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New antitumor Pd(II) compounds derived from oxygen donor ligands salicylate (SA) (1) and sulfosalicylate (SSA) (2) dianions and nitrogen donor heterocyclic ligand 2,2'-bipyridine (bpy) were synthesized and characterized by elemental analysis, UV-Vis, FT-IR, 1 H NMR and conductivity measurements. The complexes evaluated for their cytotoxicity effects towards cancer cell line of K562 using MTT assay. They are more cytotoxic than cisplatin. The dependence of their interaction modes with CT-DNA on concentration of the compounds has been discovered in this work. CT-DNA binding studies of these complexes have been investigated by UV-Vis absorption, ethidium bromide (EB) displacement, fluorescence, circular dichroism and gel electrophoresis techniques. The apparent binding constants (K app ) has been obtained 3.9 and 10.9×10 4 M -1 at lower concentration range and 1.03 and 1.59×10 4 M -1 at higher concentration range for complexes (1) and (2), respectively. These complexes effectively interact with CT-DNA in the order of (2)>(1). Fluorescence studies exhibited that the complexes quench CT-DNA-EB by simultaneous static and dynamic quenching processes. The calculated binding (K app , k q , K SV , n) and thermodynamic (ΔG°, ΔH°, ΔS°) parameters revealed that hydrophobic, van der Waals forces and hydrogen binding holds the Pd(II) complexes in the CT-DNA grooves. Gel electrophoresis supports the spectroscopic experiments., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The DNA interstrand cross-links of the antitumor drug {[cis-Pt(NH 3 ) 2 Cl] 2 (4,4'-methylenedianiline)} 2+ (1) play a prevalent role in its antitumor effects. Complex 1 forms DNA long-range interstrand cross-links uniquely in the 3'-3' direction. Conformational distortions induced by these interstrand cross-links in DNA represent a potential structural motif for recognition by high-mobility-group proteins.

Mutation 'hotspot' regions in the genome are susceptible to genetic instability, implicating them in diseases. These hotspots are not random and often co-localize with DNA sequences potentially capable of adopting alternative DNA structures (non-B DNA, e.g. H-DNA and G4-DNA), which have been identified as endogenous sources of genomic instability. There are regions that contain overlapping sequences that may form more than one non-B DNA structure. The extent to which one structure impacts the formation/stability of another, within the sequence, is not fully understood. To address this issue, we investigated the folding preferences of oligonucleotides from a chromosomal breakpoint hotspot in the human c-MYC oncogene containing both potential G4-forming and H-DNA-forming elements. We characterized the structures formed in the presence of G4-DNA-stabilizing K+ ions or H-DNA-stabilizing Mg2+ ions using multiple techniques. We found that under conditions favorable for H-DNA formation, a stable intramolecular triplex DNA structure predominated; whereas, under K+-rich, G4-DNA-forming conditions, a plurality of unfolded and folded species were present. Thus, within a limited region containing sequences with the potential to adopt multiple structures, only one structure predominates under a given condition. The predominance of H-DNA implicates this structure in the instability associated with the human c-MYC oncogene., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

DNA sequences capable of forming triplexes induce DNA double-strand breaks that have attracted attention in genome editing technologies (e.g., CRISPR/Cas9 system, TALEN, and ZFN). Therefore, novel functional tools that stabilize triplex DNA structures must be further investigated to spark renewed interest. In this study, we investigated the unique character of cationic comb-type copolymers for the selective stabilization of triplex DNA. The melting temperature (T m ) of triplex DNA increased from 24.5 to 73.0 °C (ΔT m = 48.5 °C) by the addition of poly(allylamine)-graft-dextran (PAA-g-Dex) under physiological conditions (at pH 7.0), while PAA-g-Dex did not stabilize but rather destabilized the DNA duplex. On the other hand, poly(l-lysine)-graft-dextran (PLL-g-Dex) stabilized both the duplex and triplex structures at pH 7.0. Thermodynamic parameters evaluated by isothermal titration calorimetry (ITC) revealed that the binding constant (K a ) for the intermolecular triplex formation in the presence of PAA-g-Dex was 1.1 × 10 9 M -1 at 25 °C which is more than 10 times larger than that in the presence of PLL-g-Dex (8.6 × 10 7 M -1 ). Stabilizing activity and selectivity of cationic copolymers toward DNA assemblies were successfully controlled by selecting appropriate backbone structures of the copolymer. Various functional molecules that stabilize DNA duplexes have been developed and used in biological research. However, there are few cationic polymers that stabilize triplex DNA selectively. This study indicates that PAA-g-Dex has great potential to regulate the biological activities of triplex DNA.

