs and ZnO-MFs using I. oculus-christi L. inflorescence and pappus extracts, potentially revolutionizing biopharmaceutical and catalytic applications in these fields.

The binding affinity-dependent inhibition of cell growth and viability by DNA sulfur-binding domains.

Increasing evidence suggests that DNA phosphorothioate (PT) modification serves several purposes in the bacterial host, and some restriction enzymes specifically target PT-DNA. PT-dependent restriction enzymes (PDREs) bind PT-DNA through their DNA sulfur binding domain (SBD) with dissociation constants (KD) of 5 nM~1 μM. Here, we report that SprMcrA, a PDRE, failed to dissociate from PT-DNA after cleavage due to high binding affinity, resulting in low DNA cleavage efficiency. Expression of SBDs in Escherichia coli cells with PT modification induced a drastic loss of cell viability at 25°C when both DNA strands of a PT site were bound, with one SBD on each DNA strand. However, at this temperature, SBD binding to only one PT DNA strand elicited a severe growth lag rather than lethality. This cell growth inhibition phenotype was alleviated by raising the growth temperature. An invitro assay mimicking DNA replication and RNA transcription demonstrated that the bound SBD hindered the synthesis of new DNA and RNA when using PT-DNA as the template. Our findings suggest that DNA modification-targeting proteins might regulate cellular processes involved in DNA metabolism in addition to being components of restriction-modification systems and epigenetic readers.

A novel copper(II) complex with a salicylidene carbohydrazide ligand that promotes oxidative stress and apoptosis in triple negative breast cancer cells.

We report the synthesis, characterization, anti-cancer activity and mechanism of action of a novel water-soluble Cu(II) complex with salicylidene carbohydrazide as the ligand and o-phenanthroline as the co-ligand. The synthesized complex (1) was characterized by FT-IR, EPR, and electronic spectroscopy, as well as single crystal X-ray diffraction. This compound was found to be paramagnetic from EPR spectra and X-ray crystallography revealed that the molecule crystallized in an orthorhombic crystal system. The crystal lattice was asymmetric containing two distinct binuclear copper complexes containing the Schiff base as the major ligand, o-phenanthroline as a co-ligand, two nitrate anions, and two water molecules. The Cu(II) in the first site coordinated with the enolised ligand comprising enolate O-, phenolate O-, and the imine N and N,N from o-phen. The major part of this complex exists as Cu(II) coordinated with two H2O molecules at the second site with nitrate acting as the counter anion. However, a smaller portion of the complex exists where Cu(II) is coordinated with NO3- and H2O, and the remaining water molecule acts as lattice water. It was tested for DNA binding and cleavage properties which revealed that it binds in an intercalative mode to CT-DNA with Kb value of 1.25 × 104 M-1. Furthermore, cleavage studies reveal that the complex has potential for efficient DNA cleavage under both oxidative and hydrolytic conditions. It was able to enhance the rate of cleavage by 2.8 × 108 times. The complex shows good cytotoxicity to breast cancer monolayer (2D) as well as spheroid (3D) systems. The IC50 values for MDA-MB-231 and MCF-7 monolayer culture was calculated as 1.86 ± 0.17 μM and 2.22 ± 0.08 μM, respectively, and in (3D) spheroids of MDA-MB-231 cells, the IC50 value was calculated to be 1.51 ± 0.29 μM. It was observed that the complex outperformed cisplatin in both breast cancer cell lines. The cells treated with complex 1 underwent severe DNA damage, increased oxidative stress and cell cycle arrest which finally led to programmed cell death or apoptosis in triple negative breast cancer cells through an intrinsic pathway.

