DNA Molecules Research Articles

The effects of cell displacement on DNA damages in targeted radiation therapy using Geant4-DNA

Charged particle radiation can, directly and indirectly, affect cells by breaking DNA strands. This effect includes DNA single-strand breaks (SSB) and DNA double-strand breaks (DSB), which may cause cell death and mitotic failure. Thus, using short-range charged particles such as Auger electrons (AEs) not only leads to the destruction of the target cell but also prevents the nearby healthy cells from exposing to ionizing radiation. In this study, two spherical cells (C and C2) and their cell nucleus, both made of liquid water, were modeled. An atomic DNA model constructed in the Geant4-DNA Monte Carlo (MC) simulation toolkit was placed inside the nucleus of the C and C2 cells. The number of direct and indirect SSB, DSB, and hybrid DSB (HDSB), caused by some of the most widely-used Auger electron-emitting (AEE) radionuclides, including 99mTc, 111In, 123I, 125I, and 201Tl, distributed within different compartments of the C cell, was calculated in the C and C2 cells, considering the distance between the surface of the two cells ranges from 0 to 5 μm. The present work aimed to investigate the biological effects of AEE radionuclides and their potential for cancer treatment through targeted radiation therapy. The results indicate the impact of 201Tl > 125I > 123I > 111In > 99mTc on DNA damage when the target is C (first spherical cell). On the other hand, for C2 at distances of 0 to 5 μm, the impact of 99mTc > 123I > 111In > 201Tl > 125I on DNA damage is observed.

Optimizing Tris(2-Carboxyethyl)phosphine and Mercaptohexanol Concentrations for Thiolated Oligonucleotide Immobilization on Platinum Electrodes in Microfluidic Platforms.

In this study, we propose a strategy to explore the impact of the proportion of tris(2-carboxyethyl)phosphine (TCEP) and 6-mercaptohexanol (MCH) on the efficiency of oligonucleotide functionalization on PDMS microfluidic channels equipped with pairs of homemade microfabricated platinum microelectrodes. We identified an optimal concentration of these compounds that enables the effective orientation and distribution of probes, thereby facilitating subsequent target hybridization. The experiment included optimizing sample injection into microfluidic channels. We used TCEP as a reducing agent to help the DNA probes adhere to the channel electrode better. This stopped the formation of disulfide bonds during the probe immobilization step. We found the optimal TCEP/MCH mixture ratio (5 mM TCEP and 50 mM MCH), which led to a more uniform distribution and orientation of the DNA probes on the platinum electrode. These optimized conditions resulted in a more compact DNA monolayer and enhanced detection capabilities. The biosensor's performance was evaluated by the detection of the hybridization of complementary DNA sequences in the presence of equimolar Fe(CN)63-/Fe(CN)64-. The detection of the synthetic GP8 resistance gene is facilitated by a measurable decrease in the electron transfer rate, which is directly proportional to its concentration. Under the optimized conditions, the DNA biosensor showed excellent sensitivity (with a detection limit of 10-17 M) and high specificity when tested against noncomplementary DNA strands.

Directionally co-immobilizing glucose oxidase and horseradish peroxidase on three-pronged DNA scaffold and the regulation of cascade activity

In traditional multienzyme random co-immobilization, it is difficult to precisely locate and regulate the relative positions between two enzyme molecules, resulting in low cascade efficiency between the two enzymes and limiting the application of multienzyme cascade catalysis technology. This study prepared PVAC@Y-dsDNA@GOD/HRP magnetic co-immobilized multienzyme by constructing a three-pronged DNA scaffold for co-coupling glucose oxidase (GOD) and horseradish peroxidase (HRP), which achieved directional co-immobilization of dual enzymes and precise regulation of inter-enzyme distance. Compared with traditional random co-immobilization of multienzyme, PVAC@Y-dsDNA@GOD/HRP could shorten the distance between GOD and HRP to the nanoscale and form substrate channeling, which greatly improved the cascade activity between the two enzymes. The inter-enzyme spacing between GOD and HRP could be precisely regulated by changing the length of DNA strands. When the inter-enzyme spacing was 10.08 nm, PVAC@Y-dsDNA@GOD/HRP exhibited high cascade activity of 707 U/mg. The inter-enzyme spacing that was too large or too small would reduce the cascade activity, indicating a distance-dependence of multienzyme cascade activity. PVAC@Y-dsDNA@GOD/HRP showed good reusability, indicating that the three-pronged DNA scaffold constructed by DNA double strands hybridization could firmly immobilize enzyme on carrier, with less enzyme leakage.

