Dynamic Light Scattering of DNA-Ligand Complexes.

Dynamic light scattering (DLS) enables the characterization of sizes and electrokinetic properties of colloids, polymers, and macromolecules. DNA is a charged semiflexible polyelectrolyte that is condensed or compacted by counterions, proteins, and other condensing agents in processes such as chromosome compaction and gene therapeutic applications. DNA condensation is closely related to charge screening since packaging requires effective neutralization of its surface negative charges. In this chapter, we describe in detail the protocol for DLS DNA-ligand complexes. As an example, we describe data for the condensation of DNA by chitosan and the measurement of size, zeta potential, and electrophoretic mobility of the DNA-ligand complex by DLS.

Quantitative morphological analysis of Deinococcus radiodurans elucidates complex dose-dependent nucleoid condensation during recovery from ionizing radiation.

The extremophile Deinococcus radiodurans maintains a highly organized and condensed nucleoid as its default state, possibly contributing to its high tolerance to ionizing radiation (IR). Previous studies of the D. radiodurans nucleoid were limited by reliance on manual image annotation and qualitative metrics. Here, we introduce a high-throughput approach to quantify the geometric properties of cells and nucleoids using confocal microscopy, digital reconstructions of cells, and computational modeling. We utilize this novel approach to investigate the dynamic process of nucleoid condensation in response to IR stress. Our quantitative analysis reveals that at the population level, exposure to IR induced nucleoid compaction and decreased the size of D. radiodurans cells. Morphological analysis and clustering identified six distinct sub-populations across all tested experimental conditions. Results indicate that exposure to IR induced fractional redistributions of cells across sub-populations to exhibit morphologies associated with greater nucleoid condensation and decreased the abundance of sub-populations associated with cell division. Nucleoid-associated proteins (NAPs) may link nucleoid compaction and stress tolerance, but their roles in regulating compaction in D. radiodurans are unknown. Imaging of genomic mutants of known and suspected NAPs that contribute to nucleoid condensation found that deletion of nucleic acid-binding proteins, not previously described as NAPs, can remodel the nucleoid by driving condensation or decondensation in the absence of stress and that IR increased the abundance of these morphological states. Thus, our integrated analysis introduces a new methodology for studying environmental influences on bacterial nucleoids and provides an opportunity to further investigate potential regulators of nucleoid condensation.IMPORTANCEDeinococcus radiodurans, an extremophile known for its stress tolerance, constitutively maintains a highly condensed nucleoid. Qualitative studies have described nucleoid behavior under a variety of conditions. However, a lack of quantitative data regarding nucleoid organization and dynamics has limited our understanding of the regulatory mechanisms controlling nucleoid organization in D. radiodurans. Here, we introduce a quantitative approach that enables high-throughput quantitative measurements of subcellular spatial characteristics in bacterial cells. Applying this to wild-type or single-protein-deficient populations of D. radiodurans subjected to ionizing radiation, we identified significant stress-responsive changes in cell shape, nucleoid organization, and morphology. These findings highlight this methodology's adaptability and capacity for quantitatively analyzing the cellular response to stressors for screening cellular proteins involved in bacterial nucleoid organization.

DNA-based nanosized functional ruthenium(II) complex as potential phosphorescent probe to track tumor cells.

Identifying tumor cells can be challenging due to cancer's complex and heterogeneous nature. Here, an efficacious phosphorescent probe that can precisely highlight tumor cells has been created. By combining the ruthenium(II) complex with oligonucleotides, we have developed a nanosized functional ruthenium(II) complex (Ru@DNA) with dimensions ranging from 300 to 500 nm. Our research demonstrates that Ru@DNA can readily traverse biomembranes via ATP-dependent endocytosis without carriers. Notably, the nanosized ruthenium(II) complex exhibits rapid and selective accumulation within tumor cells, possibly attributed to the nanoparticles' enhanced permeation and retention (EPR) effect. Ru@DNA can also effectively discern and label the transplanted cancer cells in the zebrafish model. Moreover, Ru@DNA is efficiently absorbed into the intestine and further distributed in the pancreas. Our findings underscore the potential of Ru@DNA as a DNA-based nanodevice derived from a functional ruthenium(II) complex. This innovative nanodevice holds promise as an efficient phosphorescent probe for both in vitro and in vivo imaging of living tumor cells.

