DNA Condensation Process Research Articles

Isothermal calorimetry studies of DNA condensation under osmotic pressure.

The physics behind DNA condensation has been a subject of research for many years. Despite this, the exact mechanism under the condensed process is still not fully understood. Isothermal calorimetry introduces a unique perspective by providing the full thermodynamics parameter that drives the condensation process. Here we report on isothermal calorimetry studies of DNA condensation by trivalent ions. We investigate this process while altering the osmotic pressure through the addition of polyethylene glycol. We use a home-built Python script to fit the two-peek signal and obtain the full thermodynamics parameter. We find a shift in the condensation peak as a function of osmotic pressure indicating osmotic pressure enhances condensation. These results are key to understanding the basic process of DNA condensation and specifically with the surrounding by osmotic pressure.

DNA Condensation Triggered by the Synergistic Self-Assembly of Tetraphenylethylene-Viologen Aggregates and CT-DNA.

Development of small organic chromophores as DNA condensing agents, which explore supramolecular interactions and absorbance or fluorescence-based tracking of condensation and gene delivery processes, is in the initial stages. Herein, we report the synthesis and electrostatic/groove binding interaction–directed synergistic self-assembly of the aggregates of two viologen-functionalized tetraphenylethylene (TPE-V) molecules with CT-DNA and subsequent concentration-dependent DNA condensation process. TPE-V molecules differ in their chemical structure according to the number of viologen units. Photophysical and morphological studies have revealed the interaction of the aggregates of TPE-V in Tris buffer with CT-DNA, which transforms the fibrous network structure of CT-DNA to partially condensed beads-on-a-string-like arrangement with TPE-V aggregates as beads via electrostatic and groove binding interactions. Upon further increasing the concentration of TPE-V, the “beads-on-a-string”-type assembly of TPE-V/CT-DNA complex changes to completely condensed compact structures with 40–50 nm in diameter through the effective charge neutralization process. Enhancement in the melting temperature of CT-DNA, quenching of the fluorescence emission of ethidium bromide/CT-DNA complex, and the formation of induced CD signal in the presence of TPE-V molecules support the observed morphological changes and thereby verify the DNA condensation abilities of TPE-V molecules. Decrease in the hydrodynamic size, increase in the zeta potential value with the addition of TPE-V molecules to CT-DNA, failure of TPE-V/cucurbit(8)uril complex to condense CT-DNA, and the enhanced DNA condensation ability of TPE-V2 with two viologen units compared to TPE-V1 with a single viologen unit confirm the importance of positively charged viologen units in the DNA condensation process. Initial cytotoxicity analysis on A549 cancer and WI-38 normal cells revealed that these DNA condensing agents are non-toxic in nature and hence could be utilized in further cellular delivery studies.

Threading Intercalator-Induced Nanocondensates and Role of Endogenous Metal Ions in Decondensation for DNA Delivery.

The interplay of condensation and decondensation of DNA plays a crucial role in chromosome maintenance and gene expression. The molecular architectonics governing the chromatin condensation-decondensation cycle are worth studying, as DNA performs unique and distinct roles in each state and switches between two states without the loss of structural and functional integrity. This phenomenon has been adapted and implemented in transfection studies. Effective gene delivery into the cells to achieve respectable transfection efficiency has remained a challenge and emphasizes the need for understanding the steps involved in DNA delivery and transfection. Especially, recognizing the factors that effectively regulate DNA decondensation can provide logical solutions to the hurdles affecting the transfection efficiency. We designed a set of small molecule-based threading intercalation ligands as model condensing agents to study various factors influencing the DNA condensation and decondensation process. This study revealed condensation of DNA into nanocondensate by the threading intercalator and endogenous stimuli induced effective decondensation. Further, DNA nanocondensates are tracked using the intrinsic fluorescence in the lower pH of endocytic pathway and were evaluated as nonviral vectors for in cellulo delivery of plasmids. The correlation of decondensation of DNA nanocondensate with endogenous metal ions at their physiological concentrations provided valuable insights and implications for intracellular DNA delivery.

Transcriptional supercoiling boosts topoisomerase II-mediated knotting of intracellular DNA.

