Target DNA Research Articles

Electrochemical sensing technology based on a ligation-initiated LAMP-assisted CRISPR/Cas12a system for high-specificity detection of EGFR E746–A750 deletion mutation

Epidermal growth factor receptor (EGFR) mutation status is pivotal in predicting the efficacy of tyrosine kinase inhibitor treatments against tumors. Among EGFR mutations, the E746–A750 deletion is particularly common and accurately quantifying it can guide targeted therapies. This study introduces a novel visual sensing technology using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system guided by ligation-initiated loop-mediated isothermal amplification (LAMP) to detect the del E746–A750 mutation in EGFR. Conventional LAMP primers were simplified by designing a pair of target-specific stem-loop DNA probes, enabling selective amplification of the target DNA. The CRISPR/Cas12a system was employed to identify the target nucleic acid and activate Cas12a trans-cleavage activity, thereby enhancing the specificity of the assay. Furthermore, the biosensor utilized high-performance nanomaterials such as triangular gold nanoparticles and graphdiyne, known for their large specific surface area, to enhance sensitivity effectively as a sensing platform. The proposed biosensor demonstrated outstanding specificity, achieving a low detection limit of 17 fM (S/N = 3). Consequently, this innovative strategy not only expands the application scope of CRISPR/Cas12a technology but also introduces a promising approach for clinical diagnostics in modern medicine.

Geometric deep learning of protein–DNA binding specificity

Predicting protein–DNA binding specificity is a challenging yet essential task for understanding gene regulation. Protein–DNA complexes usually exhibit binding to a selected DNA target site, whereas a protein binds, with varying degrees of binding specificity, to a wide range of DNA sequences. This information is not directly accessible in a single structure. Here, to access this information, we present Deep Predictor of Binding Specificity (DeepPBS), a geometric deep-learning model designed to predict binding specificity from protein–DNA structure. DeepPBS can be applied to experimental or predicted structures. Interpretable protein heavy atom importance scores for interface residues can be extracted. When aggregated at the protein residue level, these scores are validated through mutagenesis experiments. Applied to designed proteins targeting specific DNA sequences, DeepPBS was demonstrated to predict experimentally measured binding specificity. DeepPBS offers a foundation for machine-aided studies that advance our understanding of molecular interactions and guide experimental designs and synthetic biology.

Photocleavable Guide RNA for Photocontrolled CRISPR/Cas9 System

Objective: The development of CRISPR/Cas-based gene-editing systems having a higher efficacy and specificity, and capable of changing activity in response to light irradiation is an urgent problem. A promising approach to this problem is to modify CRISPR/Cas components, in particular guide RNA, by introducing photocleavable linkers. We developed an approach to the synthesis of photocleavable single guide RNA (sgRNA) for the CRISPR/Cas9 system containing linkers on the basis of 1-(2-nitrophenyl)-1,2-ethanediol. Such photomodified guide RNAs are cleaved under UV irradiation, thereby inactivating the CRISPR/Cas9 system. Methods: Automatic solid-phase phosphoramidate method was used for photomodified sgRNA synthesis. Model plasmid was used for designed system testing. Results and Discussion: We obtained three variants of photomodified sgRNA with different photolinker positions. Evidence was obtained showing that the sgRNA with the photolinker introduced in the protein Cas9 site of binding and hairpin formation is able to effectively guide Cas9 nuclease for target DNA cleavage before UV irradiation and lose its activity after irradiation. The conditions of controllable 40% cleavage of a model target DNA were chosen. Conclusions: The work presents the results of photocleavable sgRNA design and usage as a component of photoregulated CRISPR/Cas9 system. The developed approach makes possible specific inactivation of the CRISPR/Cas9 gene editing system in a specific time moment in a definite place. The photoregulation of the gene-editing system not only allows one to reduce undesirable off-target effects, but also forms the basis for genetic disease therapy.