Hepatocyte-confined CRISPR/Cas9-based nanocleaver precisely eliminates viral DNA for efficient and safe treatment of hepatitis B virus infection

Clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9)-based therapies are attractive to achieve precise and targeted disruption of the hepatitis B virus (HBV) genome for HBV infection treatment. However, unspecific in vivo distribution of the CRISPR/Cas9 vector after systemic administration not only impedes CRISPR/Cas9 expression in the liver, but also causes undesired gene editing in off-site organs, decreasing the therapeutic efficacy and leading to potential side effects. Herein, we designed a hepatocyte-confined CRISPR/Cas9-based nanocleaver (HepCCCleaver) to realize precise and efficient cleavage of HBV DNA, specifically in hepatocytes, for safe and effective HBV treatment. This HepCCCleaver was formed by encapsulating the hepatocyte-specific CRISPR/Cas9 plasmid into a galactose-modified membrane-fusogenic carrier. Our galactose-modified membrane-fusogenic carrier allowed the HepCCCleaver to specifically accumulate in the liver and enter hepatocytes through membrane fusion, bypassing lysosome entrapment to avoid degradation for superior delivery efficiency. The hepatocyte-specific CRISPR/Cas9 plasmid released in the cytoplasm could switch on the gene expression only in hepatocytes, ensuring precise cleavage on viral DNA in HBV-infected but not other types of cells. By integrating the liver-targeting membrane-fusogenic delivery system with hepatocyte-specific CRISPR/Cas9 plasmid, this HepCCCleaver could precisely and efficiently confine over 95% events of viral DNA gene editing in hepatocytes. This HepCCCleaver is a universal gene editing platform enabling safe and efficient disease treatment, specifically in the cell type of interest.

A thermostable type I-B CRISPR-Cas system for orthogonal and multiplexed genetic engineering

Thermophilic cell factories have remarkably broad potential for industrial applications, but are limited by a lack of genetic manipulation tools and recalcitrance to transformation. Here, we identify a thermophilic type I-B CRISPR-Cas system from Parageobacillus thermoglucosidasius and find it displays highly efficient transcriptional repression or DNA cleavage activity that can be switched by adjusting crRNA length to less than or greater than 26 bp, respectively, without ablating Cas3 nuclease. We then develop an orthogonal tool for genome editing and transcriptional repression using this type I-B system in both thermophile and mesophile hosts. Empowered by this tool, we design a strategy to screen the genome-scale targets involved in transformation efficiency and established dynamically controlled supercompetent P. thermoglucosidasius cells with high efficiency ( ~ 108 CFU/μg DNA) by temporal multiplexed repression. We also demonstrate the construction of thermophilic riboflavin cell factory with hitherto highest titers in high temperature fermentation by genome-scale identification and combinatorial manipulation of multiple targets. This work enables diverse high-efficiency genetic manipulation in P. thermoglucosidasius and facilitates the engineering of thermophilic cell factories.

Characterization of CoCas9 nuclease from Capnocytophaga ochracea

ABSTRACT Cas9 nucleases are widely used for genome editing and engineering. Cas9 enzymes encoded by CRISPR-Cas defence systems of various prokaryotic organisms possess different properties such as target site preferences, size, and DNA cleavage efficiency. Here, we biochemically characterized CoCas9 from Capnocytophaga ochracea, a bacterium that inhabits the oral cavity of humans and contributes to plaque formation on teeth. CoCas9 recognizes a novel 5’-NRRWC-3’ PAM and efficiently cleaves DNA in vitro. Functional characterization of CoCas9 opens ways for genetic engineering of C. ochracea using its endogenous CRISPR-Cas system. The novel PAM requirement makes CoCas9 potentially useful in genome editing applications.

Engineered circular guide RNAs boost CRISPR/Cas12a- and CRISPR/Cas13d-based DNA and RNA editing

BackgroundThe CRISPR/Cas12a and CRISPR/Cas13d systems are widely used for fundamental research and hold great potential for future clinical applications. However, the short half-life of guide RNAs (gRNAs), particularly free gRNAs without Cas nuclease binding, limits their editing efficiency and durability.ResultsHere, we engineer circular free gRNAs (cgRNAs) to increase their stability, and thus availability for Cas12a and Cas13d processing and loading, to boost editing. cgRNAs increases the efficiency of Cas12a-based transcription activators and genomic DNA cleavage by approximately 2.1- to 40.2-fold for single gene editing and 1.7- to 2.1-fold for multiplexed gene editing than their linear counterparts, without compromising specificity, across multiple sites and cell lines. Similarly, the RNA interference efficiency of Cas13d is increased by around 1.8-fold. In in vivo mouse liver, cgRNAs are more potent in activating gene expression and cleaving genomic DNA.ConclusionsCgRNAs enable more efficient programmable DNA and RNA editing for Cas12a and Cas13d with broad applicability for fundamental research and gene therapy.