Dynamics of metal/metalloid bioaccumulation and sensitivity in post-larvae shrimp (Macrobrachium rosenbergii) exposed to settleable atmospheric particulate matter from an industrial source

The metallurgy industry is a potent global source of particulate matter (PM) atmospheric emissions. A portion of this PM may settle in aquatic (SePM) carrying metal/metalloid particles and metallic nanoparticles. Surprisingly, this form of contamination has not received due attention from most environmental monitoring agencies. We analyzed the effect of exposure to SePM on shrimp post-larvae, a critical stage for the viability of shrimp populations and for the trophic chain. After acclimation, shrimp were exposed to contaminants using a randomized experimental design—a 4 × 4 factorial arrangement with 2 factors: exposure time (24, 48, 72, and 96 h) and SePM concentration (0.00, 0.01, 0.10, and 1.00 g L−1). The bioaccumulation of metals, contamination rates, mortality, and ROS-related biomarkers (lipid peroxidation – LPO; DNA strand breakage DNA SB and metallothionein content – MTs;) were evaluated. After contamination, the water contained 27 different metals/metalloids. Post-larvae accumulated metals, such as Cd, Pb, Al, As, Se, Sr, Zr, Ba, La, Ce, W, and Hg. However, the rise in SePM did not result in a proportional bioaccumulation rise, indicating that effective biological barriers may work for some metals. Although the different levels of SePM changed mortality dynamics, they resulted in a similar final lethality (60–80 %). SePM caused significant damage to lipids (increased LPO), genetic material (DNA SB), and increased Mts. Such effects may reflect a particularly deleterious ecological problem as it is present at such an early stage of life. These results identified a clear environmental risk since the lower level of exposure used was 102 times lower than that measured in the habitats affected by local industry. Consequently, our results emphasize the need for clear protocols for monitoring the effects of SePM in aquatic environments.

AuNPs-based lateral flow strip genotoxicity biosensor by sensitive quantification of 8-oxodGuo

Development of rapid and sensitive methods for screening of large number of chemical pollutants with potential genotoxicity is in urgent demands. In this work, we reported a chemical genotoxicity sensor based on the lateral flow strip (LFS) quantification of the 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo). The proposed sensing strategy introduced human 8-oxoguanine DNA glycosylase (hOGG 1) and human apyrimidinic endonuclease (APE 1) to convert 8-oxodGuo to strand break. Then, the DNA conjugated gold nanoparticles (AuNPs) complexes could not be captured on test line (T-line), showing visible red color fading. Additionally, the mean grey value of the optical density in the T-line zone was analyzed by using a mobile phone camera and Image J software to quantify 8-oxodGuo. Under the optimized experimental conditions, the LFS DNA biosensor could detect the lowest concentration of 8-oxodGuo down to 0.2 pmol. To demonstrate its universal applications, in vitro generation of 8-oxodGuo by Rose Bengal under irradiation and Fe2+-mediated Fenton reagent were successfully measured. The detection limit of oxidative DNA damage induced by Rose Bengal or Fenton reagent could be down to 0.01 μM or 1 nM Fe2+/4 nM H2O2, respectively. The 10-fold higher sensitivity for Fenton reagent was attributed to much more severe damage than Rose Bengal, producing both DNA strand break and nucleobases conversion. The results demonstrated that the LFS DNA biosensor could detect oxidative DNA damage for the first time, showing its potential application for assessment of the genotoxicity of abundant chemical pollutants.