Structural determinants of DNA cleavage by a CRISPR HNH-Cascade system

Canonical prokaryotic type I CRISPR-Cas adaptive immune systems contain a multicomponent effector complex called Cascade, which degrades large stretches of DNA via Cas3 helicase-nuclease activity. Recently, a highly precise subtype I-F1 CRISPR-Cas system (HNH-Cascade) was found that lacks Cas3, the absence of which is compensated for by the insertion of an HNH endonuclease domain in the Cas8 Cascade component. Here, we describe the cryo-EM structure of Selenomonas sp. HNH-Cascade (SsCascade) in complex with target DNA and characterize its mechanism of action. The Cascade scaffold is complemented by the HNH domain, creating a ring-like structure in which the unwound target DNA is precisely cleaved. This structure visualizes a unique hybrid of two extensible biological systems-Cascade, an evolutionary platform for programmable DNA effectors, and an HNH nuclease, an adaptive domain with a spectrum of enzymatic activity.

Elucidating the Architectural dynamics of MuB filaments in bacteriophage Mu DNA transposition

MuB is a non-specific DNA-binding protein and AAA+ ATPase that significantly influences the DNA transposition process of bacteriophage Mu, especially in target DNA selection for transposition. While studies have established the ATP-dependent formation of MuB filament as pivotal to this process, the high-resolution structure of a full-length MuB protomer and the underlying molecular mechanisms governing its oligomerization remain elusive. Here, we use cryo-EM to obtain a 3.4-Å resolution structure of the ATP(+)-DNA(+)-MuB helical filament, which encapsulates the DNA substrate within its axial channel. The structure categorizes MuB within the initiator clade of the AAA+ protein family and precisely locates the ATP and DNA binding sites. Further investigation into the oligomeric states of MuB show the existence of various forms of the filament. These findings lead to a mechanistic model where MuB forms opposite helical filaments along the DNA, exposing potential target sites on the bare DNA and then recruiting MuA, which stimulates MuB’s ATPase activity and disrupts the previously formed helical structure. When this happens, MuB generates larger ring structures and dissociates from the DNA.

Synthesis and computational studies of new pyridine, pyrazole, pyran, and pyranopyrimidine-based derivatives of potential antimicrobial activity as DNA gyrase and topoisomerase IV inhibitors

In this study, new derivatives based on pyridine, pyrazole, pyran, and pyranopyrimidine scaffolds 5–14 were designed and synthesized. The newly synthesized compounds were characterized using microanalytical and spectral data. Using amoxicillin and amphotericin B, two common antibacterial and antifungal medications, respectively, the antimicrobial activity of the new compounds was assessed against a variety of strains of both Gram-positive and Gram-negative bacteria as well as fungal infections. The most promising activity was achieved by pyridine-based derivatives 5, 7a, and 7b, which exhibited MIC values ranging from 0.95 to 18.04 µM against the examined bacterial strains and ranging from 4.65 to 33.16 µM against the examined fungal strains. The later derivatives were also evaluated as E. coli DNA gyrase B/topoisomerase IV inhibitors in comparison with novobiocin as a standard drug. Although the assessed candidates 5, 7a, and 7b exhibited a promising suppression impact against E. coli DNA gyrase B (IC50 = 3.11 ± 0.14, 3.85 ± 0.20, and 4.05 ± 0.10, respectively, IC50 novobiocin= 3.64 ± 0.10 µM), they showed detectable less suppression impact against Topoisomerase IV than that of the reference drug with IC50 values of 19.72 ± 1.00, 79.33 ± 0.25, 85.14 ± 0.10, and 13.42 ± 0.05 µM, respectively. Additionally, the safety profiles of 5, 7a, and 7b against WI38 cells were significantly superior to that of novobiocin (IC50 = 138 ± 0.33, 170 ± 0.40, 163.3 ± 0.17, and 86.2 ± 0.03 µM, respectively).Lastly, molecular docking was used to examine the most active derivatives 5, 7a, and 7b's binding capacity to the target DNA gyrase B/topoisomerase IV. The molecules exhibited various H-interactions with the amino acid residues Asp73, Arg76, and Val71 in E. coli DNA gyrase, where compound 5 exhibited H-bonding with the Asp1069 side chain of E. coli Topoisomerase IV. Additional in silico ADMET studies were conducted to represent their oral bioavailability and drug-likeness characteristics.

CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation.

RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ~20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes. We show here that Cas12a initiates DNA target recognition by bending DNA to induce transient nucleotide flipping that exposes nucleobases for DNA-RNA hybridization. Cryo-EM structural analysis of a trapped Cas12a-RNA-DNA surveillance complex and fluorescence-based conformational probing show that Cas12a-induced DNA helix destabilization enables target discovery and engagement. This mechanism of initial DNA interrogation resembles that of CRISPR-Cas9 despite distinct evolutionary origins and different RNA-DNA hybridization directionality of these enzyme families. Our findings support a model in which RNA-mediated DNA engineering begins with local helix distortion by transient CRISPR-Cas protein binding.

Load of the ash dieback pathogen hymenoscyphus fraxineus differs in soil

AbstractThe ascomycete Hymenoscyphus fraxineus causes the devastating ash dieback disease of European ash (Fraxinus excelsior L.). Spore traps are often used to measure the amount of ascospores in the environment, but the pathogen-load of the soil in ash stands has not been recorded so far. This is of particular interest with regard to the occurrence of ash stem necrosis, a decisive factor for the severe course of the disease. In order to gain a more differentiated insight into the pathogen-load in ash stands, we analysed soil samples from four ash tree sites in southern Germany, covering a clone plantation, two seed orchards and a forest. The pathogen-load was determined using a quantitative TaqMan real-time PCR assay for ten to twenty plots per stand. Results obtained by the species-specific assay highlighted that the pathogen-load is heterogeneously distributed in the ash stands. H. fraxineus DNA targets were detected in 17% of the soil samples. The pathogen-load differed according to soil depth, with the highest pathogen abundance in the top 5 cm, followed by 5–10 cm and finally 10–15 cm. Pathogen-load and thereby infection pressure were found to be highly variable for the individual trees in one stand. Overall, the study discovered detectable levels of H. fraxineus in the soil of all four study sites, which supports the hypothesis that H. fraxineus can be found in the soil of ash stands. The qPCR approach was found to be an effective method for monitoring the load of H. fraxineus in soil and for demonstrating the successful application of the method on the sample type of custom-made spore traps. Results suggest the implication of site-specific pathogen-load determination in future H. fraxineus-monitoring and selection of less susceptible ash trees for breeding and seed production.

One-Pot Detection of Proteins Using a Two-Way Extension-Based Assay with Cas12a.

Protein biomarkers are an important class of biomarkers in disease diagnosis and are traditionally detected by enzyme-linked immunosorbent assay and mass spectrometry, which involve multiple steps and a complex workflow. In recent years, many CRISPR-Cas12a-based methods for protein detection have been developed; however, most of them have not overcome the workflow complications observed in traditional assays, limiting their applicability in point-of-care testing. In this work, we designed a single-step, one-pot, and proximity-based isothermal immunoassay integrating CRISPR Cas12a for homogeneous protein target detection with a simplified workflow and high sensitivity. Probes consisting of different binders (small molecule, aptamer, and antibody) conjugated with oligonucleotides undergo two-way extension upon binding to the protein targets, leading to downstream DNA amplification by a pair of nicking enzymes and polymerases to generate target sequences for Cas12a signal generation. We used the streptavidin-biotin model to demonstrate the design of our assay and proved that all three elements of protein detection (target protein binding, DNA amplification, and Cas12a signal generation) could coexist in one pot and proceed isothermally in a single buffer system at a low reaction volume of 10 μL. The plug-and-play applicability of our assay has been successfully demonstrated using four different protein targets, streptavidin, PDGF-BB, antidigoxigenin antibody, and IFNγ, with the limit of detection ranging from fM to pM.