Cationic Residues of the HIV-1 Nucleocapsid Protein Enable DNA Condensation to Maintain Viral Core Particle Stability during Reverse Transcription.

The HIV-1 nucleocapsid protein (NC) is a multifunctional viral protein necessary for HIV-1 replication. Recent studies have demonstrated that reverse transcription (RT) completes in the intact viral capsid, and the timing of RT and uncoating are correlated. How the small viral core stably contains the ~10 kbp double stranded (ds) DNA product of RT, and the role of NC in this process, are not well understood. We showed previously that NC binds and saturates dsDNA in a non-specific electrostatic binding mode that triggers uniform DNA self-attraction, condensing dsDNA into a tight globule against extending forces up to 10 pN. In this study, we use optical tweezers and atomic force microscopy to characterize the role of NC's basic residues in dsDNA condensation. Basic residue mutations of NC lead to defective interaction with the dsDNA substrate, with the constant force plateau condensation observed with wild-type (WT) NC missing or diminished. These results suggest that NC's high positive charge is essential to its dsDNA condensing activity, and electrostatic interactions involving NC's basic residues are responsible in large part for the conformation, size, and stability of the dsDNA-protein complex inside the viral core. We observe DNA re-solubilization and charge reversal in the presence of excess NC, consistent with the electrostatic nature of NC-induced DNA condensation. Previous studies of HIV-1 replication in the presence of the same cationic residue mutations in NC showed significant defects in both single- and multiple-round viral infectivity. Although NC participates in many stages of viral replication, our results are consistent with the hypothesis that cationic residue mutations inhibit genomic DNA condensation, resulting in increased premature capsid uncoating and contributing to viral replication defects.

Liquid-liquid phase separation (LLPS) in DNA and chromatin systems from the perspective of colloid physical chemistry.

DNA is a highly charged polyelectrolyte and is prone to associative phase separation driven by the presence of multivalent cations, charged surfactants, proteins, polymers and colloids. The process of DNA phase separation induced by positively charged species is often called DNA condensation. Generally, it refers to either intramolecular DNA compaction (coil-globule transition) or intermolecular DNA aggregation with macroscopic phase separation, but the formation of a DNA liquid crystalline system is also displayed. This has traditionally been described by polyelectrolyte theory and qualitative (Flory-Huggins-based) polymer theory approaches. DNA in the cell nucleus is packed into chromatin wound around the histone octamer (a protein complex comprising two copies each of the four histone proteins H2A, H2B, H3 and H4) to form nucleosomes separated by linker DNA. During the last decade, the phenomenon of the formation of biomolecular condensates (dynamic droplets) by liquid-liquid phase separation (LLPS) has emerged as a generally important mechanism for the formation of membraneless organelles from proteins, nucleic acids and their complexes. DNA and chromatin droplet formation through LLPS has recently received much attention by in vitro as well as in vivo studies that established the importance of this for compartmentalisation in the cell nucleus. Here, we review DNA and chromatin LLPS from a general colloid physical chemistry perspective. We start with a general discussion of colloidal phase separation in aqueous solutions and review the original (pre-LLPS era) work on DNA (macroscopic) phase separation for simpler systems with DNA in the presence of multivalent cations and well-defined surfactants and colloids. Following that, we discuss and illustrate the similarities of such macroscopic phase separation with the general behaviour of LLPS droplet formation by associative phase separation for DNA-protein systems, including chromatin; we also note cases of segregative association. The review ends with a discussion of chromatin LLPS in vivo and its physiological significance.

Cytotoxicity of poly-guanidine in medulloblastoma cell lines.