The interaction between anthracenyl-methyl homospermidine conjugate (ANTMHspd) with herring sperm DNA was investigated by UV/vis absorption, fluorescent spectra, circular dichroism (CD) spectroscopy and 1 HNMR under physiological conditions (pH=7.4). The observed hypochromism effect and fluorescence quenching of ANTMHspd by DNA, and the displacement of EB from DNA-EB system by ANTMHspd suggested that ANTMHspd might interact with DNA by the combined mode of intercalation and groove binding. Further fluorescent tests at different temperatures revealed that the quenching mechanism was a static type. The quenching constant, binding constant and thermodynamic parameter obtained from fluorescence showed that the type of interaction force included mainly hydrogen bonding and van der Waals, which promoted the binding process. The CD test revealed that ANTMHspd could cause the B to A-like conformational change while ANTMHspd is not a typical DNA intercalator. The 1 H NMR tests showed that ANTMHspd partially intercalated DNA. The effect of NaCl and KI on ANTMHspd-DNA interaction provided additional evidences of intercalation. Molecular docking simulation was carried out and the docking model in silico suggested that the binding modes of ANTMHspd and DNA were groove binding and intercalation, with the anthracene moiety inserted in DNA base pairs and the polyamine chain embedded in the DNA groove., (Copyright © 2017. Published by Elsevier B.V.)

The oncogenic transcription factor forkhead box M (Foxm) is overexpressed in human colorectal cancer (CRC). Targeting the protein interaction with its cognate DNA has been established as an attractive approach to anti-CRC chemotherapy. State-of-the-art molecular dynamics (MD) simulations revealed that the Foxm adopts considerably different conformations to interact with and without its DNA partner; the holo conformation is tightly packed as a typical globulin configuration, whereas the apo form is locally unstructured that exhibits intrinsic disorder in DNA recognition helix, indicating that DNA binding can help the Foxm refolding. With this finding, the MD equilibrium structure of DNA-free Foxm was utilized to perform molecular docking virtual screening against a natural organic compound library. Consequently, six hit compounds were identified as potential small-molecule mediators of Foxm-DNA interaction; their binding affinities (K D ) to Foxm DNA-binding domain were then determined to range between 3.8 and 230μM by using isothermal titration calorimetry. These compounds were suggested to recognize and stabilize the apo conformation of Foxm, thus shifting the binding reaction equilibrium of Foxm from DNA-bound to DNA-free states to disrupt the formation of Foxm-DNA adduct., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Thymol (2-isopropyl-5-methylphenol) is a natural ingredient used as flavor or preservative agent in food products. The antibacterial mechanism of thymol against Gram-positive, Staphylococcus aureus was investigated in this work. A total of 15 membrane fatty acids were identified in S. aureus cells by gas chromatography-mass spectrometry. Exposure to thymol at low concentrations induced obvious alterations in membrane fatty acid composition, such as decreasing the proportion of branched 12-methyltetradecanoic acid and 14-methylhexadecanoic acid (from 22.4 and 17.3% to 7.9 and 10.3%, respectively). Membrane permeability assay and morphological image showed that thymol at higher concentrations disrupted S. aureus cell membrane integrity, which may decrease cell viability. Moreover, the interaction of thymol with genomic DNA was also investigated using multi-spectroscopic techniques, docking and atomic force microscopy. The results indicated that thymol bound to the minor groove of DNA with binding constant (K a ) value of (1.22 ± 0.14) × 10 4  M -1 , and this binding interaction induced a mild destabilization in the DNA secondary structure, and made DNA molecules to be aggregated. Graphical Abstract Thymol exerts its antibacterial effect throught destruction of bacterial cell membrane and binding directly to genomic DNA.

Interactions between the polyamine spermine and nucleic acids drive important cellular processes. Spermine condenses DNA and some RNAs, such as poly(rA):poly(rU). A large fraction of the spermine present in cells is bound to RNA but apparently does not condense it. Here, we study the effect of spermine binding to short duplex RNA and DNA, and compare our findings with predictions of molecular-dynamics simulations. When small numbers of spermine are introduced, RNA with a designed sequence containing a mixture of 14 GC pairs and 11 AU pairs resists condensation relative to DNA of an equivalent sequence or to 25 bp poly(rA):poly(rU) RNA. A comparison of wide-angle x-ray scattering profiles with simulation results suggests that spermine is sequestered deep within the major groove of mixed-sequence RNA. This prevents condensation by limiting opportunities to bridge to other molecules and stabilizes the RNA by locking it into a particular conformation. In contrast, for DNA, simulations suggest that spermine binds externally to the duplex, offering opportunities for intermolecular interaction. The goal of this study is to explain how RNA can remain soluble and available for interaction with other molecules in the cell despite the presence of spermine at concentrations high enough to precipitate DNA., (Copyright © 2017 Biophysical Society. All rights reserved.)