TIN2 modulates FOXO1 mitochondrial shuttling to enhance oxidative stress-induced apoptosis in retinal pigment epithelium under hyperglycemia.

Progressive dysfunction of the retinal pigment epithelium (RPE) and the adjacent photoreceptor cells in the outer retina plays a pivotal role in the pathogenesis of diabetic retinopathy (DR). Here, we observed a marked increase in oxidative stress-induced apoptosis in parallel with higher expression of telomeric protein TIN2 in RPE cells under hyperglycemia in vivo and in vitro. Delving deeper, we confirm that high glucose-induced elevation of mitochondria-localized TIN2 compromises mitochondrial activity and weakens the intrinsic antioxidant defense, thereby leading to the activation of mitochondria-dependent apoptotic pathways. Mechanistically, mitochondrial TIN2 promotes the phosphorylation of FOXO1 and its relocation to the mitochondria. Such translocation of transcription factor FOXO1 not only promotes its binding to the D-loop region of mitochondrial DNA-resulting in the inhibition of mitochondrial respiration-but also hampers its availability to nuclear target DNA, thereby undermining the intrinsic antioxidant defense. Moreover, TIN2 knockdown effectively mitigates oxidative-induced apoptosis in diabetic mouse RPE by preserving mitochondrial homeostasis, which concurrently prevents secondary photoreceptor damage. Our study proposes the potential of TIN2 as a promising molecular target for therapeutic interventions for diabetic retinopathy, which emphasizes the potential significance of telomeric proteins in the regulation of metabolism and mitochondrial function. Created with BioRender ( https://www.biorender.com/ ).

Native CRISPR-Cas-based programmable multiplex gene repression in Klebsiella variicola.

Klebsiella variicola is a Gram-negative bacterium that is frequently isolated from a wide variety of natural niches. It is a ubiquitous opportunistic pathogen that can cause diverse infections in plants, animals, and humans. It also has significant biotechnological potential. However, due to the lack of efficient genetic tools, the molecular basis contributing to the pathogenesis and beneficial activities of K. variicola remains poorly understood. In this study, we found and characterized a native type I-E CRISPR-Cas system in a recently isolated K. variicola strain KV-1. The system cannot cleave target DNA sequences due to the inactivation of the Cas3 nuclease by a transposable element but retains the activity of the crRNA-guided Cascade binding to the target DNA sequence. A targeting plasmid carrying a mini-CRISPR to encode a crRNA was designed and introduced into the KV-1 strain, which successfully repurposed the native type I-E CRISPR-Cas system to inhibit the expression of the target gene efficiently and specifically. Moreover, by creating a mini-CRISPR to encode multiple crRNAs, multiplex gene repression was achieved by providing a single targeting plasmid. This work provides the first native CRISPR-Cas-based tool for programmable multiplex gene repression in K. variicola, which will facilitate studying the pathogenic mechanism of K. variicola and enable metabolic engineering to produce valuable bioproducts.

ReadCurrent: a VDCNN-based tool for fast and accurate nanopore selective sequencing.

Nanopore selective sequencing allows the targeted sequencing of DNA of interest using computational approaches rather than experimental methods such as targeted multiplex polymerase chain reaction or hybridization capture. Compared to sequence-alignment strategies, deep learning (DL) models for classifying target and nontarget DNA provide large speed advantages. However, the relatively low accuracy of these DL-based tools hinders their application in nanopore selective sequencing. Here, we present a DL-based tool named ReadCurrent for nanopore selective sequencing, which takes electric currents as inputs. ReadCurrent employs a modified very deep convolutional neural network (VDCNN) architecture, enabling significantly lower computational costs for training and quicker inference compared to conventional VDCNN. We evaluated the performance of ReadCurrent across 10 nanopore sequencing datasets spanning human, yeasts, bacteria, and viruses. We observed that ReadCurrent achieved a mean accuracy of 98.57% for classification, outperforming four other DL-based selective sequencing methods. In experimental validation that selectively sequenced microbial DNA from human DNA, ReadCurrent achieved an enrichment ratio of 2.85, which was higher than the 2.7 ratio achieved by MinKNOW using the sequence-alignment strategy. In summary, ReadCurrent can rapidly classify target and nontarget DNA with high accuracy, providing an alternative in the toolbox for nanopore selective sequencing. ReadCurrent is available at https://github.com/Ming-Ni-Group/ReadCurrent.

