Quantification of single-strand DNA lesions caused by the topoisomerase II poison etoposide using single DNA molecule imaging.

DNA-damaging agents, such as radiation and chemotherapy, are common in cancer treatment, but the dosing has proven to be challenging, leading to severe side effects in some patients. Hence, to be able to personalize DNA-damaging chemotherapy, it is important to develop fast and reliable methods to measure the resulting DNA damage in patient cells. Here, we demonstrate how single DNA molecule imaging using fluorescence microscopy can quantify DNA-damage caused by the topoisomerase II (TopoII) poison etoposide. The assay uses an enzyme cocktail consisting of base excision repair (BER) enzymes to repair the DNA damage caused by etoposide and label the sites using a DNA polymerase and fluorescently labeled nucleotides. Using this DNA-damage detection assay we find a large variation in etoposide induced DNA-damage after in vitro treatment of blood cells from healthy individuals. We furthermore used the TopoII inhibitor ICRF-193 to show that the etoposide-induced damage in DNA was TopoII dependent. We discuss how our results support a potential future use of the assay for personalized dosing of chemotherapy.

R-Loop-Mediated ssDNA Breaks Accumulate Following Short-Term Exposure to the HDAC Inhibitor Romidepsin.

Histone deacetylase inhibitors (HDACi) induce hyperacetylation of histones by blocking HDAC catalytic sites. Despite regulatory approvals in hematological malignancies, limited solid tumor clinical activity has constrained their potential, arguing for better understanding of mechanisms of action (MOA). Multiple activities of HDACis have been demonstrated, dependent on cell context, beyond the canonical induction of gene expression. Here, using a clinically relevant exposure duration, we established DNA damage as the dominant signature using the NCI-60 cell line database and then focused on the mechanism by which hyperacetylation induces DNA damage. We identified accumulation of DNA-RNA hybrids (R-loops) following romidepsin-induced histone hyperacetylation, with single-stranded DNA (ssDNA) breaks detected by single-cell electrophoresis. Our data suggest that transcription-coupled base excision repair (BER) is involved in resolving ssDNA breaks that, when overwhelmed, evolve to lethal dsDNA breaks. We show that inhibition of BER proteins such as PARP will increase dsDNA breaks in this context. These studies establish accumulation of R-loops as a consequence of romidepsin-mediated histone hyperacetylation. We believe that the insights provided will inform design of more effective combination therapy with HDACis for treatment of solid tumors. IMPLICATIONS: Key HDAC inhibitor mechanisms of action remain unknown; we identify accumulation of DNA-RNA hybrids (R-loops) due to chromatin hyperacetylation that provokes single-stranded DNA damage as a first step toward cell death.

Obacunone protects retinal pigment epithelium cells from ultra-violet radiation-induced oxidative injury.

Ultra-violet (UV) radiation (UVR) causes significant oxidative injury to retinal pigment epithelium (RPE) cells. Obacunone is a highly oxygenated triterpenoid limonoid compound with various pharmacological properties. Its potential effect in RPE cells has not been studied thus far. Here in ARPE-19 cells and primary murine RPE cells, obacunone potently inhibited UVR-induced reactive oxygen species accumulation, mitochondrial depolarization, lipid peroxidation and single strand DNA accumulation. UVR-induced RPE cell death and apoptosis were largely alleviated by obacunone. Obacunone activated Nrf2 signaling cascade in RPE cells, causing Keap1-Nrf2 disassociation, Nrf2 protein stabilization and nuclear translocation. It promoted transcription and expression of antioxidant responsive element-dependent genes. Nrf2 silencing or CRISPR/Cas9-induced Nrf2 knockout almost reversed obacunone-induced RPE cytoprotection against UVR. Forced activation of Nrf2 cascade, by Keap1 knockout, similarly protected RPE cells from UVR. Importantly, obacunone failed to offer further RPE cytoprotection against UVR in Keap1-knockout cells. In vivo, intravitreal injection of obacunone largely inhibited light-induced retinal damage. Collectively, obacunone protects RPE cells from UVR-induced oxidative injury through activation of Nrf2 signaling cascade.

Impact of 5-formylcytosine on the melting kinetics of DNA by 1H NMR chemical exchange.