Recent studies have revealed that the DNA cross-inversion mechanism of topoisomerase II (topo II) not only removes DNA supercoils and DNA replication intertwines, but also produces small amounts of DNA knots within the clusters of nucleosomes that conform to eukaryotic chromatin. Here, we examine how transcriptional supercoiling of intracellular DNA affects the occurrence of these knots. We show that although (−) supercoiling does not change the basal DNA knotting probability, (+) supercoiling of DNA generated in front of the transcribing complexes increases DNA knot formation over 25-fold. The increase of topo II-mediated DNA knotting occurs both upon accumulation of (+) supercoiling in topoisomerase-deficient cells and during normal transcriptional supercoiling of DNA in TOP1 TOP2 cells. We also show that the high knotting probability (Pkn ≥ 0.5) of (+) supercoiled DNA reflects a 5-fold volume compaction of the nucleosomal fibers in vivo. Our findings indicate that topo II-mediated DNA knotting could be inherent to transcriptional supercoiling of DNA and other chromatin condensation processes and establish, therefore, a new crucial role of topoisomerase II in resetting the knotting–unknotting homeostasis of DNA during chromatin dynamics.

Effect of Torsion on Cisplatin-Induced DNA Condensation**Supported by the National Natural Science Foundation of China under Grant Nos 11674383, 11774407 and 11874415, the Key Research Program of Frontier Sciences of Chinese Academy of Sciences under Grant No QYZDB-SSW-SLH045, the National Key Research and Development Program under Grant No 2016YFA0301500, the Higher Educational Science and Technology Program of Shandong Province under

We investigate the effect of torsion on DNA condensation with the covalently closed circular DNA as the torsion-constrained system, using an atomic force microscope. It is found that there are two stages in the DNA condensation process under torsional constraint. At the early stage, the low torsion will accelerate DNA condensation by promoting the formation of micro-loops or intersection structures; while at the later stage, the increasing torsion will slow it down by preventing the crosslinking of cisplatin and DNA since the DNA molecule becomes more rigid. Our results show the important role of torsion in DNA condensation and sheds new light on the mechanism for DNA condensation.

DOPE–oleic acid–Ca2+ as DNA condensing agent

Abstract Phospholipid-based non-viral carriers composed of neutral phospholipid dioleoylphosphatidylethanolamine (DOPE) and the binary mixture DOPE–oleic acid (OA) are examined as potential DNA delivery vectors. The process of DNA condensation in the presence of Ca2+ ions has been monitored through changes in emmision intensity of fluorescent probe ethidium bromide. The decline in fluorescence intensity with increasing Ca2+ concentration at two different time intervals was correlated with the binding capacity of complexes and possible release of DNA from the complex. The microstructure of DOPE–OA mixtures at different OA/DOPE molar ratios and that of DOPE–OA–DNA–Ca2+ complexes were determined using synchrotron small angle X-ray diffraction (SAXD). We identified inverted hexagonal phase HII as the dominant structure. OA affects the lattice parameter of HII formed by DOPE. With the increasing OA/DOPE molar ratio, the lattice parameter decreases, which results in significantly lower fraction of DNA bound to the OA-enriched complexes.

DNA–RNA interactions are critical for chromosome condensation in Escherichia coli

Bacterial chromosome (nucleoid) conformation dictates faithful regulation of gene transcription. The conformation is condition-dependent and is guided by several nucleoid-associated proteins (NAPs) and at least one nucleoid-associated noncoding RNA, naRNA4. Here we investigated the molecular mechanism of how naRNA4 and the major NAP, HU, acting together organize the chromosome structure by establishing multiple DNA-DNA contacts (DNA condensation). We demonstrate that naRNA4 uniquely acts by forming complexes that may not involve long stretches of DNA-RNA hybrid. Also, uncommonly, HU, a chromosome-associated protein that is essential in the DNA-RNA interactions, is not present in the final complex. Thus, HU plays a catalytic (chaperone) role in the naRNA4-mediated DNA condensation process.

Doxorubicin hinders DNA condensation promoted by the protein bovine serum albumin (BSA).

In this work, we have studied the interaction between the anticancer drug doxorubicin (doxo) and condensed DNA, using optical tweezers. To perform this task, we use the protein bovine serum albumin (BSA) in the working buffer to mimic two key conditions present in the real intracellular environment: the condensed state of the DNA and the abundant presence of charged macromolecules in the surrounding medium. In particular, we have found that, when doxo is previously intercalated in disperse DNA, the drug hinders the DNA condensation process upon the addition of BSA in the buffer. On the other hand, when bare DNA is firstly condensed by BSA, doxo is capable to intercalate and to unfold the DNA condensates at relatively high concentrations. In addition, a specific interaction between BSA and doxo was verified, which significantly changes the chemical equilibrium of the DNA-doxo interaction. Finally, the presence of BSA in the buffer stabilizes the double-helix structure of the DNA-doxo complexes, preventing partial DNA denaturation induced by the stretching forces.