Empowering the on-site detection of nucleic acids by integrating CRISPR and digital signal processing

Addressing the global disparity in cancer care necessitates the development of rapid and affordable nucleic acid (NA) testing technologies. This need is particularly critical for cervical cancer, where molecular detection of human papillomavirus (HPV) has emerged as an accurate screening method. However, implementing this transition in low- and middle-income countries has been challenging due to the high costs and centralized facilities required for current NA tests. Here, we present CreDiT (CRISPR Enhanced Digital Testing) for on-site NA detection. The CreDiT platform integrates i) a one-pot CRISPR strategy that simultaneously amplifies both target NAs and analytical signals and ii) a robust fluorescent detection based on digital communication (encoding/decoding) technology. These features enable a rapid assay (<35 minutes) in a single streamlined workflow. We demonstrate the sensitive detection of cell-derived HPV DNA targets down to single copies and accurate identification of HPV types in clinical cervical brushing specimens (n = 121).

Mechanisms for HNH-mediated target DNA cleavage in type I CRISPR-Cas systems

The metagenome-derived type I-E and type I-F variant CRISPR-associated complex for antiviral defense (Cascade) complexes, fused with HNH domains, precisely cleave target DNA, representing recently identified genome editing tools. However, the underlying working mechanisms remain unknown. Here, structures of type I-FHNH and I-EHNH Cascade complexes at different states are reported. In type I-FHNH Cascade, Cas8fHNH loosely attaches to Cascade head and is adjacent to the 5' end of the target single-stranded DNA (ssDNA). Formation of the full R-loop drives the Cascade head to move outward, allowing Cas8fHNH to detach and rotate ∼150° to accommodate target ssDNA for cleavage. In type I-EHNH Cascade, Cas5eHNH domain is adjacent to the 5' end of the target ssDNA. Full crRNA-target pairing drives the lift of the Cascade head, widening the substrate channel for target ssDNA entrance. Altogether, these analyses into both complexes revealed that crRNA-guided positioning of target DNA and target DNA-induced HNH unlocking are two key factors for their site-specific cleavage of target DNA.

Transcription factor binding specificities of the oomycete Phytophthora infestans reflect conserved and divergent evolutionary patterns and predict function

BackgroundIdentifying the DNA-binding specificities of transcription factors (TF) is central to understanding gene networks that regulate growth and development. Such knowledge is lacking in oomycetes, a microbial eukaryotic lineage within the stramenopile group. Oomycetes include many important plant and animal pathogens such as the potato and tomato blight agent Phytophthora infestans, which is a tractable model for studying life-stage differentiation within the group.ResultsMining of the P. infestans genome identified 197 genes encoding proteins belonging to 22 TF families. Their chromosomal distribution was consistent with family expansions through unequal crossing-over, which were likely ancient since each family had similar sizes in most oomycetes. Most TFs exhibited dynamic changes in RNA levels through the P. infestans life cycle. The DNA-binding preferences of 123 proteins were assayed using protein-binding oligonucleotide microarrays, which succeeded with 73 proteins from 14 families. Binding sites predicted for representatives of the families were validated by electrophoretic mobility shift or chromatin immunoprecipitation assays. Consistent with the substantial evolutionary distance of oomycetes from traditional model organisms, only a subset of the DNA-binding preferences resembled those of human or plant orthologs. Phylogenetic analyses of the TF families within P. infestans often discriminated clades with canonical and novel DNA targets. Paralogs with similar binding preferences frequently had distinct patterns of expression suggestive of functional divergence. TFs were predicted to either drive life stage-specific expression or serve as general activators based on the representation of their binding sites within total or developmentally-regulated promoters. This projection was confirmed for one TF using synthetic and mutated promoters fused to reporter genes in vivo.ConclusionsWe established a large dataset of binding specificities for P. infestans TFs, representing the first in the stramenopile group. This resource provides a basis for understanding transcriptional regulation by linking TFs with their targets, which should help delineate the molecular components of processes such as sporulation and host infection. Our work also yielded insight into TF evolution during the eukaryotic radiation, revealing both functional conservation as well as diversification across kingdoms.