5-Formylcytosine (5fC) is a chemically edited, naturally occurring nucleobase which appears in the context of modified DNA strands. The understanding of the impact of 5fC on dsDNA physical properties is to date limited. In this work, we applied temperature-dependent 1H Chemical Exchange Saturation Transfer (CEST) NMR experiments to non-invasively and site-specifically measure the thermodynamic and kinetic influence of formylated cytosine nucleobase on the melting process involving dsDNA. Incorporation of 5fC within symmetrically positioned CpG sites destabilizes the whole dsDNA structure-as witnessed from the ∼2°C decrease in the melting temperature and 5-10 kJ mol-1 decrease in ΔG°-and affects the kinetic rates of association and dissociation. We observed an up to ∼5-fold enhancement of the dsDNA dissociation and an up to ∼3-fold reduction in ssDNA association rate constants, over multiple temperatures and for several proton reporters. Eyring and van't Hoff analysis proved that the destabilization is not localized, instead all base-pairs are affected and the transition states resembles the single-stranded conformation. These results advance our knowledge about the role of 5fC as a semi-permanent epigenetic modification and assist in the understanding of its interactions with reader proteins.

Single-stranded DNA damage: Protecting the single-stranded DNA from chemical attack.

Cellular processes, such as DNA replication, recombination and transcription, require DNA strands separation and single-stranded DNA is formation. The single-stranded DNA is promptly wrapped by human single-stranded DNA binding proteins, replication protein A (RPA) complex. RPA binding not only prevent nuclease degradation and annealing, but it also coordinates cell-cycle checkpoint activation and DNA repair. However, RPA binding offers little protection against the chemical modification of DNA bases. This review focuses on the type of DNA base damage that occurs in single-stranded DNA and how the damage is rectified in human cells. The discovery of DNA repair proteins, such as ALKBH3, AGT, UNG2, NEIL3, being able to repair the damaged base in the single-stranded DNA, renewed the interest to study single-stranded DNA repair. These mechanistically different proteins work independently from each other with the overarching goal of increasing fidelity of recombination and promoting error-free replication.

Assessment of DNA Susceptibility to Denaturation as a Marker of Chromatin Structure.

The susceptibility of DNA in situ to denaturation is modulated by its interactions with histone and nonhistone proteins, as well as with other chromatin components related to the maintenance of the 3D nuclear structure. Measurement of DNA proclivity to denature by cytometry provides insight into chromatin structure and thus can be used to recognize cells in different phases of the cell cycle, including mitosis, quiescence (G0 ), and apoptosis, as well as to identify the effects of drugs that modify chromatin structure. Particularly useful is the method's ability to detect chromatin changes in sperm cells related to DNA fragmentation and infertility. This article presents a flow cytometric procedure for assessing DNA denaturation based on application of the metachromatic property of acridine orange (AO) to differentially stain single- versus double-stranded DNA. This approach circumvents limitations of biochemical methods of examining DNA denaturation, in particular the fact that the latter destroy higher orders of chromatin structure and that, being applied to bulk cell populations, they cannot detect heterogeneity of individual cells. Because the metachromatic properties of AO have also found application in other cytometric procedures, such as differential staining of RNA versus DNA and assessment of lysosomal proton pump including autophagy, to avert confusion between these approaches and the use of this dye in the DNA denaturation assay, these AO applications are briefly outlined in this unit as well. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Differential staining of single- versus double-stranded DNA with acridine orange.

Bimodal Actions of a Naphthyridone/Aminopiperidine-Based Antibacterial That Targets Gyrase and Topoisomerase IV.

Gyrase and topoisomerase IV are the targets of fluoroquinolone antibacterials. However, the rise in antimicrobial resistance has undermined the clinical use of this important drug class. Therefore, it is critical to identify new agents that maintain activity against fluoroquinolone-resistant strains. One approach is to develop non-fluoroquinolone drugs that also target gyrase and topoisomerase IV but interact differently with the enzymes. This has led to the development of the "novel bacterial topoisomerase inhibitor" (NBTI) class of antibacterials. Despite the clinical potential of NBTIs, there is a relative paucity of data describing their mechanism of action against bacterial type II topoisomerases. Consequently, we characterized the activity of GSK126, a naphthyridone/aminopiperidine-based NBTI, against a variety of Gram-positive and Gram-negative bacterial type II topoisomerases, including gyrase from Mycobacterium tuberculosis and gyrase and topoisomerase IV from Bacillus anthracis and Escherichia coli. GSK126 enhanced single-stranded DNA cleavage and suppressed double-stranded cleavage mediated by these enzymes. It was also a potent inhibitor of gyrase-catalyzed DNA supercoiling and topoisomerase IV-catalyzed decatenation. Thus, GSK126 displays a similar bimodal mechanism of action across a variety of species. In contrast, GSK126 displayed a variable ability to overcome fluoroquinolone resistance mutations across these same species. Our results suggest that NBTIs elicit their antibacterial effects by two different mechanisms: inhibition of gyrase/topoisomerase IV catalytic activity or enhancement of enzyme-mediated DNA cleavage. Furthermore, the relative importance of these two mechanisms appears to differ from species to species. Therefore, we propose that the mechanistic basis for the antibacterial properties of NBTIs is bimodal in nature.

