Characteristic effect of hydroxyurea on the higher-order structure of DNA and gene expression.

Hydroxyurea (HU; hydroxycarbamide) is a chemotherapy medication used to treat various types of cancer and other diseases such as sickle cell anemia. HU inhibits DNA synthesis by targeting ribonucleotide reductase (RNR). Recent studies have suggested that HU also causes oxidative stress in living systems. In the present study, we investigated if HU could directly affect the activity and/or conformation of DNA. We measured in vitro gene expression in the presence of HU by adapting a cell-free luciferase assay. HU exhibited a bimodal effect on gene expression, where promotion or inhibition were observed at lower or higher concentrations (mM range), respectively. Using atomic force microscopy (AFM), the higher-order structure of DNA was revealed to be partially-thick with kinked-branching structures after HU was added. An elongated coil conformation was observed by AFM in the absence of HU. Single DNA molecules in bulk aqueous solution under fluctuating Brownian motion were imaged by fluorescence microscopy (FM). Both spring and damping constants, mechanical properties of DNA, increased when HU was added. These experimental investigations indicate that HU directly interacts with DNA and provide new insights into how HU acts as a chemotherapeutic agent and targets other diseases.

Unwinding mechanism of SARS-CoV helicase (nsp13) in the presence of Ca2+, elucidated by biochemical and single-molecular studies.

The recent outbreak of COVID-19 has created a serious health crisis with fatFal infectious viral diseases, such as Severe Acute Respiratory Syndrome (SARS). The nsp13, a helicase of coronaviruses is an essential element for viral replication that unwinds secondary structures of DNA and RNA, and is thus considered a major therapeutic target for treatment. The replication of coronaviruses and other retroviruses occurs in the cytoplasm of infected cells, in association with viral replication organelles, called virus-induced cytosolic double-membrane vesicles (DMVs). In addition, an increase in cytosolic Ca2+ concentration accelerates viral replication. However, the molecular mechanism of nsp13 in the presence of Ca2+ is not well understood. In this study, we applied biochemical methods and single-molecule techniques to demonstrate how nsp13 achieves its unwinding activity while performing ATP hydrolysis in the presence of Ca2+. Our study found that nsp13 could efficiently unwind double stranded (ds) DNA under physiological concentration of Ca2+ of cytosolic DMVs. These findings provide new insights into the properties of nsp13 in the range of calcium in cytosolic DMVs.

Targeted Delivery of Immunostimulatory CpG Oligodeoxynucleotides to Antigen-Presenting Cells in Draining Lymph Nodes by Stearic Acid Modification and Nanostructurization.

Polypod-like structured nucleic acids (polypodnas), which are nanostructured DNAs, are useful for delivering cytosine-phosphate guanine oligodeoxynucleotides (CpG ODNs) to antigen-presenting cells (APCs) expressing Toll-like receptor 9 (TLR9) for immune stimulation. Lipid modification is another approach to deliver ODNs to lymph nodes, where TLR9-positive APCs are abundant, by binding to serum albumin. The combination of these two methods can be useful for delivering CpG ODNs to lymph nodes in vivo. In the present study, CpG1668, a phosphodiester-type CpG ODN, was modified with stearic acid (SA) to obtain SA-CpG1668. Tripodna, a polypodna with three pods, was selected as the nanostructured DNA. Tripodnas loaded with CpG1668 or SA-CpG1668 were obtained in high yields. SA-CpG1668/tripodna bound more efficiently to plasma proteins than CpG1668/tripodna and was more efficiently taken up by macrophage-like RAW264.7 cells than CpG1668/tripodna, whereas the levels of tumor necrosis factor-α released from the cells were comparable between the two. After subcutaneous injection into mice, SA-CpG1668/tripodna induced significantly higher interleukin (IL)-12 p40 production in the draining lymph nodes than SA-CpG1668 or CpG1668/tripodna, with reduced IL-6 levels in plasma. These results indicate that the combination of SA modification and nanostructurization is a useful approach for the targeted delivery of CpG ODNs to lymph nodes.

Rhodamine 6G-Ligand Influencing G-Quadruplex Stability and Topology.

