DNA Phosphate Research Articles

DNA extraction protocol from Myrciariacaerulescens J.M.A. Braga, M.T.C. Lacerda & Stadnik (MYRTACEAE)

Myrciaria caerulescens, a fruit-bearing species endemic to Bahia, Brazil, and recently described, has ecological and economic potential. The analysis of genetic diversity and the use of molecular techniques are essential for its conservation and genetic improvement of M. caerulescens.The objective of this work was to standardize the protocol for extracting total DNA from Myrciaria caerulescens in the municipalities of Caetité and Lagoa Real in Bahia. Young leaves were collected using three collection methods and added to aluminum foil stored on ice, aluminum foil with silica and silica in a 50 mL test tube, where they received alphanumeric identification.The material was taken to the Genetic Improvement and Biotechnology Center Laboratory at UFRB, the silica was removed from the samples and stored in an ultra freezer at -80°C. For genomic DNA extraction, the three collection and extraction methods of the 40 samples, three protocols were tested for each method. The protocols of Lodhi (1994), modified by Pereira (2005), Doyle and Doyle (1987) and Murray and Tompsom (1980).Liquid nitrogen was used for crushing, helping to break down the M.caerulescens material. The isolated DNA influenced the quality and quantity of the methods and protocols tested. Silica gel method in the test tube had a greater amount of DNA compared to the others.The best result in DNA bands was described in Lodhi (1994), modified by Pereira (2005), better quality and higher concentration of extracted DNA, the test tube with silica, greater presence of bands, following the aluminum foil with silica for both locations. CTAB protocols were efficient in extraction. The increase in the amount of NaCl (0.5 to 5M) made it possible to quantify DNA bands in the agarose gel with positive ions neutralizing the negative charge of the DNA phosphate group.

Revolutionizing Quinolone Development for DNA Gyrase Targeting; Discovering the Promising Approach to Fighting Microbial Infections

Abstract: DNA gyrase is a type II topoisomerase enzyme that can cause negative supercoiling in DNA by using the energy produced by ATP hydrolysis. There are two main types of topoi-somerases: type I and type II. Type I enzymes cut a single strand of DNA and are further clas-sified as type IA if they connect to a 5′ phosphate of DNA, or type IB if they link to a 3′ phos-phate. Type II topoisomerases break both strands, creating a staggered double-strand break. Antimicrobial resistance is a major concern for the global healthcare system. Resistance is the ability of microorganisms to neutralize and withstand antimicrobial drugs previously used to treat microbial infections. Some known classes of DNA gyrase inhibitors are coumarins, cyclo-thialidines, and quinolones. Antimicrobial medicines such as quinolones have been widely used to treat microbiological diseases. However, the increased use of quinolones has led to the emer-gence of quinolone-resistant bacteria, which poses a serious risk to public health. Microorgan-isms can cause resistance due to changes in the target enzymes, DNA gyrase, and topoisomerase IV, which are responsible for transcription and DNA replication. Additionally, differences in drug entry and efflux may also play a role in resistance. Plasmids that produce the Qnr protein can mediate resistance to quinolones by protecting the quinolone targets from inhibition. This review aims to revolutionize the discovery of quinolone-based antibiotics, specifically targeting DNA gyrase, a critical enzyme in bacterial DNA replication, to enhance the efficacy and spec-ificity of anti-microbail agents against microbial infections.

Beyond the "spine of hydration": Chiral SFG spectroscopy detects DNA first hydration shell and base pair structures.

Experimental methods capable of selectively probing water at the DNA minor groove, major groove, and phosphate backbone are crucial for understanding how hydration influences DNA structure and function. Chiral-selective sum frequency generation spectroscopy (chiral SFG) is unique among vibrational spectroscopies because it can selectively probe water molecules that form chiral hydration structures around biomolecules. However, interpreting chiral SFG spectra is challenging since both water and the biomolecule can produce chiral SFG signals. Here, we combine experiment and computation to establish a theoretical framework for the rigorous interpretation of chiral SFG spectra of DNA. We demonstrate that chiral SFG detects the N-H stretch of DNA base pairs and the O-H stretch of water, exclusively probing water molecules in the DNA first hydration shell. Our analysis reveals that DNA transfers chirality to water molecules only within the first hydration shell, so they can be probed by chiral SFG spectroscopy. Beyond the first hydration shell, the electric field-induced water structure is symmetric and, therefore, precludes chiral SFG response. Furthermore, we find that chiral SFG can differentiate chiral subpopulations of first hydration shell water molecules at the minor groove, major groove, and phosphate backbone. Our findings challenge the scientific perspective dominant for more than 40 years that the minor groove "spine of hydration" is the only chiral water structure surrounding the DNA double helix. By identifying the molecular origins of the DNA chiral SFG spectrum, we lay a robust experimental and theoretical foundation for applying chiral SFG to explore the chemical and biological physics of DNA hydration.