Reactive OFF-ON type alkylating agents for higher-ordered structures of nucleic acids.

Higher-ordered structure motifs of nucleic acids, such as the G-quadruplex (G-4), mismatched and bulge structures, are significant research targets because these structures are involved in genetic control and diseases. Selective alkylation of these higher-order structures is challenging due to the chemical instability of the alkylating agent and side-reactions with the single- or double-strand DNA and RNA. We now report the reactive OFF-ON type alkylating agents, vinyl-quinazolinone (VQ) precursors with a sulfoxide, thiophenyl or thiomethyl group for the OFF-ON control of the vinyl reactivity. The stable VQ precursors conjugated with aminoacridine, which bind to the G-4 DNA, selectively reacted with a T base on the G-4 DNA in contrast to the single- and double-strand DNA. Additionally, the VQ precursor reacted with the T or U base in the AP-site, G-4 RNA and T-T mismatch structures. These VQ precursors would be a new candidate for the T or U specific alkylation in the higher-ordered structures of nucleic acids.

Synthesis, Structural Characterization, Molecular Modeling and DNA Binding Ability of CoII, NiII, CuII, ZnII, PdII and CdII Complexes of Benzocycloheptenone Thiosemicarbazone Ligand.

BACKGROUND & OBJECTIVE: Six novel complexes of transition metal namely, [CoLCl2(H2O)2]0.5H2O, [NiLCl2(H2O)2]0.5H2O, [CuLCl2]0.5H2O, [ZnLCl2], [PdLCl2]H2O and [CdLCl2]H2O, where L is benzocycloheptenone thiosemicarbazone ligand, have been obtained. The confirmation of the structures of the obtained metal chelates depends on the different spectral and physicochemical techniques including CHN analysis, infrared spectra, molar conductivity measurement, UV-vis, thermogravimetric analysis and magnetic moment. The infrared spectral results ascertained that the ligand behaved as neutral bidentate connecting the metal centers via N and S atoms of C=N and C=S groups, respectively. METHODS: The UV-Vis, molar conductivity and magnetic susceptibility results implied that the geometrical structures of the metal chelates are octahedral for Co(II) & Ni(II) complexes, tetrahedral for Zn(II) & Cd(II) complexes and square planar for Cu(II) & Pd(II) complexes which have been confirmed by molecular modeling studies. CONCLUSION: Moreover, the mode of interaction between some chosen metal complexes towards SSDNA has been thoughtful by UV-Vis spectra and viscosity measurements. The value of the intrinsic binding constant (Kb) for the examined compounds has been found to be lower than the binding affinity of the classical intercalator ethedium bromide. Also, the viscosity measurements of the complexes proved that they bind to DNA, most likely, by a non-intercalative mode like H-bonding or electrostatic interactions.

Development of a novel, high-affinity ssDNA trypsin inhibitor.

Inhibitors of serine proteases are not only extremely useful in the basic research but are also applied extensively in clinical settings. Using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) approach we developed a family of novel, single-stranded DNA aptamers capable of specific trypsin inhibition. Our most potent candidate (T24) and its short version (T59) were thoroughly characterised in terms of efficacy. T24 and T59 efficiently inhibited bovine trypsin with Ki of 176 nM and 475 nM, respectively. Interestingly, in contrast to the majority of known trypsin inhibitors, the selected aptamers have superior specificity and did not interact with porcine trypsin or any human proteases tested. These included plasmin and thrombin characterised by trypsin-like substrate specificity. Our results demonstrate that SELEX may be successfully employed in the development of potent and specific DNA based protease inhibitors.