Microscopic insight into the DNA condensation process of a zwitterion-functionalized polycation.

Zwitterion-functionalized polycations are ideal gene carriers with long circulation, high cellular uptaking and low cell viability. However, the trade-off between the DNA condensation efficiency and the cell viability must be addressed. The purpose of this study is to provide a microscopic insight into the DNA condensation process and to explore the effect of a zwitterionic block of zwitterion-functionalized polycation, which is of great significance in designing novel gene delivery systems. Poly[2-(dimethylamino)ethyl methacrylate-b-(sulfobetaine methacrylate)] (PDMAEMA-b-PSBMA) copolymers were synthesized and used as the model systems. Different from the conventional concept that the PSBMA zwitterionic block act only as the "stealthy" groups, the subtle differences in physical and colloidal characteristics between the polycation/DNA polyplexes show that the PSBMA segment is capable of wrapping DNA attributed to the quaternary ammonium cations, without compromising the DNA condensation capability. On the other hand, the incorporation of PSBMA block reduces the surface charge of the polyplexes, which substantially result in the inefficient transfection and the reduced cytotoxicity.

12]aneN3 Modified Tetraphenylethene Molecules as High-Performance Sensing, Condensing, and Delivering Agents toward DNAs.

Four [12]aneN3 modified tetraphenylethene (TPE) compounds with different numbers of polyamine units and structure configurations, namely 1, 2, 3, and 4, were designed and synthesized. All compounds showed strong aggregation-induced emission (AIE) features. Compounds 2 and 4 showed significant emission enhancement after the addition of ssDNAs and dsDNAs of different lengths as well as calf thymus DNA (ctDNA). Compounds 1 and 3 showed very poor fluorescent responses toward DNAs. Gel electrophoresis demonstrated the abilities of 1-4 to condense DNA effectively. Complete retardation of plasmid DNA can be achieved at a concentration of 25 μM (1), 8 μM (for 2 and 3) and 4 μM (4). Experiments including fluorescent contrastive titrations, scanning electron microscopy, dynamic laser scattering, EB displacement, and gel electrophoresis demonstrated that the four compounds were able to integrate with DNA through electrostatic interactions and supramolecular stacking. A vicinal configuration around TPE (2) and more triazole-[12]aneN3 recognition sites (4) evidently enhanced the sensing capability toward oligonucleotides, and the TPE unit played an important role in the plasmid DNA condensation process because of its strong binding. With the advantages of low cytotoxicity, effective DNA sensing, and DNA condensing properties, compound 4 was successfully applied as a nonviral DNA vector and fluorescent tracer for label-free gene delivery, which is the first example of a nonviral gene vector with AIE activity.

Effects of Ionic Dependence of DNA Persistence Length on the DNA Condensation at Room Temperature**Supported by National Natural Science Foundation of China under Grant Nos. 11047022, 11204045, 11464004 and 31360215; The Research Foundation from Ministry of Education of China (212152), Guizhou Provincial Tracking Key Program of Social Development (SY20123089, SZ20113069); The General Financial Grant from the China Postdoctoral Science Foundation

DNA persistence length is a key parameter for quantitative interpretation of the conformational properties of DNA and related to the bending rigidity of DNA. A series of experiments pointed out that, in the DNA condensation process by multivalent cations, the condensed DNA takes elongated coil or compact globule states and the population of the compact globule states increases with an increase in ionic concentration. At the same time, single molecule experiments carried out in solution with multivalent cations (such as spermidine, spermine) indicated that DNA persistence length strongly depends on the ionic concentration. In order to revolve the effects of ionic concentration dependence of persistence length on DNA condensation, a model including the ionic concentration dependence of persistence length and strong correlation of multivalent cation on DNA is provided. The autocorrelation function of the tangent vectors is found as an effective way to detect the ionic concentration dependence of toroidal conformations. With an increase in ion concentration, the first periodic oscillation contained in the autocorrelation function shifts, the number of segment contained in the first periodic oscillation decreases gradually. According to the experiments, the average long-axis length is defined to estimate the ionic concentration dependence of condensation process further. The relation between long-axis length and ionic concentration matches the experimental results qualitatively.