Exposure of Arabidopsis thaliana Mutants to Genotoxic Stress Provides New Insights for the Involvement of TDP1α and TDP1β genes in DNA-Damage Response.

Genotoxic stress activates the DNA-damage response (DDR) signalling cascades responsible for maintaining genome integrity. Downstream DNA repair pathways include the tyrosyl-DNA phosphodiesterase 1 (TDP1) enzyme that hydrolyses the phosphodiester bond between the tyrosine of topoisomerase I (TopI) and 3'-phosphate of DNA. The plant TDP1 subfamily contains the canonical TDP1α gene and the TDP1β gene whose functions are not fully elucidated. The current study proposes to investigate the involvement of TDP1 genes in DDR-related processes by using Arabidopsis thaliana mutants treated with genotoxic agents. The phenotypic and molecular characterization of tdp1α, tdp1β and tdp1α/β mutants treated with cisplatin (CIS), curcumin (CUR), NSC120686 (NSC), zeocin (ZEO), and camptothecin (CPT), evidenced that while tdp1β was highly sensitive to CIS and CPT, tdp1α was more sensitive to NSC. Gene expression analyses showing upregulation of the TDP2 gene in the double mutant indicate the presence of compensatory mechanisms. The downregulation of POL2A gene in the tdp1β mutant along with the upregulation of the TDP1β gene in pol2a mutants, together with its sensitivity to replication inhibitors (CIS, CTP), point towards a function of this gene in the response to replication stress. Therefore, this study brings novel information relative to the activity of TDP1 genes in plants.

Study on the electrochemical and spectroscopic characteristics of holmium ion and its interaction with DNA

Metal ion-DNA interactions play a crucial role in modulating the structure and function of genetic material in the natural environment. In this study, we report on the favorable electrochemical activity of holmium(III) (Ho3+) on a glassy carbon electrode (GCE) and its interaction with double-stranded DNA. The interaction between DNA and Ho3+ was investigated for the first time using cyclic voltammetry and differential pulse voltammetry. The electrochemical behavior of Ho3+ ions on a GCE exhibited a reversible electron transfer process, indicative of its redox activity. A linear correlation between the peak current and the square root of the scan rate was observed, suggesting a diffusion-controlled kinetic regime for the electrochemical process. Additionally, fluorescence and absorption spectroscopy were employed to confirm the binding of Ho3+ to DNA. Our findings demonstrate that, at pH 7.2, specific DNA bases and phosphate groups can interact with Ho3+ ions. Moreover, electrochemical measurements suggest that Ho3+ ions bind to DNA via a groove binding mode, with a calculated binding ratio of 1:1 between Ho3+ and DNA. Notably, under optimal conditions, an increase in the amount of DNA leads to a significant reduction in the current intensity of Ho3+ ions.

The Interaction of a Gemini Surfactant with a DNA Quadruplex.

DNA secondary structures are stabilized by mono- and divalent cations. To examine the stability of the DNA quadruplex formed from (TTAGGG)4, its interaction with a dicationic Gemini surfactant in standard phosphate buffer was investigated. The Gemini surfactant begins to form micelles in buffer at a cmc (critical micelle concentration) of 1.5 mM. In this study, solutions of DNA were prepared in buffer with surfactant concentrations ranging from 0.0 to 3.0 mM, i.e., above and below the cmc of the surfactant. In all samples of DNA and surfactant, a precipitate formed. The fraction of DNA precipitated depends upon both the initial DNA concentration and the initial concentration of the surfactant. In those samples where the DNA did not totally precipitate, the residual DNA assumed a quadruplex conformation. It was determined that two surfactant molecules per DNA phosphate are needed to completely precipitate all of the DNA in a particular sample. An estimated apparent K sp for the DNA:surfactant complex was determined.