A Tumor-Promoting Phorbol Ester Causes a Large Increase in APOBEC3A Expression and a Moderate Increase in APOBEC3B Expression in a Normal Human Keratinocyte Cell Line without Increasing Genomic Uracils.

Phorbol 12-myristate 13-acetate (PMA) promotes skin cancer in rodents. The mutations found in murine tumors are similar to those found in human skin cancers, and PMA promotes proliferation of human skin cells. PMA treatment of human keratinocytes increases the synthesis of APOBEC3A, an enzyme that converts cytosines in single-stranded DNA to uracil, and mutations in a variety of human cancers are attributed to APOBEC3A or APOBEC3B expression. We tested here the possibility that induction of APOBEC3A by PMA causes genomic accumulation of uracils that may lead to such mutations. When a human keratinocyte cell line was treated with PMA, both APOBEC3A and APOBEC3B gene expression increased, anti-APOBEC3A/APOBEC3B antibody bound a protein(s) in the nucleus, and nuclear extracts displayed cytosine deamination activity. Surprisingly, there was little increase in genomic uracils in PMA-treated wild-type or uracil repair-defective cells. In contrast, cells transfected with a plasmid expressing APOBEC3A acquired more genomic uracils. Unexpectedly, PMA treatment, but not APOBEC3A plasmid transfection, caused a cessation in cell growth. Hence, a reduction in single-stranded DNA at replication forks may explain the inability of PMA-induced APOBEC3A/APOBEC3B to increase genomic uracils. These results suggest that the proinflammatory PMA is unlikely to promote extensive APOBEC3A/APOBEC3B-mediated cytosine deaminations in human keratinocytes.

Methyl ferulic acid exerts anti-apoptotic effects on L-02 cells via the ROS-mediated signaling pathway.

The present study aimed to investigate the anti-apoptotic effects of methyl ferulic acid (MFA) on L-02 cell apoptosis induced by ethanol, and to elucidate the possible underlying mechanisms. L-02 cells were examined after being soaked in ethanol (400 mM) to allow the ethanol to permeate into the cells for 24 h. Cell survival was measured by MTT assay. Cell apoptosis was assessed by both flow cytometry and single-stranded DNA assays. Intracellular reactive oxygen species (ROS) production was determined using the 2',7'-dichlorofluorescein-diacetate dye. The protein expression levels of p38, p-p38, JNK, p-JNK, NADPH oxidase 4 (NOX4), p22, Bax and Bcl-2 were measured by western blot analysis. The mRNA expression levels of NOX4 and p22 were measured by RT-PCR. It was identified that MFA markedly suppressed the ethanol-induced apoptosis and necrosis of L-02 cells. In addition, MFA decreased the expression levels of superoxide dismutase, catalase and phospholipid hydroperoxide gluthione peroxidase, and downregulated the levels of Bax/Bcl-2 and the cleaved forms of caspase-3 in a dose- and time-dependent manner. This indicated that MFA attenuated the apoptosis of L-02 cells. MFA also decreased the elevated mRNA and protein expression levels of Nox4 and p22phox, and the production of intracellular ROS triggered by ethanol. Further analysis demonstrated that MFA significantly attenuated the phosphorylation of JNK and p38, which are major components of the mitogen-activated protein kinase (MAPK) pathways. On the whole, the findings of this study demonstrated that MFA attenuated the apoptotic cell death of L-02 cells by reducing the generation of ROS and inactivating the MAPK pathways.

Contribution of reactive oxygen species to thymineless death in Escherichia coli.

Nutrient starvation usually halts cell growth rather than causing death. Thymine starvation is exceptional, because it kills cells rapidly. This phenomenon, called thymineless death (TLD), underlies the action of several antibacterial, antimalarial, anticancer, and immunomodulatory agents. Many explanations for TLD have been advanced, with recent efforts focused on recombination proteins and replication origin (oriC) degradation. Because current proposals account for only part of TLD and because reactive oxygen species (ROS) are implicated in bacterial death due to other forms of harsh stress, we investigated the possible involvement of ROS in TLD. Here, we show that thymine starvation leads to accumulation of both single-stranded DNA regions and intracellular ROS, and interference with either event protects bacteria from double-stranded DNA breakage and TLD. Elevated levels of single-stranded DNA were necessary but insufficient for TLD, whereas reduction of ROS to background levels largely abolished TLD. We conclude that ROS contribute to TLD by converting single-stranded DNA lesions into double-stranded DNA breaks. Participation of ROS in the terminal phases of TLD provides a specific example of how ROS contribute to stress-mediated bacterial self-destruction.