RSF1 orchestrates p53 transcriptional activity by coordinating p300 acetyltransferase and FACT complex.

The transcriptional regulation of p53-dependent genes in response to DNA damage is critical for effective DNA repair and cell survival. We previously established that RSF1 (remodeling and spacing factor 1) is necessary for p53-dependent gene transcription in response to DNA strand breaks. Here, we further elucidate that the role of RSF1 in p53 regulation by demonstrating that its depletion results in a reduction in the acetylated-Lys(K)382 level of p53, which governs its transcriptional activity. RSF1 was co-precipitated with p300 acetyltransferase upon etoposide treatment. Chromatin immunoprecipitation assays on the upstream region of CDKN1A gene revealed reduced p300 and TBP accumulation, which were accompanied with low H3H27ac and H3K4me1 levels in RSF1 knockout cells. Moreover, RSF1 depletion led to a reduced accumulation of SSRP1 and SPT16, subunits of FACT complex at the promoter of CDKN1A gene. These findings suggest that RSF1 promotes p53-dependent p21 gene transcription by facilitating the accumulation of p300 acetyltransferase at the enhancer and FACT at the promoter region of CDKN1A gene, respectively.

A Comprehensive Review of Pfu DNA Polymerase: Extraction, Production and Applications

Pfu DNA Polymerase, a thermostable enzyme derived from the hyperthermophilic archaeon Pyrococcus furiosus, is widely recognized for its high fidelity and strong processivity. Its 3'-5' exonuclease activity makes it indispensable for the correct amplification of both short and complex DNA strands. These biochemical properties of Pfu DNA Polymerase have encouraged considerable advancements in its extraction and production methods. This review covers some of the traditional approaches for purification, including protein purification and affinity chromatography, and updates on recent advances in recombinant gene expression, automated systems of production, and membrane-based technologies. New ways of engineering enzymes have recently been developed, such as CRISPR-Cas9-mediated gene optimization, which raised the bar on extraction efficiency to meet emerging demand. Once challenging, Pfu DNA Polymerase production has been significantly streamlined through recombinant expression in E. coli both at a laboratory and commercial scale. The optimization techniques that are involved with IPTG concentration and Response Surface Methodology have increased the yield by up to 30%. Auto-induction means even higher biomass outputs are allowed. Today, applications of Pfu DNA Polymerase span from standard PCR up to advanced clinical diagnostics in the fields of molecular biology, forensic analysis, clinical microbiology, and biotechnology.

Veratric Acid as a Potential Antidiabetic Agent: Molecular Docking and In vivo Enzyme Inhibition Studies with Insights from STz-NA Induced Diabetic Rat Model

Streptozotocin-nicotinamide (STz-NA) has proven to be an effective agent in inducing type 2 diabetes (T2DM) in experimental models due to its genotoxic impact on pancreatic β-cells. STz reduces nicotinamide adenine dinucleotide (NAD+) through the glucose transporter GLUT2, leading to cellular damage, DNA strand breaks, and cell death. Nicotinamide (NA), a biochemical precursor of NAD+ and poly-ADP-ribose-polymerase-1 (PARP-1) inhibitor, plays a crucial protective role by modulating NAD+ levels, which are essential in ATP production and other metabolic processes. Excessive DNA damage, however, triggers PARP-1 over-activation, depleting cellular reserves and resulting in cell necrosis. This study further investigates the antidiabetic and hepatoprotective effects of Veratric Acid (VA) by comparing it to Glibenclamide. Utilizing Computer-Aided Drug Design (CADD), VA was identified as a structurally similar compound with potential antidiabetic properties. Molecular docking studies targeting proteins, such as GLUT-1 (PDB ID: 4PYP) and 1BSI, along with in vivo assays in STz-NA-induced diabetic Wistar rats, were conducted. VA demonstrated significant binding affinity and molecular interactions comparable to standard antidiabetic agents, showing inhibitory effects on liver enzymes SGOT and SGPT, and supporting its potential role in diabetes management. Docking and histopathology results revealed that VA effectively reduced blood glucose, improved insulin levels, and enhanced liver function in diabetic rats. Additionally, VA promoted insulin secretion from pancreatic β-cells and upregulated insulin-related markers, with minimal hepatotoxicity compared to Glibenclamide. VA treatment significantly improved enzymatic and non-enzymatic antioxidant levels, lowered lipid peroxidation, and improved lipid profiles, which were confirmed by histopathological liver observations. Veratric Acid at low doses effectively modulates blood glucose and diabetes-related biochemical parameters, highlighting its promise as a safer, natural therapeutic alternative for diabetes treatment.