Conformational Dynamics of the Nucleosomal Histone H2B Tails Revealed by Molecular Dynamics Simulations.

Epigenetic modifications of histone N-terminal tails play a critical role in regulating the chromatin structure and biological processes such as transcription and DNA repair. One of the key post-translational modifications (PTMs) is the acetylation of lysine residues on histone tails. Epigenetic modifications are ubiquitous in the development of diseases, such as cancer and neurological disorders. Histone H2B tails are critical regulators of nucleosome dynamics, biological processes, and certain diseases. Here, we report all-atomistic molecular dynamics (MD) simulations of the nucleosome to demonstrate that acetylation of the histone tails changes their conformational space and interaction with DNA. We perform simulations of H2B tails, critical regulators of gene regulation, in both the lysine-acetylated (ACK) and unacetylated wild type (WT) states. To explore the effects of salt concentration, we use two different NaCl concentrations to perform simulations at microsecond time scales. Salt can modulate the effects of electrostatic interactions between the DNA phosphate backbone and histone tails. Upon acetylation, H2B tails shift their secondary structure helical propensity. The number of contacts between the DNA and the H2B tail decreases. We characterize the conformational dynamics of the H2B tails by principal component analysis (PCA). The ACK tails become more compact at increased salt concentrations, but conformations from the WT tails display the most contacts with DNA at both salt concentrations. Mainly, H2B acetylation may increase the DNA accessibility for regulatory proteins to bind, which can aid in gene regulation and NCP stability.

A dual-signal ratiometric electrochemical aptasensor based on Thi/Au/ZIF-8 and catalytic hairpin assembly for ultra-sensitive detection of aflatoxin B1.

Adual-signal ratiometric electrochemical aptasensor has been developed for AFB1 detection using thionine/Au/zeolitic imidazolate framework-8 (Thi/Au/ZIF-8) nanomaterials and catalytic hairpin assembly (CHA) reaction. Thi/Au/ZIF-8 combined with DNA hairpin 2 (H2) was used as a signal probe. [Fe(CN)6]3-/4- was served as another signal probe, and the IThi/Au/ZIF-8/I[Fe(CN)6]3-/4- ratio was for the first timeutilized to quantify AFB1. AFB1-induced CHA was used to expand the ratio of electrical signals. In the presence of AFB1, H2/Thi/Au/ZIF-8 bound to the electrode via CHA, enhanced the current signal of Thi/Au/ZIF-8. H2 contained the DNA phosphate backbone hindered [Fe(CN)6]3-/4- redox reaction and resulted in a lower [Fe(CN)6]3-/4- current signal. This aptasensor exhibited high specificity for AFB1, a linear range of 0.1pgmL-1 to 100ngmL-1, and a detection limit of 0.089pgmL-1. It demonstrated favorable sensitivity, selectivity, stability, and repeatability. The aptasensor was suitable for detecting AFB1 in peanuts and black tea and holds potential for real sample applications.

Structure of the human Bre1 complex bound to the nucleosome

Histone H2B monoubiquitination (at Lys120 in humans) regulates transcription elongation and DNA repair. In humans, H2B monoubiquitination is catalyzed by the heterodimeric Bre1 complex composed of Bre1A/RNF20 and Bre1B/RNF40. The Bre1 proteins generally function as tumor suppressors, while in certain cancers, they facilitate cancer cell proliferation. To obtain structural insights of H2BK120 ubiquitination and its regulation, we report the cryo-electron microscopy structure of the human Bre1 complex bound to the nucleosome. The two RING domains of Bre1A and Bre1B recognize the acidic patch and the nucleosomal DNA phosphates around SHL 6.0–6.5, which are ideally located to recruit the E2 enzyme and ubiquitin for H2BK120-specific ubiquitination. Mutational experiments suggest that the two RING domains bind in two orientations and that ubiquitination occurs when Bre1A binds to the acidic patch. Our results provide insights into the H2BK120-specific ubiquitination by the Bre1 proteins and suggest that H2B monoubiquitination can be regulated by nuclesomal DNA flexibility.

