DNA Conformational Transitions Research Articles

Investigation into the DNA's conformations and their conversions from the phase transition theory

The functions and transformation mechanisms of nucleic acids in their various states are of great importance in physiology and pathology. Based on the Landau model, this study reveals the mechanism of the existence of chirality and the transformation rules between different chiral conformations by studying the Duffing oscillation response of the local gauge potential on the DNA chain. The research results show that normal chiral DNA and DNA configuration transition behaviors exhibit regular nonlinear periodic oscillations. Moreover, the gauge potential value approaches zero at the B-Z junction site, which may add a criterion for detecting Z-forming sites. In addition, external force and damping play a key role in the chiral gauge potential of nucleic acids, and they can influence, regulate and even control the configuration transition of nucleic acids. Finally, we verified the rationality of our theory by combining x-ray crystal diffraction data of multiple configurations of DNA. This study provides new insights into the behavior and function of chiral DNA in organisms, and provides new possibilities for regulating DNA conformational transition in the future.

Manipulating Radiation-Sensitive Z-DNA Conformation for Enhanced Radiotherapy.

DNA double-strand breaks (DSBs) yield highly determines radiotherapy efficacy. However, improving the inherent radiosensitivity of tumor DNA to promote radiation-induced DSBs remains a challenge. Using theoretical and experimental models, the underexplored impact of Z-DNA conformations on radiosensitivity, yielding higher DSBs than other DNA conformations, is discovered. Thereout, a radiosensitization strategy focused on inducing Z-DNA conformation, utilizing CBL@HfO2 nanocapsules loaded with a Z-DNA inducer CBL0137, is proposed. A hollow mesoporous HfO2 (HM-HfO2) acts as a delivery and an energy depositor to promote Z-DNA breakage. The nanocapsule permits the smart DSBs accelerator that triggers its radiosensitization with irradiation stimulation. Impressively, the CBL@HfO2 facilitates the B-Z DNA conformational transition, augmenting DSBs about threefold stronger than irradiation alone, generating significant tumor suppression with a 30% cure rate. The approach enables DSBs augmentation by improving the inherent radiosensitivity of DNA. As such, it opens up an era of Z-DNA conformation manipulation in radiotherapy.

Enhanced sampling strategies for molecular simulation of DNA

AbstractMolecular dynamics (MD) simulations can provide detailed insights into complex molecular systems, such as DNA, at high resolution in space and time. Using current computer architectures, time scales of tens of microseconds are feasible with contemporary all‐atom force fields. However, these timescales are insufficient to accurately characterize large conformational transitions in DNA and compare calculations to experimental data. This review discusses the advantages and drawbacks of two simulation approaches to overcome the timescale challenge. The first approach is based on adding biasing potentials to the system to drive transitions. Umbrella sampling, steered MD, and metadynamics are examples of these methods. A key challenge of such methods is the necessity of selecting one or a few efficient coordinates, commonly referred to as collective variables (CVs), along which to apply the biasing potential. The path‐metadynamics methodology addresses this issue by finding the optimal route(s) between states in a multi‐dimensional CV space. The second strategy is path sampling, which focuses MD simulations on the transitions. The assumption is that even though transitions between states are rare, they are generally fast. Stopping the simulations as soon as they reach a stable state can significantly increase simulation efficiency. We introduce these methods on the two‐dimensional Müller–Brown potential. DNA applications are featured for two different processes: the Watson–Crick–Franklin to Hoogsteen transition in adenine–thymine base pairs and the binding of a DNA‐binding protein domain to DNA.This article is categorized under: Molecular and Statistical Mechanics Molecular Dynamics and Monte‐Carlo Methods Molecular and Statistical Mechanics Free Energy Methods Software Simulation Methods

Mechanism of action of the low-frequency electromagnetic field on aqueous solutions of biopolymers

This paper presents the mechanism of signal transformation by which a signal from the magnetic component of the low-frequency electromagnetic field with extremely low energy is changed into chemical and biochemical signals which elicit a biological response in aqueous solutions of biopolymers based on nucleic acids and proteins. A theoretical model developed shows that the basis of oxidative DNA damage and conformational transitions of proteins is a universal mechanism associated with a change in the amount of the most long-lived form - hydrogen peroxide in a chemical oscillator of mutual transformations of reactive oxygen species under the influence of low-intensity electromagnetic field exposure. It has been experimentally established that the quantitative content of hydrogen peroxide in solutions of biopolymers resonantly depends on the frequency of applied field. Conformational changes in proteins are accompanied by an increase in the availability and activity of the nucleophilic centers that are potential targets for reactive oxygen species. Complete unfolding and denaturation of the amino acid chain of the protein under the influence of low-frequency electromagnetic field exposure do not occur. It has been shown that enhanced hydrogen peroxide formation at 3 Hz and 50 Hz leads to oxidative modification of nitrogenous bases in DNA.