Direct observation of subunit rotation during DNA strand exchange by serine recombinases

Serine recombinases are proposed to catalyse site-specific recombination by a unique mechanism called subunit rotation. Cutting and rejoining DNA occurs within an intermediate synaptic complex comprising a recombinase tetramer bound to two DNA sites. After double-strand cleavage at both sites, one half of the complex rotates 180° relative to the other, before re-ligation of the DNA ends. We used single-molecule FRET (smFRET) methods to provide compelling direct physical evidence for subunit rotation by recombinases Tn3 resolvase and Sin. Synaptic complexes containing fluorescently labelled DNA show FRET fluctuations consistent with the subunit rotation model. FRET changes were associated with the rotation steps, on a timescale of 0.4–1.1 s−1\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${{\\mbox{s}}}^{-1}$$\\end{document}, as well as opening and closing of the gap between the scissile phosphates during cleavage and ligation. Multiple rounds of recombination were observed within the ~25 s observation period, including frequent consecutive rotation events in the cleaved-DNA state without evidence of intermediate ligation.

Theory of epigenetic switching due to stochastic histone mark loss during DNA replication

How much information does a cell inherit from its ancestors beyond its genetic sequence? What are the epigenetic mechanisms that allow this? Despite the rise in available epigenetic data, how such information is inherited through the cell cycle is still not fully understood. Often, epigenetic marks can display bistable behaviour and their bistable state is transmitted to daughter cells through the cell cycle, providing the cell with a form of memory. However, loss-of-memory events also take place, where a daughter cell switches epigenetic state (with respect to the mother cell). Here, we develop a framework to compute these epigenetic switching rates, for the case when they are driven by DNA replication, i.e. the frequency of loss-of-memory events due to replication. We consider the dynamics of histone modifications during the cell cycle deterministically, except at DNA replication, where nucleosomes are randomly distributed between the two daughter DNA strands, which is therefore implemented stochastically. This hybrid stochastic-deterministic approach enables an analytic derivation of the replication-driven switching rate. While retaining great simplicity, this framework can explain experimental switching rate data, establishing its biological importance as a framework to quantitatively study epigenetic inheritance.

Manipulation and analysis of large DNA molecules by controlling their dynamics using micro- and nano-gaps.

Manipulation and analysis methods for large DNAs are critical for epidemiological, clinical, diagnostic, and fundamental research on bacteria, membrane vesicles, plants, yeast, and human cells. However, the physical properties of large DNAs often challenge their manipulation and analysis with high accuracy and speed using the conventional methods such as gel electrophoresis and column-based methods. This review presents the approaches that leverage micrometer- and nanometer-sized gaps within microchannels to control the dynamics and conformations of large DNAs, thereby overcoming these challenges. By designing gap structures and migration conditions based on the relationship between gap parameters and the physical characteristics of large DNAs-such as diameter and persistence length-these methods enable swifter and more precise manipulation and analysis of large DNAs, including size separation, concentration, purification, and single-molecule analysis.

Real-Space Spectral Determination of Short Single-Stranded DNA Sequence Structures.