Unusual bending patterns of spermidine3+ bound to DNA double helix

Natural polyamines play a fundamental role in the cell cycle. Despite being recognized as the most abundant organic counterions of DNA in the cell nucleus, their interactions with DNA have not been fully characterized. In a recent work [S. Perepelytsya, T. Vasiliu, A. Laaksonen, L. Engelbrecht, G. Brancato, and F. Mocci, J. Molec. Liq.389, 122828 (2023)], we have shown how the interactions between spermidine3+ and the DNA double helix induce significant conformational variations in the polyamine molecule. Specifically, we found that DNA induces conformations that are not observed in solution. Following that study, we present here a detailed investigation of the most compact conformation of the polyamine, analyzing its connection to the interaction with the DNA duplex. The analysis reveals that anomalous bent conformations of the spermidine3+ molecule result from the interaction of all three amino groups of the polyamine with the DNA phosphate groups on the minor groove side of the double helix. The changes in dihedral angles of the bent spermidine3+ molecule can be explained in terms of conformational transformations of six- and seven-membered rings, analogous to cyclohexane and cycloheptane. The analysis of the position of spermidine3+ molecule along the DNA surface reveals a sequence specificity of this binding mode with a marked preference for the narrow minor groove of A-tracts. The formation of the anomalous bent conformations of spermidine3+ in the complex with the DNA double helix is expected to be of paramount importance in understanding the mechanisms underlying DNA’s biological function.

Synthesis, Characterization, and Evaluation of Sulfonium Lipids as Potential Nonviral Gene Vectors

Introduction: Non-viral gene vectors have attracted much attention in the last few decades because of their potential activity and fewer side effects. Headgroup chemistry is a key aspect of lipid design. Methods: In this study, a group of sulfonium lipids were designed and constructed by combining tetrahydrothiophene or tetrahydrothiopyran with an ethoxy linker and carbon aliphatic chains and were evaluated in terms of their ability as potential gene vectors. The sulfonium lipids were synthesized and characterized by 1H NMR, 13C NMR, and Mass. Condensates of sulfonium lipids (SL) and DNA were examined by gel electrophoresis and particle size and zeta potential were measured. Sulfonium compounds were tested on HepG2 cells for cytotoxicity. SL/DNA condensates were studied in cellular uptake and distribution using fluorescent microscopy. Results: 12 sulfonium lipids were obtained. Gel electrophoresis experiments showed that sulfonium cation can effectively interact with phosphorous in DNA. Compounds containing longer lipid chains can effectively retard DNA at an S/P ratio higher than 10/1 and can condense DNA into nano size particles with particle size in the range of 150 nm ~ 300 nm and zeta potential in the range of +20~+40. Sulfonium compounds were calculated against HepG2 cells in the range of 0.035 μg/mL to 1.64 μg/mL. The intracellular uptake experiments revealed that SL/DNA nanoparticle was taken into the cell at low efficiency. Conclusion: Sulfonium head group can interact with the phosphates of DNA. The structural environment of sulfonium ions influences the DNA bonding effect. The designed cyclic sulfonium ion was buried in the middle of the structure and thus hindered interaction with DNA. This type of molecule is worthy of further modification to increase DNA capacity and reduce cell cytotoxicity.

Structural characterization of DNA amplicons by ATR-FTIR spectroscopy as a guide for screening metainflammatory disorders in blood plasma

Attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy is a promising rapid, reagent-free, and low-cost technique considered for clinical translation. It allows to characterize biofluids proteome, lipidome, and metabolome at once. Metainflammatory disorders share a constellation of chronic systemic inflammation, oxidative stress, aberrant adipogenesis, and hypoxia, that significantly increased cardiovascular and cancer risk. As a result, these patients have elevated concentration of cfDNA in the bloodstream. Considering this, DNA amplicons were analyzed by ATR-FTIR at 3 concentrations with 1:100 dilution: (IU/mL): 718, 7.18, and 0.0718. The generated IR spectrum was used as a guide for variable selection. The main peaks in the biofingerprint (1800–900 cm−1) give important information about the base, base-sugar, phosphate, and sugar-phosphate transitions of DNA. To validate our method of selecting variables in blood plasma, 38 control subjects and 12 with metabolic syndrome were used. Using the wavenumbers of the peaks in the biofingerprint of the DNA amplicons, was generated a discriminant analysis model with Mahalanobis distance in blood plasma, and 100 % discrimination accuracy was obtained. In addition, the interval 1475–1188 cm−1 showed the greatest sensitivity to variation in the concentration of DNA amplicons, so curve fitting with Gaussian funcion was performed, obtaining adjusted-R2 of 0.993. PCA with Mahalanobis distance in the interval 1475–1188 cm−1 obtained an accuracy of 96 % and PLS-DA modeling in the interval 1475–1088 cm−1 obtained AUC = 0.991 with sensitivity of 95 % and specificity of 100 %. Therefore, ATR-FTIR spectroscopy with variable selection guided by DNA IR peaks is a promising and efficient method to be applied in metainflammatory disorders.