Pharmacological Prospects of Morin Conjugated Selenium Nanoparticles-Evaluation of Antimicrobial, Antioxidant, Thrombolytic, and Anticancer Activities.

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.

Necessity of HuR/ELAVL1 for activation-induced cytidine deaminase-dependent decrease in topoisomerase 1 in antibody diversification.

Activation-induced cytidine deaminase (AID)-dependent DNA cleavage are 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.

Orthogonal light-triggered multiple effects based on photochromic nanoparticles for DNA cleavage and beyond.

Efficient and spatiotemporally controllable cleavage of deoxyribonucleic acid (DNA) is of great significance for both disease treatment (e.g. tumour, bacterial infection, etc) and molecular biology applications (e.g. gene editing). The recently developed light-induced cleavage strategy based on catalytic nanoparticles has been regarded as a promising strategy for DNA controllable cleavage. Although the regulation based on orthogonal light in biomedical applications holds more significant advantages than that based on single light, nanoparticle-mediated DNA cleavage based on orthogonal light has yet to be reported. In this article, for the first time, we demonstrated an orthogonal light-regulated nanosystem for efficient and spatiotemporal DNA cleavage. In this strategy, tungsten oxide (WO3) nanoparticles with photochromic properties were used as nano-antennae to convert the photoenergy from the orthogonal visible light (405 nm) and near-infrared light (808 nm) into chemical energy for DNA cleavage. We verified that only the orthogonal light can trigger high cleavage efficiency on different types of DNA. Moreover, such an orthogonal light-response nano-system can not only induce significant apoptosis of tumour cells, but also effectively eliminate bacterial biofilms.

Structure- and Content-Dependent Efficiency of Cas9-Assisted DNA Cleavage in Genome-Editing Systems.

Genome-editing systems, being some of the key tools of molecular biologists, represent a reasonable hope for progress in the field of personalized medicine. A major problem with such systems is their nonideal accuracy and insufficient selectivity. The selectivity of CRISPR-Cas9 systems can be improved in several ways. One efficient way is the proper selection of the consensus sequence of the DNA to be cleaved. In the present work, we attempted to evaluate the effect of formed non-Watson-Crick pairs in a DNA duplex on the efficiency of DNA cleavage in terms of the influence of the structure of the formed partially complementary pairs. We also studied the effect of the location of such pairs in DNA relative to the PAM (protospacer-adjacent motif) on the cleavage efficiency. We believe that the stabilization of the Cas9-sgRNA complex with a DNA substrate containing noncomplementary pairs is due to loop reorganization in the RuvC domain of the enzyme. In addition, PAM-proximal mismatches in the DNA substrate lower enzyme efficiency because the "seed" region is involved in binding and cleavage, whereas PAM-distal mismatches have no significant impact on target DNA cleavage. Our data suggest that in the case of short duplexes with mismatches, the stages of recognition and binding of dsDNA substrates by the enzyme determine the reaction rate and time rather than the thermodynamic parameters affected by the "unwinding" of DNA. The results will provide a theoretical basis for predicting the efficiency and accuracy of CRISPR-Cas9 systems at cleaving target DNA.

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.

Mononuclear cobalt(II) complexes with polypyridyl ligands: Synthesis, characterization, DNA interactions and in vitro cytotoxicity towards human cancer cells