Resolving the sequence and structure of flexible biomolecules such as DNA is crucial to understanding their biological mechanisms and functions. Traditional structural biology methods remain challenging for the analysis of small and disordered biomolecules, especially those that are difficult to label or crystallize. Recent development of single-molecule tip-enhanced Raman spectroscopy (TERS) offers a label-free approach to identifying nucleobases in a single DNA chain. However, a clear demonstration of sequencing both spatially and spectrally at single-base resolution is still elusive due to the challenges caused by weak Raman signals and the flexibility of DNA molecules. Here, we report a proof-of-principle demonstration to this end, spectrally resolving in real space individual nucleobases and their sequence structures within a short, single-stranded DNA molecule artificially designed. This breakthrough is achieved through the development of subnanometer-resolved low-temperature TERS methodology for such thermally unstable flexible biomolecules. Further TERS mapping over individual nucleobases provides additional structural information about the molecular configurations and even the locations of functional groups, offering a way to track modification types and binding sites in biomolecules.

Proton-Mediated Dynamic Nestling of DNA Payloads Within Size-Matched MOFs Nanochannels for Smart Intracellular Delivery.

With sequence-programmable biological functions and excellent biocompatibility, synthetic functional DNA holds great promise for various biological applications. However, it remains a challenge to simultaneously retain their biological functions while protecting these fragile oligonucleotides from the degradation by nucleases abundant in biological circumstances. Herein, a smart delivery system for functional DNA payloads is developed based on proton-mediated dynamic nestling of cytosine-rich DNA moieties within the precisely size-matched nanochannels of highly crystalline metal-organic frameworks (MOFs): At neutral pH, cytosine-rich DNA strands exhibit a flexible single-stranded state and can be accommodated by MOFs nanochannels with a size of ca. 2.0nm; while at acidic conditions, the protonation of cytosine-rich strands weakens their interaction with the nanochannels, and the tendency to form four-strandedstructures drives these DNA strands out of the nanochannels. Results confirm the successful protection of DNA payloads from enzymatic hydrolysis by the MOFs nanochannels, and the delicate coupling of the endocytosis processes and the proton-responsive Cytosine-rich DNA/MOFs systems realized the efficient intracellular delivery of DNA payloads. Furthermore, with a complementary sequence to the telomere overhangs, direct imaging of telomeres and the nucleus is successfully achieved with the proton-mediated DNA/MOFs system.

Binding interaction of food preservatives with glycosylated DNA: In vitro assessment and computational studies

This work aims to evaluate the interaction between sodium benzoate (SB), potassium sorbate (PS), and sodium dihydrogen citrate (CIT) as food preservatives, individually, as well as in combinations of two and three, with DNA and glycosylation DNA to determine the allowable amount of food additive consumption to mitigate potential harm to human health. Various techniques including UV-Vis absorption, fluorescence spectroscopy, DNA amadori product formation, and agarose gel electrophoresis, were employed to observe these interactions. Furthermore, pioneering molecular docking and molecular dynamics simulations were performed to gain deeper insights into the interactions between these preservative compounds and DNA. It is also noteworthy that there have been no documented biological or computational studies concerning citrate. The results revealed that all three food preservatives could interact with DNA, leading to conformational changes as indicated by increased absorption and fluorescence intensity. Both SB and PS were found to damage the DNA structure and promote the formation of DNA Amadori products. Moreover, all treatment groups exhibited a decrease in migration speed on the agarose gel, suggesting binding to DNA and alterations in the supercoiled form of DNA structure. Interestingly, the groups treated with citrate showed a smeared appearance, DNA strand fragmentation, and cleavage. Molecular docking complemented the in vitro achievement that SB, PS, and CIT could interact with DNA. DFT analysis showed that CIT exhibited the best thermodynamic and kinetic stability compared to other food additives.