Compelling DNA intercalation through 'anion-anion' anti-coulombic interactions: boron cluster self-vehicles as promising anticancer agents.

Anticancer drugs inhibit DNA replication by intercalating between DNA base pairs, forming covalent bonds with nucleotide bases, or binding to the DNA groove. To develop safer drugs, novel molecular structures with alternative binding mechanisms are essential. Stable boron hydrides offer a promising alternative for cancer therapy, opening up additional options like boron neutron capture therapy based on 10B and thermal neutron beams or proton boron fusion therapy using 11B and proton beams. These therapies are more efficient when the boron compound is ideally located inside cancer cells, particularly in the nucleus. Current cancer treatments often utilize small, polycyclic, aromatic, planar molecules that intercalate between ds-DNA base pairs, requiring only a spacing of approximately 0.34 nm. In this paper, we demonstrate another type of intercalation. Notably, [3,3'-Fe(1,2-C2B9H11)2]-, ([o-FESAN]-), a compact 3D molecule measuring 1.1 nm × 0.6 nm, can as well intercalate by strong non-bonding interactions preferentially with guanine. Unlike known intercalators, which are positive or neutral, [o-FESAN]- is a negative species and when an [o-FESAN]- molecule approaches the negatively charged DNA phosphate chain an anion-anion interaction consistently anti-electrostatic via Ccluster-H⋯O-P bonds occurs. Then, when more molecules approach, an elongated outstandingly self-assembled structure of [o-FESAN]--[o-FESAN]- forms moving anions towards the interthread region to interact with base pairs and form aggregates of four [o-FESAN]- anions per base pair. These aggregates, in this environment, are generated by Ccluster-H⋯O-C, N-H⋯H-B and Ccluster-H⋯H-B interactions. The ferrabis(dicarbollide) boron-rich small molecules not only effectively penetrate the nucleus but also intercalate with ds-DNA, making them promising for cancer treatment. This amphiphilic anionic molecule, used as a carrier-free drug, can enhance radiotherapy in a multimodal perspective, providing healthcare professionals with improved tools for cancer treatment. This work demonstrates these findings with a plethora of techniques.

Magnetic composite microspheres with a controlled mesoporous shell for highly efficient DNA extraction and fragment screening.

Rapid extraction and screening of high-purity DNA fragments is an indispensable technology in advanced molecular biology. In this article, mesoporous magnetic composite microspheres (MSP@mTiO2) with tunable pore sizes were successfully fabricated for high-purity DNA extraction and fragment screening. Owing to the strong complexation ability of Ti ions with DNA phosphate groups and the high specific surface area of mesoporous microspheres, the MSP@mTiO2 microspheres possess excellent adsorption performance, where the saturated loading capacity of MSP@mTiO2 with a specific surface area of 122 m2 g-1 is as high as 575 μg mg-1 for a salmon sperm specimen. ITC experiments demonstrated that DNA adsorption on MSP@mTiO2 microspheres is mainly driven by entropy, which gives us more potential ways to regulate the balance of adsorption and desorption. Meanwhile, the mesoporous MSP@mTiO2 microspheres exhibit a much higher extraction efficiency compared with non-porous MSP@TiO2 for whole genome DNA from Arabidopsis thaliana plants. Interestingly, DNA fragments with different lengths could be screened by simply regulating the pore size of MSP@mTiO2 or the concentration of Na3PO4 in the eluent. A small pore size and low phosphate concentration are advantageous for the extraction of short-stranded DNA fragments, and DNA fragments (≤1000 bp) can be efficiently extracted when the mesopore size of MSP@mTiO2 is lower than 7.6 nm. The extraction results from the mesoporous composite microspheres provide new promising insights into the purification and screening of DNA from complex biological samples.