A New Concept of Enhancing the Anticancer Activity of Manganese Terpyridine Complex by Oxygen-Containing Substituent Modification.

Eleven manganese 4'-substituted-2,2':6',2″-terpyridine complexes (1a-1c and 2a-2h) with three non-oxygen-containing substituents (L1a-L1c: phenyl, naphthalen-2-yl and naphthalen-1-yl, L1a-L1c) and eight oxygen-containing substituents (L2a-L2h: 4-hydroxyl-phenyl, 3-hydroxyl-phenyl, 2-hydroxyl-phenyl, 4-methoxyl-phenyl, 4-carboxyl-phenyl, 4-(methylsulfonyl)phenyl, 4-nitrophenyl and furan-2-yl) were prepared and characterized by IR, elemental analysis or single crystal X-ray diffraction. In vitro data demonstrate that all of these show higher antiproliferative activities than cisplatin against five human carcinoma cell lines: A549, Bel-7402, Eca-109, HeLa and MCF-7. Compound 2d presents the strongest antiproliferative effect against A549 and HeLa cells, with IC50 values being 0.281 μM and 0.356 μM, respectively. The lowest IC50 values against Bel-7402 (0.523 μM) Eca-109 (0.514 μM) and MCF-7 (0.356 μM) were obtained for compounds 2h, 2g and 2c, respectively. Compound 2g with a nitro group showed the best results on the whole, with relevantly low IC50 values against all the tested tumor cells. The DNA interactions with these compounds were studied by circular dichroism spectroscopic and molecular modeling methods. Spectrophotometric results revealed that the compounds have strong affinities in binding with DNA as intercalators, and the binding induces DNA conformational transition. Molecular docking studies indicate that the binding is contributed by the π-π stacking and hydrogen bonds. The anticancer activities of the compounds are correlated with their DNA binding ability, and the modification of oxygen-containing substituents significantly enhanced the anticancer activity, which could provide a new rationale for the future design of terpyridine-based metal complexes with antitumor potential.

Raman Research on Bleomycin-Induced DNA Strand Breaks and Repair Processes in Living Cells.

Even several thousands of DNA lesions are induced in one cell within one day. DNA damage may lead to mutations, formation of chromosomal aberrations, or cellular death. A particularly cytotoxic type of DNA damage is single- and double-strand breaks (SSBs and DSBs, respectively). In this work, we followed DNA conformational transitions induced by the disruption of DNA backbone. Conformational changes of chromatin in living cells were induced by a bleomycin (BLM), an anticancer drug, which generates SSBs and DSBs. Raman micro-spectroscopy enabled to observe chemical changes at the level of single cell and to collect hyperspectral images of molecular structure and composition with sub-micrometer resolution. We applied multivariate data analysis methods to extract key information from registered data, particularly to probe DNA conformational changes. Applied methodology enabled to track conformational transition from B-DNA to A-DNA upon cellular response to BLM treatment. Additionally, increased expression of proteins within the cell nucleus resulting from the activation of repair processes was demonstrated. The ongoing DNA repair process under the BLM action was also confirmed with confocal laser scanning fluorescent microscopy.

Conformational plasticity of DNA secondary structures: probing the conversion between i-motif and hairpin species by circular dichroism and ultraviolet resonance Raman spectroscopies.