Intracellular positive ions neutralize negative charges on the phosphates of a DNA strand, conferring greater strength on the hydrogen bonds that connect complementary strands into a double helix and so confer enhanced stability. Beyond a certain value of salt concentration, the DNA molecule displays an unstable nature in vivo as well as in vitro. We consider a wide range of salt concentrations and study the stability of the DNA double helix using a statistical model. Through numerical calculations, we attempt to explain the different behavior exhibited by DNA molecules in this range. We compare our results with experimental data and find a close agreement.

Relocation of a bioactive photosensitizer, namely phenosafranin (PSF), from the phenazinium family, has been demonstrated from non-ionic micellar carrier to the DNA. For the purpose, interaction of micelle-bound PSF with calf thymus DNA (ctDNA) has been investigated vividly exploiting various spectroscopic techniques like absorption, steady state and time resolved emission, fluorescence anisotropy, circular dichroism etc. Experimental outcomes reveal that PSF binds strongly with both the micelle as well as the DNA. In the presence of DNA, however, relocation of the micelle-carried PSF occurs from the micelle to the DNA. Competitive binding of the probe between micelle and the DNA is assigned responsible for this relocation. Circular dichroism spectral measurements reflect that the DNA conformation remains intact in the presence of the micelle advocating that the non-ionic micelles can safely be used for the drug delivery purpose. The work is expected to encourage development of newer carriers for DNA targeted drug delivery., (Copyright © 2016 Elsevier B.V. All rights reserved.)

trans-BMPyP induced the B-Z transition for alternated AT oligonucleotides as it was evident by inversed CD spectrum. The transition occurred simultaneously with appearance of the extensive stacking of porphyrin. Complete B-Z transition required at least 14 base-pairs long. Insertion of one or two GC base pairs prevented the B-Z transition., (Copyright © 2016. Published by Elsevier B.V.)

Telomestatin, a natural product isolated from Streptomyces anulatus, stabilizes telomeric DNA G-quadruplexes. Treatment with this ligand induces apoptosis of various cancer cells with a relatively low effect on somatic cells because of its high selectivity toward G-quadruplex over duplex DNA. A high-resolution structure of a G-quadruplex in complex with telomestatin does not yet exist because of its low solubility, and the binding nature of this ligand remains elusive. In this study, we utilized molecular binding simulations and MMGBSA binding energy analysis to decipher the nature of the binding of telomestatin to a telomeric G-quadruplex. We identified three major binding poses: bottom intercalation, top stacking, and groove binding. The top stacking mode resembles the pose observed in an NMR complex of the same G-quadruplex with the telomestatin analogue L2H. The bottom intercalation and groove binding poses were not observed in the previous studies of L2H. The bottom intercalation mode exhibited the most favorable binding energy among the three modes, while also partially intercalating into the telomeric quadruplex. The dynamic and energetic properties of these three binding modes are thoroughly examined. "Flip insertion" and "slide insertion" were observed in the bottom intercalation mode. Our findings also provide insight into the design of more selective DNA quadruplex ligands as anticancer agents in the future.

Developing the Wnt pathway inhibitors has been considered as a therapeutic approach for cancers and other Wnt-related diseases. Previously we found that the G-rich sequence of WNT1 promoter is capable of forming G-quadruplex structure and stabilizing agents for Wnt1-mediated signaling pathway. Using a established cell-based drug screen system that enabled the evaluation of WNT1 expression activity in a G-quadruplex structure dependent manner, we evaluated a series of 6-substituted 9-chloro-11H-indeno[1,2-c]quinolin-11-one derivatives that potentially inhibit the Wnt1-mediated signaling pathway. The most potent compound SJ26 showed repression of WNT1 activity in a G-quadruplex structure-dependent manner. Moreover, compound SJ26 inhibited the WNT1-mediated downstream signaling pathway and suppressed migration activity of cancer cells. Thus, we have identified a tetracyclic azafluorenone, SJ26, that is capable of binding to G-quadruplex DNA structure, repressing WNT1 expression, and inhibiting cell migration.