Smart enzyme-free amplification dual-mode self-powered platform designed on two-dimensional networked graphdiyne and DNA nanorods for ultra-sensitive detection of breast cancer biomarkers

Research has shown that microRNAs exhibit regular dysregulation in cancers, making them potential biomarkers for cancer diagnosis. However, achieving specific and sensitive detection of microRNAs has been a challenging task. To address this issue, two-dimensional networked graphdiyne is used to fabricate a self-powered biosensor and establish a new approach for ultra-responsive dual-mode detection of miRNA-141, a breast cancer biomarker. This method detects miRNA-141 using both electrochemical and colorimetric modes by measuring the output electrical signal of an enzyme-based biofuel cell and the RGB blue value of the electrolyte solution. Tetrahedral DNA and DNA nanorods also are immobilized on the electrode as a biocathode and methylene blue is used as the electron acceptor, which is fixed in the DNA phosphate backbone through electrostatic adsorption. The bioanode catalyzes the oxidation of glucose to produce electrons, which reduces methylene blue to its reduced form, resulting in a high open-circuit voltage (EOCV) and a highger RGB Blue value, enabling dual-mode detection. A reliable linear correlation is observed between EOCV values and miRNA-141 concentrations ranging from 0.0001 to 100 pM, with a detection limit of 21.9 aM (S/N = 3). Additionally, the colorimetric mode also demonstrates a reliable linear correlation with a concentration range of 0.0001–10000 pM, and this method can detect a concentration of 22.2 aM (S/N = 3). This innovative research realizes sensitive and accurate determination of miRNA-141 and provides an important new method for cancer diagnosis.

Backbone Conformational Equilibrium in Mismatched DNA Correlates with Enzyme Activity.

T:G mismatches in mammals arise primarily from the deamination of methylated CpG sites or the incorporation of improper nucleotides. The process by which repair enzymes such as thymine DNA glycosylase (TDG) identify a canonical DNA base in the incorrect pairing context remains a mystery. However, the abundant contacts of the repair enzymes with the DNA backbone suggest a role for protein-phosphate interaction in the recognition and repair processes, where conformational properties may facilitate the proper interactions. We have previously used 31P NMR to investigate the energetics of DNA backbone BI-BII interconversion and the effect of a mismatch or lesion compared to canonical DNA and found stepwise differences in ΔG of 1-2 kcal/mol greater than equivalent steps in unmodified DNA. We have currently compared our results to substrate dependence for TDG, MBD4, M. HhaI, and CEBPβ, testing for correlations to sequence and base-pair dependence. We found strong correlations of our DNA phosphate backbone equilibrium (Keq) to different enzyme kinetics or binding parameters of these varied enzymes, suggesting that the backbone equilibrium may play an important role in mismatch recognition and/or conformational rearrangement and energetics during nucleotide flipping or other aspects of enzyme interrogation of the DNA substrate.

Water-Dispersible Carbon Nano-Test Tubes as a Container for Concentrated DNA Molecules.

Water-dispersible carbon nano-test tubes (CNTTs) with an inner and outer diameter of about 25 and 35 nm, respectively, were prepared by the template technique and then their inner carbon surface was selectively oxidized to introduce carboxy groups. The adsorption behavior of DNA molecules on the oxidized CNTTs (Ox-CNTTs) was examined in the presence of Ca2+ cations. Many DNA molecules are attracted to the inner space of Ox-CNTTs based on the Ca2+ -mediated electrostatic interaction between DNA phosphate groups and carboxylate anions on the inner carbon surface. Moreover, the total net charge of the DNA adsorbed was found to be equal to the total charge of the carboxylate anions. This selective adsorption into the interior of Ox-CNTTs can be explained from the fact that the electrostatic interaction onto the inner concave surface is much stronger than that on the outer convex surface. On the other hand, the desorption of DNA easily occurs whenever Ca2+ cations are removed by washing with deionized water. Thus, each of Ox-CNTTs works well as a nano-container for a large amount of DNA molecules, thereby resulting in the occurrence of DNA enrichment in the nanospace.

Sulfonium Lipids: Synthesis and Evaluation as DNA Delivery Vectors.