The promoter regions of important oncogenes such as BCL2 and KRAS contain GC-rich sequences that can form distinctive noncanonical DNA structures involved in the regulation of transcription: G-quadruplexes on the G-rich strand and i-motifs on the C-rich strand. Interestingly, BCL2 and KRAS promoter i-motifs are highly dynamic in nature and exist in a pH-dependent equilibrium with hairpin and even with hybrid i-motif/hairpin species. Herein, the effects of pH and presence of cell-mimicking molecular crowding conditions on conformational equilibria of the BCL2 and KRAS i-motif-forming sequences were investigated by ultraviolet resonance Raman (UVRR) and circular dichroism (CD) spectroscopies. Multivariate analysis of CD data was essential to model the presence and identity of the species involved. Analysis of UVRR spectra measured as a function of pH, performed also by the two-dimensional correlation spectroscopy (2D-COS) technique, showed the role of several functional groups in the DNA conformational transitions, and provided structural and dynamic information. Thus, the UVRR investigation of intramolecular interactions and of local and environmental dynamics in promoting the different species induced by the solution conditions provided valuable insights into i-motif conformational transitions. The combined use of the two spectroscopic tools is emphasized by the relevant possibility of working in the same DNA concentration range and by the heterospectral UVRR/CD 2D-COS analysis. The results of this study shed light on the factors that can influence at the molecular level the equilibrium between the different conformational species putatively involved in the oncogene expression.

Structural switching/polymorphism by sequential base substitution at quasi-palindromic SNP site (G → A) in LCR of human β-globin gene cluster

The human β-globin gene Locus Control Region (LCR), a dominant regulator of globin gene expression contains five tissue-specific DNase I-hypersensitive sites (HSs). A single nucleotide polymorphism (SNP) (A → G) present in HS4 region of locus control region (LCR), have shown a notable association between the G allele and the occurrence of β-thalassemia. This SNP site exhibiting a hairpin − duplex equilibrium manifested in A → B like DNA transition has previously been reported from this laboratory. Since, DNA is a dynamic and adaptable molecule, so any change of a single base within a primary DNA sequence can produce major biological consequences commonly manifested in genetic disorders such as sickle cell anemia and β-thalassemia. Herein, the differential behavior of sequential single base substitutions G → A on the quasi-palindromic sequence (d-TGGGGGCCCCA; HPG11) has been explored. A combination of native gel electrophoresis, circular dichroism (CD), and UV-thermal denaturation (Tm) techniques have been used to investigate the structural polymorphism associated with various variants of HPG11 i.e. HPG11A2 to HPG11A5. The CD spectra confirmed that all the HPG11 variants exhibit a hairpin − duplex equilibrium. Oligomer concentration dependence on CD spectra has been correlated with A → B DNA conformational transition. However, as revealed in gel electrophoresis, HPG11A2 → A5 exhibit the formation of a tetramolecular structure (four-way junction) at higher oligomer concentration. UV-melting studies also supported the melting of hairpin, duplex and four-way junction structure. This polymorphism pattern may possibly be significant for DNA-protein recognition, in the process of regulation of LCR in the β-globin gene.

Cytotoxic Effects of 5-Azacytidine on Primary Tumour Cells and Cancer Stem Cells from Oral Squamous Cell Carcinoma: An In Vitro FTIRM Analysis.

In the present study, the cytotoxic effects of 5-azacytidine on primary Oral Squamous Cell Carcinoma cells (OSCCs) from human biopsies, and on Cancer Stem Cells (CSCs) from the same samples, were investigated by an in vitro Fourier Transform InfraRed Microscospectroscopy (FTIRM) approach coupled with multivariate analysis. OSCC is an aggressive tumoral lesion of the epithelium, accounting for ~90% of all oral cancers. It is usually diagnosed in advanced stages, and this causes a poor prognosis with low success rates of surgical, as well as radiation and chemotherapy treatments. OSCC is frequently characterised by recurrence after chemotherapy and by the development of a refractoriness to some employed drugs, which is probably ascribable to the presence of CSCs niches, responsible for cancer growth, chemoresistance and metastasis. The spectral information from FTIRM was correlated with the outcomes of cytotoxicity tests and image-based cytometry, and specific spectral signatures attributable to 5-azacytidine treatment were identified, allowing us to hypothesise the demethylation of DNA and, hence, an increase in the transcriptional activity, together with a conformational transition of DNA, and a triggering of cell death by an apoptosis mechanism. Moreover, a different mechanism of action between OSSC and CSC cells was highlighted, probably due to possible differences between OSCCs and CSCs response.