Single molecular study on interactions between avidin and DNA

Avidin is a common basic protein, widely used for connecting DNA and modified surface in single-molecule techniques of biophysics, and it can also be used as a DNA vector in gene therapy. Avidin is highly positively charged and can condense DNA in solution. Understanding the physical mechanism of its condensing DNA is a key factor to promote avidin-DNA complex to be used for many purposes, such as a probe of biomacromlecules, signal enhancer or carrier of disease diagnosis.In the present study, we use atomic force microscope (AFM), dynamic light scattering (DLS), and single molecular magnetic tweezers (MT) to systematically investigate the interaction between DNA and avidin and the underlying mechanism of DNA condensation by avidin. The conformation of DNA-avidin complex is observed and measured by AFM and we find that the condensation includes two types: one is toroidal condensation of DNA induced by avidin, the other is the condensing structure by avidin compaction. Quantitative analysis shows that the size of avidin-DNA complex decreases monotonically with the concentration of avidin increasing. However, when the concentration of avidin reaches up to a critical value of 2 ngL-1, the size of complex begins to increase suddenly with avidin concentration increasing. The phenomenon is also confirmed by the corresponding DLS measurements. For example, when the concentration of avidin increases from 0 to 2 ngL-1, the size of condensed avidin-DNA complex reduces from 170 nm to about 125 nm. In the mean while, its electrophoretic mobility changes from -2.76 (10-4cm2V-1s-1) to -0.1 (10-4 cm2V-1s-1). The negative charge of DNA is mostly neutralized by avidin. From their force spectroscopy measured by MT, it is found that the extension of DNA varies almost linearly and a few stairlike jumps appear occasionally. For example, its characteristic trend is quite similar to the one by histones. The condensing force of DNA by avidin grows up with the concentration of avidin increasing. The statistics of force-extension curves by MT shows that the peak of unraveling steps of avidin-DNA complex is around 160 nm, which corresponds to the typical toroidal structure of DNA.In DNA condensation by avidin, electrostatic interaction plays a key role due to the neutralization of negatively charged phosphate groups of DNA by cationic avidin. From the comprehensive data by AFM, DLS and MT, we conclude that the process of DNA condensation induced by avidin consists of two mechnisms: the predominant DNA-avidin electrostatic attraction and the ancillary avidin aggregation.

Dependence of a DNA globule size in a gas phase on the chain length

Modern trends in the use of DNA in nanotechnology and biotechnology are aiming to develop new methods of analysis of DNA molecules based on the developing instrument base. We have developed a method of mild nondestructive ablation to transfer DNA molecules into an aerosol phase using terahertz radiation. In this paper, the size of DNA nanoparticles in the gas phase was measured using the diffusion aerosol spectrometer.?The changes taking place with DNA in the gas phase were visualized using the atomic force microscopy (AFM). A comparison of the measurements of the diffusion sizes of the plasmid pUC18 aerosol particles with measurements using AFM gives grounds to assume that the process of condensation of the DNA molecules takes place in the gas phase. A model was constructed according to modern concepts of the process of DNA condensation and formation of globules. The theoretical calculations are in good agreement with the experimental results. The experimentally estimated persistence length of the DNA in the gas phase was about 0.5 nm, which indicates the absence of a distributed charge on the surface of the DNA in the gas phase and the nonionizing nature of terahertz radiation. The study of DNA conformation in the gas phase will expand knowledge of DNA compaction in natural and artificial conditions.

Improved DNA condensation, stability, and transfection with alkyl sulfonyl-functionalized PAMAM G2

In this work, we have used a second-generation PAMAM grafted with octadecyl sulfonyl chains to condense plasmid DNA. The influence of this modification at different levels was investigated by comparison with original PAMAM G2. The condensation process and temporal stability of the complexes was studied with DLS, finding that the aliphatic chains influence DNA compaction via hydrophobic forces and markedly improve the formation and temporal stability of a single populated system with a hydrodynamic diameter below 100 nm. Interaction with a cell membrane model was also evaluated with a pendant drop tensiometer, resulting in further incorporation of the C18-PAMAM dendriplexes onto the interface. The improvement observed in transfection with our C18 grafted PAMAM is ascribed to the size, stability, and interfacial behavior of the complexes, which in turn are consequence of the DNA condensation process and the interactions involved.