The involvement of G-quadruplex (G4) structures in nucleic acids in various molecular processes in cells such as replication, gene-pausing, the expression of crucial cancer-related genes and DNA damage repair is well known. The compounds targeting G4 usually bind directly to the G4 structure, but some ligands can also facilitate the G4 folding of unfolded G-rich sequences and stabilize them even without the presence of monovalent ions such as sodium or potassium. Interestingly, some G4-ligand complexes can show a clear induced CD signal, a feature which is indirect proof of the ligand interaction. Based on the dichroic spectral profile it is not only possible to confirm the presence of a G4 structure but also to determine its topology. In this study we examine the potential of the commercially available Rhodamine 6G (RhG) as a G4 ligand. RhG tends to convert antiparallel G4 structures to parallel forms in a manner similar to that of Thiazole Orange. Our results confirm the very high selectivity of this ligand to the G4 structure. Moreover, the parallel topology of G4 can be verified unambiguously based on the specific induced CD profile of the G4-RhG complex. This feature has been verified on more than 50 different DNA sequences forming various non-canonical structural motifs.

Targeting a noncanonical, hairpin-containing G-quadruplex structure from the MYCN gene.

The MYCN gene encodes the transcription factor N-Myc, a driver of neuroblastoma (NB). Targeting G-quadruplexes (G4s) with small molecules is attractive strategy to control the expression of undruggable proteins such as N-Myc. However, selective binders to G4s are challenging to identify due to the structural similarity of many G4s. Here, we report the discovery of a small molecule ligand (4) that targets the noncanonical, hairpin containing G4 structure found in the MYCN gene using small molecule microarrays (SMMs). Unlike many G4 binders, the compound was found to bind to a pocket at the base of the hairpin region of the MYCN G4. This compound stabilizes the G4 and has affinity of 3.5 ± 1.6 µM. Moreover, an improved analog, MY-8, suppressed levels of both MYCN and MYCNOS (a lncRNA embedded within the MYCN gene) in NBEB neuroblastoma cells. This work indicates that the approach of targeting complex, hybrid G4 structures that exist throughout the human genome may be an applicable strategy to achieve selectivity for targeting disease-relevant genes including protein coding (MYCN) as well as non-coding (MYCNOS) gene products.

Towards Profiling of the G-Quadruplex Targeting Drugs in the Living Human Cells Using NMR Spectroscopy.

Recently, the 1H-detected in-cell NMR spectroscopy has emerged as a unique tool allowing the characterization of interactions between nucleic acid-based targets and drug-like molecules in living human cells. Here, we assess the application potential of 1H and 19F-detected in-cell NMR spectroscopy to profile drugs/ligands targeting DNA G-quadruplexes, arguably the most studied class of anti-cancer drugs targeting nucleic acids. We show that the extension of the original in-cell NMR approach is not straightforward. The severe signal broadening and overlap of 1H in-cell NMR spectra of polymorphic G-quadruplexes and their complexes complicate their quantitative interpretation. Nevertheless, the 1H in-cell NMR can be used to identify drugs that, despite strong interaction in vitro, lose their ability to bind G-quadruplexes in the native environment. The in-cell NMR approach is adjusted to a recently developed 3,5-bis(trifluoromethyl)phenyl probe to monitor the intracellular interaction with ligands using 19F-detected in-cell NMR. The probe allows dissecting polymorphic mixture in terms of number and relative populations of individual G-quadruplex species, including ligand-bound and unbound forms in vitro and in cellulo. Despite the probe's discussed limitations, the 19F-detected in-cell NMR appears to be a promising strategy to profile G-quadruplex-ligand interactions in the complex environment of living cells.

Peroxynitrite-induced conformational changes in DNA that lead to cell death: UV, CD spectral, molecular dynamics simulation and FACS analysis.

Peroxynitrite is known to react with biomolecules leading to their structural and function alteration. Structural alteration in DNA induced by peroxynitrite is not clearly known. The current study was carried out to decipher the changes induced by peroxynitrite using UV-Vis spectra, circular dichrometry, molecular dynamics simulation followed by restriction digestion. Apoptotic markers Bax, Bcl-2 and caspase genes were also studied by FACS in peroxynitrite induced PC12 cells. The results obtained showed that PXN binds to DNA leading to hyperchromicity of DNA in the presence of PXN over a period of time and the same was established by In silico studies where PXN modifies the DNA to accommodate itself into the stacking and brings about the significant structural alterations. Further, FACS studies reveal that Bcl-2 gene expression was down regulated whereas BAXand caspase genes were up regulated compared to control concluding that PXN induces apoptotic cell death in PC12 cells.