Mononuclear cobalt(II) complexes [CoII(L1)Cl2]; 1, [CoII(L1)(bpy)Cl]PF6; 2, [CoII(L1)(phen)Cl]PF6; 3 and [CoII(L2)Cl2]; 4 (where L1 = N,N-bis(pyridin-2-ylmethyl)aniline, L2 = (2,4,6-trimethyl-N,N-bis(pyridin-2-ylmethyl)aniline, bpy = 2,2/−bipyridine, phen = 1,10-phenanthroline) were synthesized and characterized by different analytical and spectroscopic methods. All the complexes were structurally identified by single-crystal X-ray crystallography. Penta-coordinated complex 1 adopted distorted trigonal bipyramidal and hexacoordinated complexes 2 and 3 having distorted octahedral geometry whereas tetra-coordinated complex 4 has distorted tetrahedral geometry. The interactions of salmon sperm DNA (ss-DNA) with complexes (1-4) were investigated by absorbance, fluorescence spectroscopy and molecular docking studies. All the complexes are very susceptible to DNA binding and the binding affinity (Kb) follows the order 3 (2.05 × 104 M −1) > 4 (1.40 × 104 M −1) > 2 (1.36 × 104 M −1) > 1 (1.34 × 104 M −1) indicating they have superior DNA binding ability. The Stern-Volmer constant (Ksv) ranges from 1.10 × 104 M −1 to 1.95 × 104 M −1 suggesting weak or moderate binding with DNA. DNA cleavage study in plasmid DNA reveals very efficient DNA cleavage factors even in the absence of any external agents. Using multiple biochemical assays, we have demonstrated that 1–4 induces apoptosis of human cancer cells with IC50 values of 26.48 ± 1.45 μM, 10.89 ± 0.55 μM, 7.63 ± 0.4 μM and 37.67 ± 2.06 μM, respectively in A549 lung adenocarcinoma cells and 14.45 ± 0.73 μM, 1.97 ± 0.1 μM, 0.98 ± 0.05 μM and 24.43 ± 1.22 μM, respectively in MDA-MB-231 breast adenocarcinoma cells.

Engineering efficient artificial nanozyme based on chitosan grafted Fe-doped-carbon dots for bacteria biofilm eradication

Bacterial biofilms have evoked worldwide attention owing to their serious threats to public health, but how to effectively eliminate bacterial biofilms still remains great challenges. Here, we rationally designed a novel and vigorous chitosan grafted Fe-doped-carbon dots (CS@Fe/CDs) as an efficient artificial nanozyme to combat rigid bacterial biofilms through the selective activation of Fenton-like reaction-triggered peroxidase-like catalytic activity and the synergistic antibacterial activity of CS. On the one hand, the peroxidase-like catalytic activity made CS@Fe/CDs catalyze H2O2 for producing hydroxyl radicals (•OH), resulting in efficient cleavage of extracellular DNA (eDNA). On the other hand, CS was capable of binding with the negatively charged cell membrane through electrostatic interaction, changing the cell membrane permeability and causing cell death within bacterial biofilms. Based on their synergistic effects, the fragments of bacterial biofilm and exposed bacteria were persistently eradicated. Remarkably, CS@Fe/CDs-based nanozyme not only enabled the effective destroying of gram-positive Staphylococcus aureus (S. aureus) biofilms, but also completely eliminated gram-negative Pseudomonas aeruginosa (P. aeruginosa) biofilms, showing great potential as a promising anti-biofilm agent against bacteria biofilms. This proposed synergistic strategy for bacterial biofilm eradication might offer a powerful modality to manage of bacterial biofilm fouling in food safety and environmental protection.

Biochemical Study of Some New Cephems and Selenacephems Based on 6H-1,3-Thiazines and 6H-1,3-selenazines

Several new and know 6-(4-substituted phenyl)-4-(4-substituted phenyl)-2-phenyl-6H-1,3-thiazine (or selenazine) (Z4B7, Z4D5, Z4B7' and Z4D5') were prepared by the 1,4-Michael addition reaction of chalcone derivatives with thiobenzamide or phenylselenocarboxamide in basic medium (where the chalcones was formed by Claisen-Schimidt condensation of aromatic aldehydes with 4-substituted acetophenone in presence of sodium hydroxide). These 6H-1,3-thia- or selenazine were used to a new series of cephem and selenacephem compounds (i.e. 7-chloro-4-(4-substituted phenyl)-2-(4-substituted phenyl)-6-phenyl-5-thia (or 5-selena)-1-azabicyclo[4.2.0]oct-2-en-8-one; AZ4B7, AZ4D5, AZ4B7' and AZ4D5'). All new compound derivatives were characterized by IR, 1H NMR, 13C NMR, mass spectroscopic techniques and elemental analysis. The toxicity of new compounds was assayed via the determination of their LD50 value by using Dixon's up and down method. The antibacterial activity of cephem and selenacephem compounds were tested in vitro against Staphylococcus aureus, Bacillus, Escherichia coli and Pseudomonas aeruginosa. Furthermore, the antioxidant, anticancer and DNA cleavage efficiency of compounds were evaluated.