Characterization of a Salmonella abortus equi phage 4FS1 and its depolymerase

The significant economic losses caused by S. abortus equi in donkey husbandry have increased interest in exploring the potential of phages and their enzymes as control strategies. In this study, a S. abortus equi phage, designated 4FS1, was isolated from sewage at a donkey farm. Transmission electron microscopy (TEM) revealed a typical icosahedral head and a long, non-contractile tail. It exhibited a short latent period of 20 min and a burst size of 160 plaque-forming units (PFU) per cell. It demonstrated a broad host range, infecting 36 out of 60 salmonella strains, with an optimal multiplicity of infection (MOI) of 0.01 for S. abortus equi S1. The phage titer remained stable at 109 PFU/mL between 37°C and 50°C and exceeded 108 PFU/mL at pH from 5.0 to 10.0. After 1 h of UV exposure, the titer remained at 107 PFU/mL and showed no significant variation across NaCl concentrations from 2.5 to 15%. The genome of phage 4FS1 consists of a 42,485 bp linear double-stranded DNA molecule with a G + C content of 49.07%. Of the 56 predicted open reading frames (ORFs), 32 were functional annotated, with no virulence or drug resistance genes identified. ORF36 was predicted to encode a depolymerase responsible for endorhamnosidase activity. Recombinant expression of the Dpo36 protein in prokaryotes significantly reduced biofilm formation and removal. Combined with healthy donkey serum, Dpo36 inhibited bacterial growth in vitro and enhanced the survival rates of mice infected with S. abortus equi. These findings highlight the promising potential of phages and their depolymerases as novel therapeutic agents against S. abortus equi.

Contribution of HPV Status for Neutrophil Extracellular Traps Release in Oropharyngeal Cancer.

Oropharyngeal squamous cell carcinoma (OP-SCC) represents a public health problem and human papillomavirus (HPV) is one of the risk factors. Neutrophil extracellular traps (NET) are meshes of DNA strands and granule proteins. NET has been identified in diverse cancers, whether associated with viruses or not. However, there is no information on NET in OP-SCC. We aimed to evaluate the NET release by neutrophils in the OP-SCC microenvironment, stratified by HPV status. This cross-sectional study analyzed OP-SCC biopsy specimens diagnosed from 1997 to 2021. HPV status was determined by p16 immunohistochemistry and "in situ" hybridization. Neutrophils were detected by CD66b immunohistochemistry. Immunofluorescence was used to identify NET by co-localization of myeloperoxidase (MPO) and citrullinated histone H3 (H3Cit). Bivariate statistics, Kaplan-Meier survival analysis, and the log-rank test were performed. HPV-positive and HPV-negative OP-SCC had similar CD66b + neutrophil infiltration (p > 0.05), but the release of NET was significantly increased in HPV-positive compared to HPV-negative OP-SCC samples (p < 0.05). Overall survival was not impacted by NET indexes (p > 0.05). The presence of HPV may stimulate NET release in the OP-SCC microenvironment.

Dual-site responsive module-triggered CRISPR/Cas12a switch with enhanced reaction kinetics for specific detection of PSA

Despite advances in DNA-based nanodevices for tumor biomarker detection, the pursuit of enhanced sensitivity and specificity remains a profound challenge. In this study, we designed an integrated dual-site responsive module as the AND logic gate that responded to both prostate-specific antigen (PSA) and apurinic/apyrimidinic endonuclease 1 (APE1), triggering a tetrahedral DNA nanostructure (TDN)-accelerated CRISPR/Cas12a signal switch for PSA detection. In detail, the dual-site responsive module was a T-shaped probe comprised of three DNA strands, which contained PSA aptamer and apurinic/apyrimidinic (AP) sites, significantly improving the specificity of prostate cancer screening by orthogonal recognition of PSA and APE1. When both PSA and APE1 were present, PSA could specifically bind with the aptamer, while APE1 catalyzed and cleaved the AP sites, releasing a part of one auxiliary strand as the mimic target (MT). To enhance the trans-cleavage efficiency of the CRISPR/Cas12a signal switch, we parallelly constructed the TDN with extended non-target strands (NTS) at each vertex, which could capture the MT to form double-stranded DNA (dsDNA) activators at the TDN vertices (TDN-activators) for triggering the CRISPR/Cas12a signal switch. Benefiting from the rigid framework of TDN, the TDN-activators not only increased the local concentration of Cas12a but also enabled a dominant conformation that was more favorable for recognizing and activating the Cas12a-crRNA complex, which demonstrated a 4.06-fold increase in trans-cleavage efficiency compared to that of dsDNA-activated Cas12a. This work applies the T-shaped probes and TDN-accelerated CRISPR/Cas12a signal switch to achieve the specific and sensitive electrochemiluminescence (ECL) detection of PSA from 1.0 × 10−5 to 10 μg/mL with a low limit of detection (LOD) of 4.5 × 10−6 μg/mL, offering a promising tool for early clinical screening of prostate cancer.