Investigating the thermodynamics of conformational transitions in DNA oligomers

I first met Professor Wilhelm when I joined the Board of Directors of the North American Calorimetry Conference (CalCon) in 2009. Since that time, I have had the pleasure of seeing him every year at CalCon. Although I am formally trained in organic chemistry, my research has evolved to nucleic acid biophysics. None the less, I would attend Prof. Wilhelm’s lecture whenever possible at the CalCon meetings. The first lecture I attended focused on fugacity and afterwards, I thought to myself: “I could learn some physical chemistry from this man!” I am honored to contribute my story to this special issue of The Journal of Chemical Thermodynamics. The work reviewed below focuses on biophysical approaches by my research group to study conformational transitions in DNA using designed DNA oligomers and monitoring temperature or salt induced transition by optical melting and calorimetric determinations. Further, the influences on DNA sequence context and environmental conditions on these transitions are also discussed.

Removal of extracellular antibiotic resistance genes using magnetic biochar/quaternary phosphonium salt in aquatic environments: A mechanistic study

The proliferation and spread of antibiotic resistance genes (ARGs) is becoming a worldwide crisis. Extracellular DNA encoding ARGs (eARGs) in aquatic environment plays a critical role in the dispersion of antimicrobial resistance genes. Strategies to control the dissemination of eARGs are urgently required for ecological safety and human health. Towards this goal, magnetic biochar/quaternary phosphonium salt (MBQ), was used to investigate the efficiency and removal mechanism for eARGs. Magnetic biochar modified by quaternary phosphonium salt enhanced the adsorption capacity of extracellular DNA to approximately 9 folds, compared to that of the unmodified. DNA adsorption by MBQ was mainly dominated by chemisorption in heterogeneous systems and was promoted in acidic and low-salt environment. The generation of •OH and MBQ colloid jointly cleaved DNA into fragments, facilitating the adsorption of the phosphate backbone of DNA onto MBQ through electrostatic force as well as the conformational transition of DNA. Furthermore, quantification of extracellular DNA after MBQ was applied in water demonstrated that over 92.7% of resistance genes were removed, indicating a significantly reduced risk of propagation of antimicrobial resistance in aquatic environments. These findings have a practical significance in the application of MBQ in mitigating the spread of ARGs in aquatic environment.

Marked Difference in the Conformational Transition of DNA Caused by Propanol Isomer

We measured the changes in the higher-order structure of DNA molecules (λ phage DNA, 48 kbp) at different concentrations of 1- and 2-propanol through single-molecular observation. It is known that 2-propanol is usually adapted for the procedure to isolate genomic DNA from living cells/organs in contrast to 1-propanol. In the present study, it was found that with an increasing concentration of 1-propanol, DNA exhibits reentrant conformational transitions from an elongated coil to a folded globule, and then to an unfolded state. On the other hand, with 2-propanol, DNA exhibits monotonous shrinkage into a compact state. Stretching experiments under direct current (DC) electrical potential revealed that single DNA molecules intermediately shrunk by 1- and 2-propanol exhibit intrachain phase segregation, i.e., coexistence of elongated and compact parts. The characteristic effect of 1-propanol causing the reentrant transition is argued in terms of the generation of water-rich nanoclusters.

Observation of Ethanol-Induced Condensation and Decondensation Processes at a Single-DNA Molecular Level in Microfluidic Devices Equipped with a Rapid Solution Exchange System

Conformational transitions from secondary (e.g., B- to A-form DNA) to higher-order (e.g., coil to globule) transitions play important roles in genome expression and maintenance. Several single-molecule approaches using microfluidic devices have been used to determine the kinetics of DNA chromatin assembly because microfluidic devices can afford stretched DNA molecules through laminar flow and rapid solution exchange. However, some issues, particularly the uncertainty of time 0 in the solution exchange process, are encountered. In such kinetic experiments, it is critical to determine when the target solution front approaches the target DNA molecules. Therefore, a new design for a microfluidic device is developed that enables the instantaneous exchange of solutions in the observation channel, allowing accurate measurements of DNA conformational transitions; stepwise, ethanol-induced conformational transitions are revealed. Although full DNA contraction from coil to globule is observed with >50% ethanol, no outstanding change is observed at concentrations <40% in 10 min. With 50% ethanol solution, the DNA conformational transition passes through two steps: (i) fast and constant-velocity contraction and (ii) relatively slow contraction from the free end. The first process is attributed to the B to A conformational transition by gradual dehydration. The second process is due to the coil-globule transition as the free end of DNA starts the contraction. This globular structure formation counteracts the shear force from the microfluids and decelerates the contraction velocity. This real-time observation system can be applied to the kinetic analysis of DNA conformational transitions such as kinetics of chromatin assembly and gene expression.