Structural analysis of the BisI family of modification dependent restriction endonucleases.

The BisI family of restriction endonucleases is unique in requiring multiple methylated or hydroxymethylated cytosine residues within a short recognition sequence (GCNGC), and in cleaving directly within this sequence, rather than at a distance. Here, we report that the number of modified cytosines that are required for cleavage can be tuned by the salt concentration. We present crystal structures of two members of the BisI family, NhoI and Eco15I_Ntd (N-terminal domain of Eco15I), in the absence of DNA and in specific complexes with tetra-methylated GCNGC target DNA. The structures show that NhoI and Eco15I_Ntd sense modified cytosine bases in the context of double-stranded DNA (dsDNA) without base flipping. In the co-crystal structures of NhoI and Eco15I_Ntd with DNA, the internal methyl groups (G5mCNGC) interact with the side chains of an (H/R)(V/I/T/M) di-amino acid motif near the C-terminus of the distal enzyme subunit and arginine residue from the proximal subunit. The external methyl groups (GCNG5mC) interact with the proximal enzyme subunit, mostly through main chain contacts. Surface plasmon resonance analysis for Eco15I_Ntd shows that the internal and external methyl binding pockets contribute about equally to sensing of cytosine methyl groups.

Pyrimidine-Dependent UV-Mediated Cross-Linking Magnifies Minor Genetic or Epigenetic Changes in Clinical Samples.

Detection of minor DNA allele alterations is becoming increasingly important for early detection and monitoring of cancer. We describe a new method that uses ultraviolet light to eliminate wild-type DNA alleles and enables improved detection of minor genetic or epigenetic changes. Pyrimidine-dependent UV-based minor-allele enrichment (PD-UVME) employed oligonucleotide probes that incorporated a UVA-sensitive 3-cyanovinylcarbazole (CNVK), placed directly opposite interrogated pyrimidines, such as thymine (T) or cytosine (C) in wild-type (WT) DNA. Upon UVA-illumination, CNVK cross-linked with T/C, preventing subsequent amplification. Mutations that removed the T/C escaped cross-linking and were amplified and detected. Similarly, CNVK discriminated between methylated and unmethylated cytosine in CpG dinucleotides, enabling direct enrichment of unmethylated DNA targets. PD-UVME was combined with digital droplet PCR (ddPCR) to detect serine/threonine-protein kinase B-Raf (BRAF) V600E mutations in model systems, thyroid patient cancer tissue samples, and circulating DNA of tumor origin (ctDNA) from melanoma patients. One thyroid cancer sample out of 9, and 6 circulating-DNA samples out of 7 were found to be BRAF V600E-positive via PD-UVME while classified as negative by conventional ddPCR. Positive samples via conventional ddPCR were also found positive via PD-UVME. All 10 circulating cell-free DNA (cfDNA) samples obtained from normal volunteers were negative via both approaches. Furthermore, preferential enrichment of unmethylated alleles in MAGEA1 promoters using PD-UVME was demonstrated. PD-UVME mutation/methylation enrichment performed prior to ddPCR magnifies low-level mutations or epigenetic changes and increases sensitivity and confidence in the results. It can assist with clinical decisions that hinge on the presence of trace alterations like BRAF V600E.