Medulloblastoma (MB) is the most common pediatric brain tumor. The therapy frequently causes serious side effects, and new selective therapies are needed. MB expresses hyper sialylation, a possible target for selective therapy. The cytotoxic efficacy of a poly guanidine conjugate (GuaDex) incubated with medulloblastoma cell cultures (DAOY and MB-LU-181) was investigated. The cells were incubated with 0.05-8 µM GuaDex from 15 min to 72 h. A fluorometric cytotoxicity assay (FMCA) measured the cytotoxicity. Labeled GuaDex was used to study tumor cell interaction. FITC-label Sambucus nigra confirmed high expression of sialic acid (Sia). Immunofluorescence microscopy was used to visualize the cell F-actin and microtubules. The cell interactions were studied by confocal and fluorescence microscopy. Annexin-V assay was used to detect apoptosis. Cell cycle analysis was done by DNA content determination. A wound-healing migration assay determined the effects on the migratory ability of DAOY cells after GuaDex treatment. IC50 for GuaDex was 223.4 -281.1 nM. FMCA showed potent growth inhibition on DAOY and MB-LU-181 cells at 5 uM GuaDex after 4 h of incubation. GuaDex treatment induced G2/M phase cell cycle arrest. S. nigra FITC-label lectin confirmed high expression of Sia on DAOY medulloblastoma cells. The GuaDex treatment polymerized the cytoskeleton (actin filaments and microtubules) and bound to DNA, inducing condensation. The Annexin V assay results were negative. Cell migration was inhibited at 0.5 µM GuaDex concentration after 24 h of incubation. GuaDex showed potent cytotoxicity and invasion-inhibitory effects on medulloblastoma cells at low micromolar concentrations. GuaDex efficacy was significant and warrants further studies.

Packaging of DNA Integrated with Metal Nanoparticles in Solution.

The transformation of high-molecular DNA from a random swollen coil in a solution to a discrete nanosized particle with the ordered packaging of a rigid and highly charged double-stranded molecule is one of the amazing phenomena of polymer physics. DNA condensation is a well-known phenomenon in biological systems, yet its molecular mechanism is not clear. Understanding the processes occurring in vivo is necessary for the usage of DNA in the fabrication of new biologically significant nanostructures. Entropy plays a very important role in DNA condensation. DNA conjugates with metal nanoparticles are useful in various fields of nanotechnology. In particular, they can serve as a basis for creating multicomponent nanoplatforms for theranostics. DNA must be in a compact state in such constructions. In this paper, we tested the methods of DNA integration with silver, gold and palladium nanoparticles and analyzed the properties of DNA conjugates with metal nanoparticles using the methods of atomic force microscopy, spectroscopy, viscometry and dynamic light scattering. DNA size, stability and rigidity (persistence length), as well as plasmon resonance peaks in the absorption spectra of systems were studied. The methods for DNA condensation with metal nanoparticles were analyzed.

DNA Morphology: Global Alteration of DNA Topological States Induced by Chemotherapeutic Agents and Its Implication in Cancer.

The dynamic topological states of chromosomal DNA regulate many cellular fundamental processes universally in all three domains of life, that is, bacteria, archaea, and eukaryotes. DNA-binding proteins maintain the regional and global supercoiling of the chromosome and thereby regulate the chromatin architecture that ultimately influences the gene expression network and other DNA-centric molecular events in various microenvironments and growth phases. DNA-binding small molecules are pivotal weapons for treating a wide range of cancers. Recent advances in single-molecule biophysical tools have uncovered the fact that many DNA-binding ligands not only alter the regional DNA supercoiling but also modulate the overall morphology of DNA. Here we provide insight into recent advances in atomic force microscopy (AFM) acquired DNA structural change induced by therapeutically important mono- and bis-intercalating anticancer agents as well as DNA-adduct-forming anticancer drugs. We also emphasize the growing evidence of the mechanistic relevance of changes in DNA topology in the anticancer cellular responses of DNA-targeting chemotherapeutic agents.

Spermine enhances antiviral and anticancer responses by stabilizing DNA binding with the DNA sensor cGAS.