Unraveling the B to A Conformational Transition in Duplex DNA at Single Base Pair Resolution

DNA shows conformational heterogeneity and the interest in non‐canonical DNA conformations is increasing as these conformers are found to be involved in many important biological process. Although triple helices, quadruple helices and supercoils are the most studied structures, the conformational transition of the standard B‐form DNA helix to A‐form DNA was the first observed one. The transition of B‐DNA double helix to A‐form is essential for biological functions as recognized by the presence of A‐form DNA in many protein‐DNA complexes. Even though it is known that the B to A conformational transition occurs, the specifics like where in the DNA it originates, how it propagates, and the detailed step‐by‐step mechanism involved is still unknown. By using site specifically positioned fluorescent oligonucleotides, we explored the local and global conformational changes in this transition. Our results showed that by using 2‐Aminopurine (2‐AP), a fluorescent analogue of Adenine, we could monitor the local and global conformational change simultaneously. As a next step our aim is to introduce spermine or spermidine to the system at different ethanol percentages and monitor the spectral changes at various intervals of this transition. This will help us to understand the fundamentals of the long known B to A DNA conformational transition.

Zinc(II) Terpyridine Complexes: Substituent Effect on Photoluminescence, Antiproliferative Activity, and DNA Interaction.

A series of ZnCl2 complexes (compounds 1–10) with 4′-(substituted-phenyl)-2,2′:6′,2′′-terpyridine that bears hydrogen (L1), p-methyl (L2), p-methoxy (L3), p-phenyl (L4), p-tolyl (L5), p-hydroxyl (L6), m-hydroxyl (L7), o-hydroxyl (L8), p-carboxyl (L9), or p-methylsulfonyl (L10) were prepared and then characterized by 1H NMR, electrospray mass-spectra (ESI-MS), IR, elemental analysis, and single crystal X-ray diffraction. In vitro cytotoxicity assay was used to monitor the antiproliferative activities against tumor cells. Absorption spectroscopy, fluorescence titration, circular dichroism spectroscopy, and molecular modeling studied the DNA interactions. All of the compounds display interesting photoluminescent properties and different maximal emission peaks due to the difference of the substituent groups. The cell viability studies indicate that the compounds have excellent antiproliferative activity against four human carcinoma cell lines, A549, Bel-7402, MCF-7, and Eca-109, with the lowest IC50 values of 0.33 (10), 0.66 (6), 0.37 (7), and 1.05 (7) μM, respectively. The spectrophotometric results reveal that the compounds have strong affinity binding with DNA as intercalator and induce DNA conformational transition. Molecular docking studies indicate that the binding is contributed by the π…π stacking and hydrogen bonds, providing an order of nucleotide sequence binding selectivity as ATGC > ATAT > GCGC. These compounds intercalate into the base pairs of the DNA of the tumor cells to affect their replication and transcription, and the process is supposed to play an important role in the anticancer mechanism.

Amyloid β and Tumorigenesis: Amyloid β-Induced Telomere Dysfunction in Tumor Cells

Amyloid β and Tumorigenesis: Amyloid β-Induced Telomere Dysfunction in Tumor Cells

Groove binding between ferulic acid and calf thymus DNA: spectroscopic methodology combined with chemometrics and molecular docking studies