Predicting DNA damage response in non-small cell lung cancer organoids via simultaneous label-free autofluorescence multiharmonic microscopy

The DNA damage response (DDR) is a fundamental readout for evaluating efficacy of cancer therapeutics, many of which target DNA associated processes. Current techniques to evaluate DDR rely on immunostaining for phosphorylated histone H2AX (γH2AX), which is an indicator of DNA double-strand breaks. While γH2AX immunostaining can provide a snapshot of DDR in fixed cell and tissue samples, this method is technically cumbersome due to temporal monitoring of DDR requiring timepoint replicates, extensive assay development efforts for 3D cell culture samples such as organoids, and time-consuming protocols for γH2AX immunostaining and its evaluation. The goal of this current study is to reduce overall burden on assay duration and development in non-small cell lung cancer (NSCLC) organoids by leveraging label-free multiphoton imaging. In this study, simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy was used to provide rich intracellular information based on endogenous contrasts. SLAM microscopy enables imaging of live samples eliminating the need to generate sacrificial sample replicates and has improved image acquisition in 3D space over conventional confocal microscopy. Predictive modeling between label-free SLAM microscopy and γH2AX immunostained images confirmed strong correlation between SLAM image features and γH2AX signal. Across multiple DNA targeting chemotherapeutics and multiple patient-derived NSCLC organoid lines, the optical redox ratio and third harmonic generation channels were used to robustly predict DDR. Imaging via SLAM microscopy can be used to more rapidly predict DDR in live 3D NSCLC organoids with minimal sample handling and without labeling.

Targeting DNA junction sites by bis-intercalators induces topological changes with potent antitumor effects.

Targeting inter-duplex junctions in catenated DNA with bidirectional bis-intercalators is a potential strategy for enhancing anticancer effects. In this study, we used d(CGTATACG)2, which forms a tetraplex base-pair junction that resembles the DNA-DNA contact structure, as a model target for two alkyl-linked diaminoacridine bis-intercalators, DA4 and DA5. Cross-linking of the junction site by the bis-intercalators induced substantial structural changes in the DNA, transforming it from a B-form helical end-to-end junction to an over-wounded side-by-side inter-duplex conformation with A-DNA characteristics and curvature. These structural perturbations facilitated the angled intercalation of DA4 and DA5 with propeller geometry into two adjacent duplexes. The addition of a single carbon to the DA5 linker caused a bend that aligned its chromophores with CpG sites, enabling continuous stacking and specific water-mediated interactions at the inter-duplex contacts. Furthermore, we have shown that the different topological changes induced by DA4 and DA5 lead to the inhibition of topoisomerase 2 activities, which may account for their antitumor effects. Thus, this study lays the foundations for bis-intercalators targeting biologically relevant DNA-DNA contact structures for anticancer drug development.

Synthesis, Characterization, DNA Binding, Biological Significance, and Molecular Docking Approaches of a Palladium(II) Complex with Ciprofloxacin for More Efficient Therapy.

To evaluate the biotransformation and the mechanism of binding as well as the biological impact of metal-based- drugs involving Pd(II), known to have high potency and low toxicity for use as anticancer therapeutics, in the present study, a newly synthesized palladium (II) complex, [Pd(CPF)(OH2)2]2+ (where CPF is ciprofloxacin), has been synthesized and characterized and thoroughly evaluated for its antimicrobial properties. The interaction of the diaqua complex with CT-DNA and BSA was studied through various techniques, including UV-vis spectroscopy, thermal denaturation, viscometry, gel electrophoresis, ethanol precipitation, and molecular docking studies. The results indicate that the complex exhibits a robust binding interaction with CT-DNA, possibly via minor groove binding and (or) electrostatic interactions. Furthermore, the complex displays good binding affinity towards BSA, indicating its potential as a target for DNA and BSA in biological media. The invitro cytotoxicity assay reveals that this complex can be classified as a promising cell growth inhibitor against MCF-7, HT-29, and A549. Thus, this newly synthesized palladium (II) complex is a promising candidate for further exploration as a potential anticancer therapeutic.