Self-nonself discrimination is vital for the immune system to mount responses against pathogens while maintaining tolerance toward the host and innocuous commensals during homeostasis. Here, we investigated how indiscriminate DNA sensors, such as cyclic GMP-AMP synthase (cGAS), make this self-nonself distinction. Screening of a small-molecule library revealed that spermine, a well-known DNA condenser associated with viral DNA, markedly elevates cGAS activation. Mechanistically, spermine condenses DNA to enhance and stabilize cGAS-DNA binding, optimizing cGAS and downstream antiviral signaling. Spermine promotes condensation of viral, but not host nucleosome, DNA. Deletion of viral DNA-associated spermine, by propagating virus in spermine-deficient cells, reduced cGAS activation. Spermine depletion subsequently attenuated cGAS-mediated antiviral and anticancer immunity. Collectively, our results reveal a pathogenic DNA-associated molecular pattern that facilitates nonself recognition, linking metabolism and pathogen recognition.

Apoptosis Detection Assays.

Many apoptosis assays are available since there are many proteins regulated at multiple points and involved in apoptosis signaling cascade. To detect apoptosis accurately, two or more assays should be used since there are many overlapped features between apoptosis and necrosis. There are six major groups of available assays to detect apoptosis: membrane alteration, mitochondrial assays, cytomorphological alterations, DNA fragmentation, detection of caspase, cleaved substrate, inhibitors and regulators, and detection of apoptosis in whole mounts. Among those assay, early apoptosis could be detected through annexin V, which is based on the loss of the cellular membrane integrity. Also, there are many assays that can detect midphase of apoptosis using caspase activation and molecular processing including PARP degradation. Late phase of apoptosis could be detected with DNA fragmentation assays. Combinations of these assays allow us to identify the mechanisms of apoptosis induction after specific stimulus. This chapter will introduce three apoptosis detection assays including annexin assay, DNA/chromatin condensation assays, and TUNEL assay.

Steering DNA Condensation on Engineered Nanointerfaces.

DNA has received increasing attention in nanotechnology due to its ability to fold into prescribed structures. Different from the commonly adopted base-pairing strategy, an emerging class of amorphous DNA materials are formed by DNA's abiological interactions. Despite the great successes, a lack of nanoscale nucleation/growth control disables more advanced considerations. This work aims at harnessing the heterogeneous nucleation of metal-ion-glued DNA condensates on nanointerfaces. Upon unveiling key orthogonal factors including solution pH, ionic cross-linkers, and surface functionalities, chemically programmable DNA condensation on nanoparticle seeds is achieved, resembling a famous Stöber process for silica coating. The nucleation rules discovered on individual nanoseeds can be passed on to their dimeric assemblies, where broken spherical symmetry and the existence of interparticle gaps help a regiospecific DNA gelation. The steerable DNA condensation, and the multifunctions from DNA, metal ions, and nanocores, hold a great promise in noncanonical DNA nanotechnology toward novel applications.

Application of Special Electron Microscopy Techniques to the Study of DNA - Protein Complexes in E. coli Cells.

Various electron microscopy techniques were applied recently to the study of DNA condensation in dormant bacterial cells. Here, we describe, in detail, the preparation of dormant Escherichia coli cells for electron microscopy studies and electron tomography and energy dispersive spectroscopy (EDS) approaches, which were used to reveal the structures of DNA-protein complexes in dormant Escherichia coli cells.

Condensation and Protection of DNA by the Myxococcus xanthus Encapsulin: A Novel Function.

Encapsulins are protein nanocages capable of harboring smaller proteins (cargo proteins) within their cavity. The function of the encapsulin systems is related to the encapsulated cargo proteins. The Myxococcus xanthus encapsulin (EncA) naturally encapsulates ferritin-like proteins EncB and EncC as cargo, resulting in a large iron storage nanocompartment, able to accommodate up to 30,000 iron atoms per shell. In the present manuscript we describe the binding and protection of circular double stranded DNA (pUC19) by EncA using electrophoretic mobility shift assays (EMSA), atomic force microscopy (AFM), and DNase protection assays. EncA binds pUC19 with an apparent dissociation constant of 0.3 ± 0.1 µM and a Hill coefficient of 1.4 ± 0.1, while EncC alone showed no interaction with DNA. Accordingly, the EncAC complex displayed a similar DNA binding capacity as the EncA protein. The data suggest that initially, EncA converts the plasmid DNA from a supercoiled to a more relaxed form with a beads-on-a-string morphology. At higher concentrations, EncA self-aggregates, condensing the DNA. This process physically protects DNA from enzymatic digestion by DNase I. The secondary structure and thermal stability of EncA and the EncA-pUC19 complex were evaluated using synchrotron radiation circular dichroism (SRCD) spectroscopy. The overall secondary structure of EncA is maintained upon interaction with pUC19 while the melting temperature of the protein (Tm) slightly increased from 76 ± 1 °C to 79 ± 1 °C. Our work reports, for the first time, the in vitro capacity of an encapsulin shell to interact and protect plasmid DNA similarly to other protein nanocages that may be relevant in vivo.