Drugging the "undruggable" microRNAs.

As a naturally occurring class of gene regulators, microRNAs (miRNAs) have attracted much attention as promising targets for therapeutic development. However, RNAs including miRNAs have long been considered undruggable, and most efforts have been devoted to using synthetic oligonucleotides to regulate miRNAs. Encouragingly, recent findings have revealed that miRNAs can also be drugged with small molecules that directly target miRNAs. In this review paper, we give a summary of recently emerged small-molecule inhibitors (SMIs) and small-molecule degraders (SMDs) for miRNAs. SMIs are small molecules that directly bind to miRNAs to inhibit their biogenesis, and SMDs are bifunctional small molecules that upon binding to miRNAs induce miRNA degradation. Strategies for discovering SMIs and developing SMDs were summarized. Applications of SMIs and SMDs in miRNA inhibition and cancer therapy were also introduced. Overall, SMIs and SMDs introduced here have high potency and specificity in miRNA inhibition. We envision that these small molecules will pave the way for developing novel therapeutics toward miRNAs that were previously considered undruggable.

Human Papillomavirus G-Rich Regions as Potential Antiviral Drug Targets.

Herein, we report, for the first time, the screening of several ligands in terms of their ability to bind and stabilize G-quadruplexes (G4) found in seven human Papillomavirus (HPV) genomes. Using a variety of biophysical assays, HPV G-quadruplexes were shown to possess a high degree of structural polymorphism upon ligand binding, which may have an impact on transcription, replication, and viral protein production. A sequence found in high-risk HPV16 genotype folds into multiple non-canonical DNA structures; it was converted into a major G4 conformation upon interaction with a well-characterized highly selective G4 ligand, PhenDC3, which may have an impact on the viral infection. Likewise, HPV57 and 58, which fold into multiple G4 structures, were found to form single stable complexes in the presence of two other G4 ligands, C8 and pyridostatin, respectively. In addition, one of the selected compounds, the acridine derivative C8, demonstrated a significant antiviral effect in HPV18-infected organotypic raft cultures. Altogether, these results indicate that targeting HPV G4s may be an alternative route for the development of novel antiviral therapies.

Stabilization of an intermolecular RNA triplex by two novel binders Lys- and Arg-rich Ru(II) polypyridyl metallopeptides.

In this work, using [Ru(bpy)2(pip)]2+ (bpy = 2,2'-bipyridine, pip = 2-phenyl-1H-imidazo[4,5-f]-[1,10]-phenanthroline) as chromophores and neutral amino acid glycine as spacers, two novel Arg- and Lys-rich Ru(II) polypyridyl metallopeptides as an intermolecular triplex RNA stabilizers, namely [Ru(bpy)2(pic-Lys2-Gly-Lys2-Gly-Lys2)]8+ (Ru1; pic = 2-(4-carboxy-phenyl)imidazo-[4,5-f] [1,10] phenanthroline, Gly = glycine, Lys = lysine) and [Ru(bpy)2(pic-Arg2-Gly-Arg2-Gly-Arg2)]8+ (Ru2; Arg = arginine), have been synthesized and characterized. The binding properties of Ru1 and Ru2 with poly(U)·poly(A)∗poly(U) triplex have been studied by UV-Vis spectroscopy, fluorescence spectroscopy, viscosity measurements as well as circular dichroism and thermal denaturation. The obtained results suggest that attaching cationic peptides to a Ru(II) polypyridyl complex can obviously enhance the triplex stabilization. Considering the structure natures of Ru1 and Ru2, conceivably besides electrostatic interaction, the forces stabilizing the triplex should also involve hydrophobic interaction and hydrogen binding. Compared with the Lys-rich metallopeptide (Ru1), however, the third-strand stabilizating effect of the Arg-rich one (Ru2) is slightly more marked, which may be due to differences in the interactions of arginine and lysine residues with the third strand of the triplex. The results obtained here may be useful for understanding the interaction of triplex RNA poly(U)·poly(A)∗poly(U) with small molecule, particularly ruthenium(II) complexes.

Synthesis and nucleic acid binding evaluation of a thyminyl l-diaminobutanoic acid-based nucleopeptide.