On the Effects of Intercalators in DNA Condensation: A Force Spectroscopy and Gel Electrophoresis Study

In this work we have characterized the effects of the intercalator ethidium bromide (EtBr) on the DNA condensation process by using force spectroscopy and gel electrophoresis. We have tested two condensing agents: spermine (spm(4+)), a tetravalent cationic amine which promotes cation-induced DNA condensation, and poly(ethylene glycol) (PEG), a neutral polymer which promotes DNA ψ-condensation. Two different types of experiments were performed. In the first type, bare DNA molecules disperse in solution are first treated with EtBr for intercalation, and then the condensing agent is added to the sample with the purpose of verifying the effects of the intercalator in hindering DNA condensation. In the second experiment type, the bare DNA molecules are first condensed, and then the intercalator is added to the sample in order to verify its influence on the previously condensed DNA. The results obtained with the two different experimental techniques used agree very well, indicating that previously intercalated EtBr can hinder both cation-induced and ψ-condensation, being more efficient in the first case. On the other hand, EtBr has little effect on the previously formed cation-induced condensates, but is efficient in unfolding the ψ-condensates.

A fluoride-sensor for kink structure in DNA condensation process

Bloomfield has pointed out that the kink structure occurs for sharp bending during DNA condensation process, until now, which has not been proved by experiments. Using UV Spectrophotometer, the effects of fluoride and chlorine on the polyamine-DNA condensation system can be detected. Fluoride and chlorine both belong to the halogen family, but their effects on spermine-DNA condensation system are totally different. Fluoride ions make blue-shift and hyperchromicity appear in the spermine-DNA condensation system, but chlorine ions only make insignificant hyperchromicity happen in this system. Both fluoride ions and chlorine ions only make insignificant hyperchromicity happen in spermidine-DNA condensation system. Based on the distinguished character of fluoride, a fluoride-sensor for "kink" structure in DNA condensation was developed and the second kind of "kink" structure only appear in the spermine-DNA condensation system.

Syntheses of bifunctional molecules containing [12]aneN3 and carbazol moieties as effective DNA condensation agents

Bifunctional molecules containing macrocyclic polyamine [12]aneN3 and carbazol units, 1–4, have been efficiently synthesized and fully characterized. Through gel electrophoresis, atomic force microscopy, and dynamic light scattering experiments, compounds 3 and 4b bearing both [12]aneN3 and carbazol moieties showed effective DNA condensation ability at the concentration of 80 μM. Investigations from EB displacement fluorescence spectra, viscosity titration, and ionic strength effects revealed that the effective DNA condensation comes from the appropriate combination of carbazol and [12]aneN3 units in the bifunctional molecules, and the DNA condensation process is reversible. The incorporation of triazole units in the molecules clearly reduced the cytotoxicity.

Processes of DNA condensation induced by multivalent cations: Approximate annealing experiments and molecular dynamics simulations

The condensation of DNA induced by spermine is studied by atomic force microscopy (AFM) and molecular dynamics (MD) simulation in this paper. In our experiments, an equivalent amount of multivalent cations is added to the DNA solutions in different numbers of steps, and we find that the process of DNA condensation strongly depends on the speed of adding cations. That is, the slower the spermine cations are added, the slower the DNA aggregates. The MD and steered molecular dynamics (SMD) simulation results agree well with the experimental results, and the simulation data also show that the more steps of adding multivalent cations there are, the more compact the condensed DNA structure will be. This investigation can help us to control DNA condensation and understand the complicated structures of DNA—cation complexes.

DNA condensation and size effects of DNA condensation agent

Based on the model of the strong correlation of counterions condensed on DNA molecule, by tailoring interaction potential, interduplex spacing and correlation spacing between condensed counterions on DNA molecule and interduplex spacing fluctuation strength, toroidal configuration, rod-like configuration and two-hole configurations are possible. The size effects of counterion structure on the toroidal structure can be detected by this model. The autocorrelation function of the tangent vectors is found as an effective way to detect the structure of toroidal conformations and the generic pathway of the process of DNA condensation. The generic pathway of all of the configurations involves an initial nucleation loop, and the next part of the DNA chain is folded on the top of the initial nucleation loop with different manners, in agreement with the recent experimental results.

Nanostructure-induced DNA condensation

The control of the DNA condensation process is essential for compaction of DNA in chromatin, as well as for biological applications such as nonviral gene therapy. This review endeavours to reflect the progress of investigations on DNA condensation effects of nanostructure-based condensing agents (such as nanoparticles, nanotubes, cationic polymer and peptide agents) observed by using atomic force microscopy (AFM) and other techniques. The environmental effects on structural characteristics of nanostructure-induced DNA condensates are also discussed.