Synthesis of derived Cu(II) complex from Schiff base of Vitamin B6 and antihypertensive hydralazine: DNA interaction, biological application and molecular docking studies

A novel Schiff base ligand (E)-5-(hydroxymethyl)-2-methyl-4-((2-(phthalazin-1-yl)hydrazineylidene)methyl)pyridin-3-ol was synthesized through the condensation of antihypertensive hydralazine and Pyridoxal (Vitamin B6) ligands. This fabricated Schiff base (L) was a precursor for developing a unique Cu(II) complex. TheSchiff base ligand and Cu(II) complex structureswere characterized using various analytical tools, including IR, UV–Vis Spectroscopy and NMR. An octahedron geometry of this complex was proposed for the assigned molecular formula [Cu(L)2].3H2O. The molecular structures of theSchiff base ligand and its Cu(II) complex have been optimized using the B3LYP functional and the 6-311G++(d,p) basis set. Investigating the interaction of this prepared Cu(II) complex with calf thymus DNA (CT-DNA) molecules was operated using UV–Vis spectrophotometry, viscosity measurement, electrochemical studies, gel electrophoresis, and molecular docking. In vitro cytotoxicity was conducted using the MTT assay on four human tumor cell lines including hepatocellular carcinoma, estrogen-dependent breast cancer, melanoma, triple-negative breast cancer as well as healthy human skin fibroblasts.

Design principles for accurate folding of DNA origami

We describe design principles for accurate folding of three-dimensional DNA origami. To evaluate design rules, we reduced the problem of DNA strand routing to the known problem of shortest-path finding in a weighted graph. To score candidate DNA strand routes we used a thermodynamic model that accounts for enthalpic and entropic contributions of initial binding, hybridization, and DNA loop closure. We encoded and analyzed new and previously reported design heuristics. Using design principles emerging from this analysis, we redesigned and fabricated multiple shapes and compared their folding accuracy using electrophoretic mobility analysis and electron microscopy imaging. Redesigned shapes showed 6- to 30-fold improvements in yield compared to original designs. We demonstrate accurate folding can be achieved by optimizing staple routes using our model and provide a computational framework for applying our methodology to any design.

L1-ORF1p nucleoprotein can rapidly assume distinct conformations and simultaneously bind more than one nucleic acid.

LINE-1 (L1) is a parasitic retrotransposable DNA element, active in primates for the last 80-120 Myr. L1 has generated nearly one-third of the human genome by copying its transcripts, and those of other genetic elements (e.g. Alu and SVA), into genomic DNA by target site-primed reverse transcription (TPRT) and remains active in modern humans. L1 encodes two proteins that bind their encoding transcript (cis preference) to form an L1 ribonucleoprotein (RNP) that mediates retrotransposition. ORF2p provides reverse transcriptase and endonuclease activity. ORF1p, its major component, is a homo-trimeric phospho-protein that binds single-stranded nucleic acid (ssNA) with high affinity and exhibits nucleic acid (NA) chaperone activity. We used optical tweezers to examine ORF1p binding to individual single-stranded DNA (ssDNA) molecules and found that the arrangement of ORF1p on the ssDNA depends on their molar ratio. When the concentration of ORF1p is just sufficient to saturate the entire NA molecule, the nucleoprotein (NP) is compact and stable. However, additional ORF1p binds and destabilizes the compacted NP, allowing it to engage a second ssDNA. Our results suggest that ORF1p displaced from its RNA template during TPRT could bind and destabilize remaining downstream L1 RNP, making them susceptible to hijacking by non-L1 templates, and thereby enable retrotransposition of non-L1 transcripts.