Herein we present the synthesis of a l-diaminobutanoic acid (DABA)-based nucleopeptide (3), with an oligocationic backbone, realized by solid phase peptide synthesis using thymine-bearing DABA moieties alternating in the sequence with free ones. CD studies evidenced the ability of this oligothymine nucleopeptide, well soluble in aqueous solution, to alter the secondary structure particularly of complementary RNA (poly rA vs poly rU) and inosine-rich RNAs, like poly rI and poly rIC, and showed its preference in binding double vs single-stranded DNAs. Furthermore, ESI mass spectrometry revealed that 3 bound also G-quadruplex (G4) DNAs, with either parallel or antiparallel topologies (adopted in our experimental conditions by c-myc and tel22, respectively). However, it caused detectable changes only in the CD of c-myc (whose parallel G4 structure was also thermally stabilized by ~3 °C), while leaving unaltered the antiparallel structure of tel22. Interestingly, CD and UV analyses suggested that 3 induced a hybrid mixed parallel/antiparallel G4 DNA structure in a random-coil tel22 DNA obtained under salt-free buffer conditions. Titration of the random-coil telomeric DNA with 3 gave quantitative information on the stoichiometry of the obtained complex. Overall, the findings of this work suggest that DABA-based nucleopeptides are synthetic nucleic acid analogues potentially useful in antigene and antisense strategies. Nevertheless, the hexathymine DABA-nucleopeptide shows an interesting behaviour as molecular tool per se thanks to its efficacy in provoking G4 induction in random coil G-rich DNA, as well as for the possibility to bind and stabilize c-myc oncogene in a G4 structure.

Hydrated electrons induce the formation of interstrand cross-links in DNA modified by cisplatin adducts.

Double-stranded oligonucleotides containing cisplatin adducts, with and without a mismatched region, were exposed to hydrated electrons generated by gamma-rays. Gel electrophoresis analysis demonstrates the formation of cisplatin-interstrand crosslinks from the cisplatin-intrastrand species. The rate constant per base for the reaction between hydrated electrons and the double-stranded oligonucleotides with and without cisplatin containing a mismatched region was determined by pulse radiolysis to be 7 × 109 and 2 × 109 M-1 s-1, respectively. These results provide a better understanding of the radiosensitizing effect of cisplatin adducts in hypoxic tumors and of the formation of interstrand crosslinks, which are difficult for cells to repair.

Signal-off/on electrogenerated chemiluminescence deoxyribosensors for assay of early lung cancer biomarker (NAP2) based on target-caused DNA charge transfer.

Two novel electrochemiluminescence (ECL) deoxyribosensors are designed for assay of early lung cancer biomarker (NAP2) using the DNA three-way junction (DNA-TWJ) inserted NAP2 binding aptamer between two double-helical stems and labeled with ruthenium (II) complex (Ru) (NBAT-Ru) taken as molecular recognition element. The signal-off ECL deoxyribosensor was fabricated by covalently coupling the 5'-NH2-(CH2)6-NBAT-Ru to glassy carbon electrode surface modified with 4-aminobenzoic acid (4-ABA). After combining NAP2 and NBAT-Ru, the changed conformation of NBAT-Ru altered the distance between Ru complex and electrode, resulting in a low ECL signal. The signal-on deoxyribosensor was fabricated by self-assembling the 5'-SH-(CH2)6-NBAT-Ru onto the Au electrode. The introduction of NAP2 triggered the conformational change in the aptamer domain, which induces the interhelical stacking of the two double-helical stems of NBAT-Ru. This stacking constitutes "electrical contact," which promotes transmission of electron-holes through the stems of NBAT-Ru, and produces high ECL intensity. Both deoxyribosensors show high sensitivity and selectivity. The biosensors have been successfully applied to clinical plasma detection. The approaches we describe represent unique principles based on DNA-TWJ inserted target special binding domain as molecular recognition element and different immobilization types for the fabrication of biosensors, which are greatly promising for the detection of protein, metal ions, bacteria, and cells.

A New Look into the Mode of Action of Metal-Based Anticancer Drugs.