An insight into the morphology of DNA compaction induced by homobinuclear Ru(II) polypyridyl complexes.

Binuclear Ru(II) polypyridyl complexes [Ru2(NN)4(BIPMB)]4+ (1-4), where N-N = 2,2'-bipyridine (bpy), 1,10-phenanthroline (phen), dipyrido [3,2-d:2',3'-f] quinoxaline (dpq), and dipyrido[3,2-a:2',3'-c]phenazine (dppz), have been synthesized using suitable precursors and bridging ligand (BIPMB), where BIPMB = 3,3'-bis-{(imidazol-1-yl)-[4,5-f]-1,10-phenanthroline) methyl}-1,1'-biphenyl. The binding mode and affinity of complexes 1-4 with Calf Thymus DNA (CT-DNA) were determined by absorption and steady-state fluorescence spectroscopy. The decrease in viscosity of CT-DNA on sequential addition of these complexes indicated DNA condensation and the result was corroborated by circular dichroism (CD). The nanosized globular aggregates of CT-DNA induced by complexes 1-4 were observed by dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements. The gel electrophoretic mobility studies revealed the small orderly particles of supercoiled plasmid pBR322 DNA induced by these complexes. Additionally, the morphology and size of compact plasmid DNA condensates were studied by DLS and atomic force microscopy (AFM). The complexes were moderately cytotoxic against MCF-7 cells.

Shelterin Components Modulate Nucleic Acids Condensation and Phase Separation in the Context of Telomeric DNA.

Telomeres are nucleoprotein complexes that protect the ends of chromosomes and are essential for chromosome stability in Eukaryotes. In cells, individual telomeres form distinct globules of finite size that appear to be smaller than expected for bare DNA. Moreover, telomeres can cluster together, form telomere-induced-foci or co-localize with promyelocytic leukemia (PML) nuclear bodies. The physical basis for collapse of individual telomeres and coalescence of multiple ones remains unclear, as does the relationship between these two phenomena. By combining single-molecule force spectroscopy measurements, optical microscopy, turbidity assays, and simulations, we show that the telomere scaffolding protein TRF2 can condense individual DNA chains and drives coalescence of multiple DNA molecules, leading to phase separation and the formation of liquid-like droplets. Addition of the TRF2 binding protein hRap1 modulates phase boundaries and tunes the specificity of solution demixing while simultaneously altering the degree of DNA compaction. Our results suggest that the condensation of single telomeres and formation of biomolecular condensates containing multiple telomeres are two different outcomes driven by the same set of molecular interactions. Moreover, binding partners, such as other telomere components, can alter those interactions to promote single-chain DNA compaction over multiple-chain phase separation.

HIV-1 Nucleocapsid Protein Binds Double-Stranded DNA in Multiple Modes to Regulate Compaction and Capsid Uncoating.

The HIV-1 nucleocapsid protein (NC) is a multi-functional protein necessary for viral replication. Recent studies have demonstrated reverse transcription occurs inside the fully intact viral capsid and that the timing of reverse transcription and uncoating are correlated. How a nearly 10 kbp viral DNA genome is stably contained within a narrow capsid with diameter similar to the persistence length of double-stranded (ds) DNA, and the role of NC in this process, are not well understood. In this study, we use optical tweezers, fluorescence imaging, and atomic force microscopy to observe NC binding a single long DNA substrate in multiple modes. We find that NC binds and saturates the DNA substrate in a non-specific binding mode that triggers uniform DNA self-attraction, condensing the DNA into a tight globule at a constant force up to 10 pN. When NC is removed from solution, the globule dissipates over time, but specifically-bound NC maintains long-range DNA looping that is less compact but highly stable. Both binding modes are additionally observed using AFM imaging. These results suggest multiple binding modes of NC compact DNA into a conformation compatible with reverse transcription, regulating the genomic pressure on the capsid and preventing premature uncoating.