Replication stress affects the fidelity of nucleosome-mediated epigenetic inheritance.

The fidelity of epigenetic inheritance or, the precision by which epigenetic information is passed along, is an essential parameter for measuring the effectiveness of the process. How the precision of the process is achieved or modulated, however, remains largely elusive. We have performed quantitative measurement of epigenetic fidelity, using position effect variegation (PEV) in Schizosaccharomyces pombe as readout, to explore whether replication perturbation affects nucleosome-mediated epigenetic inheritance. We show that replication stresses, due to either hydroxyurea treatment or various forms of genetic lesions of the replication machinery, reduce the inheritance accuracy of CENP-A/Cnp1 nucleosome positioning within centromere. Mechanistically, we demonstrate that excessive formation of single-stranded DNA, a common molecular abnormality under these conditions, might have correlation with the reduction in fidelity of centromeric chromatin duplication. Furthermore, we show that replication stress broadly changes chromatin structure at various loci in the genome, such as telomere heterochromatin expanding and mating type locus heterochromatin spreading out of the boundaries. Interestingly, the levels of inheritable expanding at sub-telomeric heterochromatin regions are highly variable among independent cell populations. Finally, we show that HU treatment of the multi-cellular organisms C. elegans and D. melanogaster affects epigenetically programmed development and PEV, illustrating the evolutionary conservation of the phenomenon. Replication stress, in addition to its demonstrated role in genetic instability, promotes variable epigenetic instability throughout the epigenome.

Highly efficient preparation of single-stranded DNA rings by T4 ligase at abnormally low Mg(II) concentration.

Preparation of large amount of single-stranded circular DNA in high selectivity is crucial for further developments of nanotechnology and other DNA sciences. Herein, a simple but practically useful methodology to prepare DNA rings has been presented. One of the essential factors is to use highly diluted T4 ligase buffer for ligase reactions. This strategy is based on our unexpected finding that, in diluted T4 buffers, intermolecular polymerization of DNA fragments is greatly suppressed with respect to their intramolecular cyclization. This promotion of cyclization is attributable to abnormally low concentration of Mg2+ ion (0.5-1.0 mM) but not ATP in the media for T4 ligase reactions. The second essential factor is to add DNA substrate intermittently to the mixture and maintain its temporal concentration low. By combining these two factors, single-stranded DNA rings of various sizes (31-74 nt) were obtained in high selectivity (89 mol% for 66-nt DNA) and in satisfactorily high productivity (∼0.2 mg/ml). A linear 72-nt DNA was converted to the corresponding DNA ring in nearly 100% selectivity. The superiority of this new method was further substantiated by the fact that small-sized DNA rings (31-42 nt), which were otherwise hardly obtainable, were successfully prepared in reasonable yields.

Cylindrospermopsin induces biochemical changes leading to programmed cell death in plants.

In the present study we provide cytological and biochemical evidence that the cyanotoxin cylindrospermopsin (CYN) induces programmed cell death (PCD) symptoms in two model vascular plants: the dicot white mustard (Sinapis alba) and the monocot common reed (Phragmites australis). Cytological data include chromatin fragmentation and the increase of the ratio of TUNEL-positive cells in roots, the latter being detected in both model systems studied. The strongest biochemical evidence is the elevation of the activity of several single-stranded DNA preferring nucleases-among them enzymes active at both acidic and alkaline conditions and are probably directly related to DNA breaks occurring at the initial stages of plant PCD: 80 kDa nucleases and a 26 kDa nuclease, both having dual (single- and double-stranded nucleic acid) specificity. Moreover, the total protease activity and in particular, a 53-56 kDa alkaline protease activity increases. This protease could be inhibited by PMSF, thus regarded as serine protease. Serine proteases are detected in all organs of Brassicaceae (Arabidopsis) having importance in differentiation of specialized plant tissue through PCD, in protein degradation/processing during early germination and defense mechanisms induced by a variety of biotic and abiotic stresses. However, knowledge of the physiological roles of these proteases and nucleases in PCD still needs further research. It is concluded that CYN treatment induces chromatin fragmentation and PCD in plant cells by activating specific nucleases and proteases. CYN is proposed to be a suitable molecule to study the mechanism of plant apoptosis.