The mode of action of Pt- and Pd-based anticancer agents (cisplatin and Pd2Spm) was studied by characterising their impact on DNA. Changes in conformation and mobility at the molecular level in hydrated DNA were analysed by quasi-elastic and inelastic neutron scattering techniques (QENS and INS), coupled to Fourier transform infrared (FTIR) and microRaman spectroscopies. Although INS, FTIR and Raman revealed drug-triggered changes in the phosphate groups and the double helix base pairing, QENS allowed access to the nanosecond motions of the biomolecule's backbone and confined hydration water within the minor groove. Distinct effects were observed for cisplatin and Pd2Spm, the former having a predominant effect on DNA´s spine of hydration, whereas the latter had a higher influence on the backbone dynamics. This is an innovative way of tackling a drug´s mode of action, mediated by the hydration waters within its pharmacological target (DNA).

Study of the intracellular nanoparticle-based radiosensitization mechanisms in F98 glioma cells treated with charged particle therapy through synchrotron-based infrared microspectroscopy.

The use of nanoparticles (NP) as dose enhancers in radiotherapy (RT) is a growing research field. Recently, the use of NP has been extended to charged particle therapy in order to improve the performance in radioresistant tumors. However, the biological mechanisms underlying the synergistic effects involved in NP-RT approaches are not clearly understood. Here, we used the capabilities of synchrotron-based Fourier Transform Infrared Microspectroscopy (SR-FTIRM) as a bio-analytical tool to elucidate the NP-induced cellular damage at the molecular level and at a single-cell scale. F98 glioma cells doped with AuNP and GdNP were irradiated using several types of medical ion beams (proton, helium, carbon and oxygen). Differences in cell composition were analyzed in the nucleic acids, protein and lipid spectral regions using multivariate methods (Principal Component Analysis, PCA). Several NP-induced cellular modifications were detected, such as conformational changes in secondary protein structures, intensity variations in the lipid CHx stretching bands, as well as complex DNA rearrangements following charged particle therapy irradiations. These spectral features seem to be correlated with the already shown enhancement both in the DNA damage response and in the reactive oxygen species (ROS) production by the NP, which causes cell damage in the form of protein, lipid, and/or DNA oxidations. Vibrational features were NP-dependent due to the NP heterogeneous radiosensitization capability. Our results provided new insights into the molecular changes in response to NP-based RT treatments using ion beams, and highlighted the relevance of SR-FTIRM as a useful and precise technique for assessing cell response to innovative radiotherapy approaches.

Sequence specificity in DNA-drug intercalation: MD simulation and density functional theory approaches.

DNA is an essential target for the treatment of various pathologies, especially cancer. Hence targeting DNA double helix for alteration of its function has been attempted by several ways. Drug-DNA intercalation, one such biophysical process, could not be studied extensively as this requires significant deformation of the receptor DNA. Here we report thorough theoretical investigation of intercalation process in daunomycin-DNA interaction, by performing molecular dynamics simulations of the drug-DNA complexes for various DNA sequences, followed by Free-energy analysis and density functional theory (DFT) based studies to understand the binding preference. The classical energy based analyses indicate that the drug prefers to bind to TC/GA sequence over others. The DFT based energies of supra-molecular complexes are always contaminated with basis set superposition error (BSSE), which can be corrected by counterpoise method. This method is quite effective for systems containing two molecular fragments but is not appropriate for studying interaction between two base pair fragments and the drug intercalated between them. We have adopted an extension of the counterpoise method for BSSE corrected interaction energy calculation. These interaction energies, along with the energy penalty due to un-stacking of the base pairs, also indicate TC/GA sequence is the most preferred sequence for binding.

Structures of 1:1 and 2:1 complexes of BMVC and MYC promoter G-quadruplex reveal a mechanism of ligand conformation adjustment for G4-recognition.

BMVC is the first fluorescent probe designed to detect G-quadruplexes (G4s) in vivo. The MYC oncogene promoter forms a G4 (MycG4) which acts as a transcription silencer. Here, we report the high-affinity and specific binding of BMVC to MycG4 with unusual slow-exchange rates on the NMR timescale. We also show that BMVC represses MYC in cancer cells. We determined the solution structures of the 1:1 and 2:1 BMVC-MycG4 complexes. BMVC first binds the 5'-end of MycG4 to form a 1:1 complex with a well-defined structure. At higher ratio, BMVC also binds the 3'-end to form a second complex. In both complexes, the crescent-shaped BMVC recruits a flanking DNA residue to form a BMVC-base plane stacking over the external G-tetrad. Remarkably, BMVC adjusts its conformation to a contracted form to match the G-tetrad for an optimal stacking interaction. This is the first structural example showing the importance of ligand conformational adjustment in G4 recognition. BMVC binds the more accessible 5'-end with higher affinity, whereas sequence specificity is present at the weaker-binding 3'-site. Our structures provide insights into specific recognition of MycG4 by BMVC and useful information for design of G4-targeted anticancer drugs and fluorescent probes.