DNA condensation and formation of ultrathin nanosheets via DNA assisted self-assembly of an amphiphilic fullerene derivative.

DNA nanotechnology propose various assembly strategies to develop novel functional nanostructures utilizing unique interactions of DNA with small molecules, nanoparticles, polymers, and other biomolecules. Although, well defined nanostructures of DNA and amphiphilic small molecules were achieved through hybridization of covalently modified DNA, attaining precise organization of functional moieties through non-covalent interactions remain as a challenging task. Herein, we report mutually assisted assembly of an amphiphilic fullerene derivative and various DNA structures through non-covalent interactions, which leads to initial DNA condensation and subsequent assembly yielding ordered fullerene-DNA nanosheets. The molecular design of the cationic, amphiphilic fullerene derivative (FPy) ensures molecular solubility in the 10% DMSO-PBS buffer system and facile interactions with DNA through groove binding and electrostatic interactions of fullerene moiety and positively charged pyridinium moiety, respectively. The formation of FPy/DNA nanostructures were thoroughly investigated in the presence of λ-DNA, pBR322 plasmid DNA, and single and double stranded 20-mer oligonucleotides using UV-visible spectroscopy, AFM and TEM analysis. λ-DNA and pBR322 plasmid DNA readily condense in presence of FPy leading to micrometer sized few layer nanosheets with significant crystallinity due to ordered arrangement of fullerenes. Similarly, single and double stranded 20-mer oligonucleotides also interact efficiently with FPy and form highly crystalline nanosheets, signifying the role of electrostatic interaction and subsequent charge neutralization in the condensation triggered assembly. However, there is significant differences in the crystallinity and ordered arrangements of fullerenes between these two cases, where longer DNA form condensed structures and less ordered nanosheets while short oligonucleotides lead to more ordered and highly crystalline nanosheets, which could be attributed to the differential DNA condensation. Finally, we have demonstrated the addressability of the assembly using a cyanine modified single strand DNA, which also forms highly crystalline nanosheets and exhibit efficient quenching of the cyanine fluorescence upon self-assembly. These results open up new prospects in the development of functional DNA nanostructures through non-covalent interactions and hence have potential applications in the context of DNA nanotechnology.

Allyl Isothiocyanate Induces DNA Damage and Impairs DNA Repair in Human Breast Cancer MCF-7 Cells.

BACKGROUND/AIM: Ally lisothiocyanate (AITC), a constituent of naturally occurring isothiocyanates (ITCs) found in some Brassica vegetables, has been previously demonstrated to have anti-carcinogenic activity. However, there is no available information showing that AITC induces DNA damage and alters DNA damage repair proteins in human breast cancer MCF-7 cells. MATERIALS AND METHODS: In the present study, we investigated the effects of AITC on DNA damage and repair responses in human breast cancer MCF-7 cells in vitro. Cell viability was measured by flow cytometric assay. DNA condensation (apoptotic cell death) and DNA fragmentation (laddered DNA) were assayed by DAPI staining and DNA gel electrophoresis assays, respectively. Furthermore, DNA damage (comet tail) was measured by the comet assay. Western blotting was used to measure the expression of DNA damage- and repair-associated proteins. RESULTS: AITC decreased cell viability in a dose-dependent and induced apoptotic cell death (DNA condensation and fragmentation) and DNA damage in MCF-7 cells. AITC increased p-ATMSer1981, p-ATRSer428, p53, p-p53Ser15, p-H2A.XSer139, BRCA1, and PARP at 10-30 µM at 24 and 48 h treatments. However, AITC decreased DNA-PK at 24 and 48 h treatment, and decreased MGMT at 48 h in MCF-7 cells. CONCLUSION: AITC induced cytotoxic effects (decreased viable cell number) through induction of DNA damage and condensation and altered DNA damage and repair associated proteins in MCF-7 cells in vitro.

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.