Biological properties of novel mono and double-decker hexadeca-substituted metal phthalocyanines

This study reports chemical agents that exhibit efficient antibacterial photodynamic, antimicrobial, antioxidant, biofilm inhibition, and DNA cleavage activities.

Efficient target cleavage by Type V Cas12a effectors programmed with split CRISPR RNA.

CRISPR RNAs (crRNAs) that direct target DNA cleavage by Type V Cas12a nucleases consist of constant repeat-derived 5′-scaffold moiety and variable 3′-spacer moieties. Here, we demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by a Cas12a ortholog from Acidaminococcus sp. (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer moiety only. crRNAs split into separate scaffold and spacer RNAs catalyzed highly specific and efficient cleavage of target DNA by AsCas12a in vitro and in lysates of human cells. In addition to dsDNA target cleavage, AsCas12a programmed with split crRNAs also catalyzed specific ssDNA target cleavage and non-specific ssDNA degradation (collateral activity). V-A effector nucleases from Francisella novicida (FnCas12a) and Lachnospiraceae bacterium (LbCas12a) were also functional with split crRNAs. Thus, the ability of V-A effectors to use split crRNAs appears to be a general property. Though higher concentrations of split crRNA components are needed to achieve efficient target cleavage, split crRNAs open new lines of inquiry into the mechanisms of target recognition and cleavage and may stimulate further development of single-tube multiplex and/or parallel diagnostic tests based on Cas12a nucleases.

Spectroanalytical, computational, DNA/BSA binding and in vitro cytotoxic activity studies of new transition metal complexes of novel aryl hydrazone

The ligand ((1-ethyl-Nʹ-furan-2-yl)-methylene)-1,4-dihydro-7-methyl-4-oxo-1,8- napthyridine-3-carbohydrazide (NFMHMONC) and its transition metal (II) complexes were synthesized and characterized by various spectroanalytical techniques. The quantum chemical calculations and physicochemical properties of NFMHMONC were evaluated by HyperChem 7.5 software tools. The binding ability of metal complexes with CT-DNA has been determined by electronic absorption spectral titrations and competitive DNA binding fluorescence measurements showed a relatively high binding constant value in [Cu(II)(NFMHMONC)2]Cl2 compared to [Co(II)(NFMHMONC)2H2O]Cl2. The DNA binding behaviors were investigated by the study of viscosity measurement and the results indicated the intercalative mode of binding. The binding efficiency of metal complexes with BSA was also determined by electron absorption spectroscopy. Hydrolytic cleavage activity against supercoiled pBR322 plasmid DNA of NFMHMONC and its metal (II) complexes carried out and inferred more efficient double-strand DNA cleavage with [Cu(II)(NFMHMONC)2]Cl2 complex. The radical scavenging activity study of NFMHMONC and its metal complexes evaluated by their interaction with the DPPH free radical on electronic absorption spectroscopy has shown remarkable antioxidant activity. In vitro cytotoxicity studies revealed a lower percentage of cell viability and IC50 value in both HeLa and MCF-7 cell lines with [Cu(II)(NFMHMONC)2]Cl2 compared to [Co(II)(NFMHMONC)2H2O]Cl2 and NFMHMONC.

Metallonucleases encompassing curcumin, 2-aminobenzothiazole and o-phenylenediamine: a search for new metallonucleases

A few novelmetallonucleases were synthesized from curcumin derived Schiff base incorporating 2-aminobenzothiazoleand o-phenylenediamine. By characterization, these complexes were found to have 1:1:1 stoichiometry [M:L:o-Ph]. They were inferred to be electrolytic in nature in accordance to the values of molar conductance. From the spectral data the proposed geometry of all the complexes was deduced to be square planar. The complexes were screened against a few pathogens for their antimicrobial activities which reveal that they are efficient antimicrobial activity than the free ligands. The interactions of CT-DNA were studied by UV spectrophotometric, viscosity and cyclic voltammetric techniques. The complexes with higher values of Kb are optional to have greater DNA binding propensity. They have potent interaction with the CT-DNA and hence they are proficient DNA cleavable linkers due to their importance in biotechnology and drug design. Furthermore, the copper(II) complex exhibits efficient DNA cleavage with supercoiled pBR322 involving hydrolytic cleavage pathway.