The Effect of Dimethyl Sulfoxide on Supercoiled DNA Relaxation Catalyzed by Type I Topoisomerases.

The effects of dimethyl sulfoxide (DMSO) on supercoiled plasmid DNA relaxation catalyzed by two typical type I topoisomerases were investigated in our studies. It is shown that DMSO in a low concentration (less than 20%, v/v) can induce a dose-related enhancement of the relaxation efficiency of Escherichia coli topoisomerase I (type IA). Conversely, obvious inhibitory effect on the activity of calf thymus topoisomerase I (type IB) was observed when the same concentration of DMSO is used. In addition, our studies demonstrate that 20% DMSO has an ability to reduce the inhibitory effect on EcTopo I, which was induced by double-stranded oligodeoxyribonucleotides while the same effect cannot be found in the case of CtTopo I. Moreover, our AFM examinations suggested that DMSO can change the conformation of negatively supercoiled plasmid by creating some locally loose regions in DNA molecules. Combining all the lines of evidence, we proposed that DMSO enhanced EcTopo I relaxation activity by (1) increasing the single-stranded DNA regions for the activities of EcTopo I in the early and middle stages of the reaction and (2) preventing the formation of double-stranded DNA-enzyme complex in the later stage, which can elevate the effective concentration of the topoisomerase in the reaction solution.

Ultrasensitive SERS detection of mercury based on the assembled gold nanochains.

Mercuric ions (Hg(2+)) mediate the transformation of single-stranded DNA to form double helical DNA by T-Hg(2+)-T interaction between base pairs. With this strategy, DNA modified gold nanoparticles (Au NPs) were assembled into chains which were displayed remarkable surface-enhanced Raman scattering (SERS) signal. Under optimized conditions, the length of gold nanochains was directly proportional to the mercuric ions concentrations over 0.001-0.5 ng mL(-1) and the limit of detection (LOD) in drinking water was as low as 0.45 pg mL(-1). With ultrasensitivity and excellent selectivity, this feasible and simple method is potentially as a promising tool for monitoring of mercury ions in food safety and environmental applications.

BRG1 promotes the repair of DNA double-strand breaks by facilitating the replacement of RPA with RAD51.

DNA double-strand breaks (DSBs) are a type of lethal DNA damage. The repair of DSBs requires tight coordination between the factors modulating chromatin structure and the DNA repair machinery. BRG1, the ATPase subunit of the chromatin remodelling complex Switch/Sucrose non-fermentable (SWI/SNF), is often linked to tumorigenesis and genome instability, and its role in DSB repair remains largely unclear. In the present study, we show that BRG1 is recruited to DSB sites and enhances DSB repair. Using DR-GFP and EJ5-GFP reporter systems, we demonstrate that BRG1 facilitates homologous recombination repair rather than nonhomologous end-joining (NHEJ) repair. Moreover, the BRG1-RAD52 complex mediates the replacement of RPA with RAD51 on single-stranded DNA (ssDNA) to initiate DNA strand invasion. Loss of BRG1 results in a failure of RAD51 loading onto ssDNA, abnormal homologous recombination repair and enhanced DSB-induced lethality. Our present study provides a mechanistic insight into how BRG1, which is known to be involved in chromatin remodelling, plays a substantial role in the homologous recombination repair pathway in mammalian cells.

Understanding the guanidine-like cationic moiety for optimal binding into the DNA minor groove.

Based on our previous positive results with bis-guanidine-like diaromatic compounds as DNA minor groove binders, we propose a new family: bis-2-amino-1,4,5,6-tetrahydropyrimidines. According to calculated parameters, these dicationic systems would have a more suitable size and lipophilicity for binding into the minor groove than previous series. Moreover, their DFT-optimised structures and docking into an AT oligomer model show that they would bind in the minor groove with good strength and without energy penalty. Hence, we prepared compounds 4 a-c and evaluated their binding to ssDNA and poly(dA-dT)2 by thermal denaturation experiments. The results showed that 4 a (CO) and 4 d (NH) were the best DNA binders. Compared to the previous series, 4 a-d are better binders than bis-guanidiniums but poorer than bis-2-aminoimidazolinium derivatives. Moreover, circular dichroism experiments using ssDNA and poly(dA-dT)2 confirmed binding into the minor groove. Based on our computational design as well as biophysical studies, we have been able to determine that the optimal interaction of guanidine-like dications in the minor grove occurs with bis-2-aminoimidazolinium systems.