Specific effects of antitumor active norspermidine on the structure and function of DNA.

We compared the effects of trivalent polyamines, spermidine (SPD) and norspermidine (NSPD), a chemical homologue of SPD, on the structure of DNA and gene expression. The chemical structures of SPD and NSPD are different only with the number of methylene groups between amine groups, [N-3-N-4-N] and [N-3-N-3-N], respectively. SPD plays vital roles in cell function and survival, including in mammals. On the other hand, NSPD has antitumor activity and is found in some species of plants, bacteria and algae, but not in humans. We found that both polyamines exhibit biphasic effect; enhancement and inhibition on in vitro gene expression, where SPD shows definitely higher potency in enhancement but NSPD causes stronger inhibition. Based on the results of AFM (atomic force microscopy) observations together with single DNA measurements with fluorescence microscopy, it becomes clear that SPD tends to align DNA orientation, whereas NSPD induces shrinkage with a greater potency. The measurement of binding equilibrium by NMR indicates that NSPD shows 4-5 times higher affinity to DNA than SPD. Our theoretical study with Monte Carlo simulation provides the insights into the underlying mechanism of the specific effect of NSPD on DNA.

Mechanistic Aspects of Fungicide-Induced DNA Damage: Spectroscopic and Molecular Dynamics Simulation Studies.

The non-targeted action of fungicides leads to DNA damage of organisms, which causes several serious diseases such as cancer, behavioral change, and nausea. The mechanistic aspects of DNA damage by fungicides are not much known, irrespective of its importance. Spectroscopic and molecular dynamics simulation techniques have been employed to bring out the key aspects of the mechanism of DNA damage by an important fungicide, namely, n-dodecylguanidine acetate (dodine). The hydrocarbon and guanidinium groups of dodine interact in the adenine (A)-thymine (T) region of the minor groove of DNA via electrostatic and hydrophobic interactions. The accommodation of dodine in the minor groove of DNA interrupts the cone of hydration of DNA by removing water from its surface. The interaction of dodine in the minor groove of DNA perturbs inclination, twist, roll, and slide of base pairs in the A-T region, which broadens the minor groove as well as elongates the first strand of DNA, causing the loss of its helicity and base stacking. The detailed understanding of dodine-induced DNA damage may guide the development of fungicide with minimal non-targeted genotoxic effect.

Electrochemical behaviour of anticancer drug lomustine and in situ evaluation of its interaction with DNA.

Electrochemical techniques were used to investigate the behavior of lomustine (CCNU) and its degradation in aqueous solution at a glassy carbon electrode (GCE). The in situ interaction of CCNU and chemically degraded CCNU (cdCCNU) with dsDNA was then investigated in dsDNA incubated solutions, using dsDNA electrochemical biosensors and comet assays. CCNU undergoes electrochemical reduction in two irreversible, diffusion-controlled, and pH-dependent redox processes, each with transfer of two electrons and one proton. At pH ≥ 10.1, the peak potential for the two processes was essentially pH-independent and involved only one electron. A mechanism was proposed for the reduction of CCNU in a neutral medium. In addition, it was found that CCNU underwent spontaneous degradation during incubation in aqueous solution, without the formation of electroactive degradation products. The degradation process was faster in basic media. Moreover, this pro-drug interacted with the DNA. Its metabolite(s) initially caused condensation of the double helix chains, followed by the unwinding of these chains. In addition, free guanine (Gua) was released from the dsDNA and oxidative damage to the DNA by the CCNU metabolite(s) was evidenced from the detection of 8-oxoGua and 2,8-oxoAde. These results were confirmed by the poly(dA)- and poly(dG)-polyhomonucleotide biosensors, which revealed the oxidative damage caused to both bases (guanine and adenine) of the dsDNA by the CCNU metabolite(s). The comet assay indicated breaks in the single strand DNA, complementing the results of the studies using differential pulse voltammetry. Conformational changes of dsDNA caused by CCNU and cdCCNU were confirmed using comet assays.