Cationic lipids can be used as nonviral vectors in gene delivery therapy. Most cationic lipids contain quaternary ammonium that can bind to negative phosphates of the plasmid. In this study, sulfonium-a trialkylated sulfur cation was adopted in the synthesis of a series of cationic lipids which were evaluated for their ability to function as gene delivery vectors. The sulfonium lipids were synthesized by condensing cyclic thioether and aliphatic carbon chains with ethoxy linkage and the structure was characterized by NMR and mass. The DNA condensing abilities of sulfonium lipids were evaluated using a gel retardation experiment. Sulfonium lipids/ DNA condensates were measured for particle size and Zeta potential. The cytotoxicity of sulfoniums was evaluated with the MTT assay. The intracellular uptake of sulfonium lipid/DNA complexes was observed with a fluorescence microscope. The results showed that the sulfonium head can effectively bind to the phosphate of DNA. When the S/P ratio is larger than 10/1, sulfonium lipids with longer carbon chains can completely condense DNA to form a nanoparticle with particle size ranging from 135 nm to 155 nm and zeta potential ranging from 28 mV to 42 mV. The IC50 of sulfonium lipids on HepG2 cells ranged from 2.37 μg/mL to 3.67 μg/mL. Cellular uptake experiments showed that sulfonium lipids/DNA condensate can be taken into cells. Sulfonium lipids can effectively condense DNA and transfer DNA into cells. The sulfonium compound is worth further development to reduce the cytotoxicity and increase the transfection rate as gene vectors.

Structures of CTCF-DNA complexes including all 11 zinc fingers.

The CCCTC-binding factor (CTCF) binds tens of thousands of enhancers and promoters on mammalian chromosomes by means of its 11 tandem zinc finger (ZF) DNA-binding domain. In addition to the 12-15-bp CORE sequence, some of the CTCF binding sites contain 5' upstream and/or 3' downstream motifs. Here, we describe two structures for overlapping portions of human CTCF, respectively, including ZF1-ZF7 and ZF3-ZF11 in complex with DNA that incorporatesthe CORE sequence together with either 3' downstream or 5' upstream motifs. Like conventional tandem ZF array proteins, ZF1-ZF7 followthe right-handed twist of the DNA, with each finger occupying and recognizing one triplet of three basepairs in the DNA major groove. ZF8 plays a unique role, acting as a spacer across the DNA minor groove and positioning ZF9-ZF11 to make cross-strand contacts with DNA. We ascribe the difference between the two subgroups of ZF1-ZF7 and ZF8-ZF11 to residues at the two positions -6 and -5 within each finger, with small residues for ZF1-ZF7 and bulkier and polar/charged residues for ZF8-ZF11. ZF8 is also uniquely rich in basic amino acids, which allows salt bridges to DNA phosphates in the minor groove. Highly specific arginine-guanine and glutamine-adenine interactions, used to recognize G:C or A:T base pairs at conventional base-interacting positions of ZFs, also apply to the cross-strand interactions adopted by ZF9-ZF11. The differences between ZF1-ZF7 and ZF8-ZF11 can be rationalized structurally and may contribute to recognition of high-affinity CTCF binding sites.

Integrative Transcriptomics Data Mining to Explore the Functions of TDP1α and TDP1β Genes in the Arabidopsis thaliana Model Plant.

The tyrosyl-DNA phosphodiesterase 1 (TDP1) enzyme hydrolyzes the phosphodiester bond between a tyrosine residue and the 3'-phosphate of DNA in the DNA-topoisomerase I (TopI) complex, being involved in different DNA repair pathways. A small TDP1 gene subfamily is present in plants, where TDP1α has been linked to genome stability maintenance, while TDP1β has unknown functions. This work aimed to comparatively investigate the function of the TDP1 genes by taking advantage of the rich transcriptomics databases available for the Arabidopsis thaliana model plant. A data mining approach was carried out to collect information regarding gene expression in different tissues, genetic backgrounds, and stress conditions, using platforms where RNA-seq and microarray data are deposited. The gathered data allowed us to distinguish between common and divergent functions of the two genes. Namely, TDP1β seems to be involved in root development and associated with gibberellin and brassinosteroid phytohormones, whereas TDP1α is more responsive to light and abscisic acid. During stress conditions, both genes are highly responsive to biotic and abiotic treatments in a time- and stress-dependent manner. Data validation using gamma-ray treatments applied to Arabidopsis seedlings indicated the accumulation of DNA damage and extensive cell death associated with the observed changes in the TDP1 genes expression profiles.