Ferulic acid (FA), a dietary phenolic acid compound, is proved to possess numerous biological activities. Hence, this study was devoted to explore the interaction between FA and calf thymus DNA (ctDNA) by UV − vis absorption, fluorescence, circular dichroism (CD) spectroscopy combined with multivariate curve resolution-alternating least-squares (MCR − ALS) and molecular docking studies. The concentration curves and the pure spectra of compositions (FA, ctDNA and FA − ctDNA complex) were obtained by MCR − ALS approach to verify and monitor the interaction of FA with ctDNA. The groove binding mode between FA and ctDNA was confirmed by the results of melting analysis, viscosity measurements, single-stranded DNA experiments, and competitive studies. The binding constant of FA − ctDNA complex was 4.87 × 104 L mol−1 at 298 K. The values of enthalpy (ΔH°) and entropy (ΔS°) changes in the interaction were −16.24 kJ mol−1 and 35.02 J mol−1 K−1, respectively, indicating that the main binding forces were hydrogen bonds and hydrophobic interactions. The result of CD spectra suggested that a decrease in right-handed helicity of ctDNA was induced by FA and the DNA conformational transition from the B-form to the A-form. The results of docking indicated that FA binding with ctDNA in the minor groove. These findings may be conducive to understand the interaction mechanism of FA with ctDNA and the pharmacological effects of FA. Communicated by Ramaswamy H. Sarma

Сhiral and Racemic Fields Concept for Understanding of the Homochirality Origin, Asymmetric Catalysis, Chiral Superstructure Formation from Achiral Molecules, and B-Z DNA Conformational Transition

The four most important and well-studied phenomena of mirror symmetry breaking of molecules were analyzed for the first time in terms of available common features and regularities. Mirror symmetry breaking of the primary origin of biological homochirality requires the involvement of an external chiral inductor (environmental chirality). All reviewed mirror symmetry breaking phenomena were considered from that standpoint. A concept of chiral and racemic fields was highly helpful in this analysis. A chiral gravitational field in combination with a static magnetic field (Earth’s environmental conditions) may be regarded as a hypothetical long-term chiral inductor. Experimental evidences suggest a possible effect of the environmental chiral inductor as a chiral trigger on the mirror symmetry breaking effect. Also, this effect explains a conformational transition of the right-handed double DNA helix to the left-handed double DNA helix (B-Z DNA transition) as possible DNA damage.

Changes in Microenvironment Modulate the B- to A-DNA Transition.

B- to A-DNA transition is known to be sensitive to the macroscopic properties of the solution, such as salt and ethanol concentrations. Microenvironmental effects on DNA conformational transition have been broadly studied. Providing an intuitive picture of how DNA responds to environmental changes is, however, still needed. Analyzing the chemical equilibrium of B-to-A DNA transition at critical concentrations, employing explicit-solvent simulations, is envisioned to help understand such microenvironmental effects. In the present study, free-energy calculations characterizing the B- to A-DNA transition and the distribution of cations were carried out in solvents with different ethanol concentrations. With the addition of ethanol, the most stable structure of DNA changes from the B- to A-form, in agreement with previous experimental observation. In 60% ethanol, a chemical equilibrium is found, showing reversible transition between B- and A-DNA. Analysis of the microenvironment around DNA suggests that with the increase of ethanol concentration, the cations exhibit a significant tendency to move toward the backbone, and mobility of water molecules around the major groove and backbone decreases gradually, leading eventually to a B-to-A transition. The present results provide a free-energy view of DNA microenvironment and of the role of cation motion in the conformational transition.

Eu3+ as a luminescence probe in DNA studies: Structural and conformational implications.

Lanthanide ions are widely used as luminescent probes for structural studies of various biomolecules, including DNA. Latest developments of circularly polarized luminescence (CPL) methodology further boosted interest to luminescence techniques. However, an effect of the lanthanide probes themselves on the DNA structure and conformation was investigated only partially and not for all lanthanides. In the present work, we performed a detailed spectroscopic study of Eu3+ complexes with native double-stranded DNA and compared them to the relevant complexes with single-stranded DNA. We employed infrared (IR), vibrational circular dichroism (VCD) and electronic circular dichroism (ECD) spectroscopic methods to investigate Eu3+ effect on DNA structure and conformational transitions. It was shown that Eu3+ ions can induce significant alteration of the native DNA structure at the concentrations often used in luminescence studies. While no DNA denaturation was observed at these metal ion concentrations, significant unstacking of the base pairs and disordering of the sugar-phosphate backbone, partial appearance of the A-form backbone geometry, and DNA transition into condensed ψ-type form took place. Eu3+ binding to single-stranded DNA was more pronounced than the binding to double-stranded DNA. We detected the main Eu3+ binding sites and determined the metal ion concentration range in which DNA geometry remains largely unaltered. The results obtained in the current study could be used for tuning the luminescence and CPL structural studies of DNA utilizing Eu3+ ions as probes.