Unlabelled: Campylobacter jejuni is the leading cause of bacterial gastroenteritis in humans, but relatively little is known about the global regulation of virulence factors during infection of chickens or humans. This study identified DNA supercoiling as playing a key role in regulating motility and flagellar protein production and found that this supercoiling-controlled regulon is induced by growth in chicken mucus. A direct correlation was observed between motility and resting DNA supercoiling levels in different strains of C. jejuni, and relaxation of DNA supercoiling resulted in decreased motility. Transcriptional analysis and Western immunoblotting revealed that a reduction in motility and DNA supercoiling affected the two-component regulatory system FlgRS and was associated with reduced FlgR expression, increased FlgS expression, and aberrant expression of flagellin subunits. Electron microscopy revealed that the flagellar structure remained intact. Growth in the presence of porcine mucin resulted in increased negative supercoiling, increased motility, increased FlgR expression, and reduced FlgS expression. Finally, this supercoiling-dependent regulon was shown to be induced by growth in chicken mucus, and the level of activation was dependent on the source of the mucus from within the chicken intestinal tract. In conclusion, this study reports for the first time the key role played by DNA supercoiling in regulating motility in C. jejuni and indicates that the induction of this supercoiling-induced regulon in response to mucus from different sources could play a critical role in regulating motility in vivo, Importance: Although Campylobacter jejuni is the leading cause of bacterial gastroenteritis, very little is understood about how this pathogen controls the expression of genes involved in causing disease. This study for the first time identifies DNA supercoiling as a key regulator of motility in C. jejuni, which is essential for both pathogenesis and colonization. Altering the level of DNA supercoiling results in changes in motility levels, as well as changes in the expression of genes involved in flagellar gene regulation. Furthermore, spontaneous clones of the organism with different motility profiles have altered DNA supercoiling levels. Finally, mucus was identified as a key stimulator of changes in DNA supercoiling, and it was shown that mucus from different sites in the chicken intestine induced different levels of DNA supercoiling. In conclusion, this study implicates DNA supercoiling as a key regulator of motility in C. jejuni in vivo during colonization of the mucus layer., (Copyright © 2016 Shortt et al.)

Aims: The studies were aimed to contribute to the elucidation of the relationships between structure of the double-headed cationic surfactants-N,N-bis[3,3'-(dimethylamine)- propyl]alkylamide dihydrochlorides and N,N-bis[3,3'-(trimethylammonio)propyl]alkylamide dibromides (alkyl: n-C9 H19 , n-C11 H23 , n-C13 H27 , n-C15 H31 ) and their antibacterial and biofilm preventing activity., Methods and Results: The minimal inhibitory and bactericidal concentrations (MIC and MBC) of dicephalic surfactants against Staphylococcus epidermidis and Pseudomonas aeruginosa were tested using standard methods. Pseudomonas aeruginosa was resistant to studied compounds but MBC values against Staph. epidermidis reached 0·48-0·01 mmol l(-1) . The influence of dicephalic surfactants on bacterial biofilm and adhesion to the various surfaces was investigated with crystal violet staining or colony counting. The reduction in bacterial adhesion was observed, especially in the case of glass and stainless steel. The condensation of the DNA was shown in the ethidium bromide intercalation assay., Conclusions: Dicephalic surfactants exhibited antibacterial activity against Staph. epidermidis. The activity of studied compounds depended on the hydrocarbon chain length and the counterion. Surfactants deposited on different materials reduced Staph. epidermidis adhesion, dependently on the surfactant structure and the substratum. Dicephalic surfactants showed the ability of DNA compaction., Significance and Impact of the Study: This study points the possibility of application of dicephalic surfactants as the surface-coating agents to prevent biofilm formation. These compounds efficiently condensed DNA and are potential candidates for further studies towards the transfection., (© 2016 The Society for Applied Microbiology.)

Electrostatics are central to all aspects of nucleic acid behavior, including their folding, condensation, and binding to other molecules, and the energetics of these processes are profoundly influenced by the ion atmosphere that surrounds nucleic acids. Given the highly complex and dynamic nature of the ion atmosphere, understanding its properties and effects will require synergy between computational modeling and experiment. Prior computational models and experiments suggest that cation occupancy in the ion atmosphere depends on the size of the cation. However, the computational models have not been independently tested, and the experimentally observed effects were small. Here, we evaluate a computational model of ion size effects by experimentally testing a blind prediction made from that model, and we present additional experimental results that extend our understanding of the ion atmosphere. Giambasu et al. developed and implemented a three-dimensional reference interaction site (3D-RISM) model for monovalent cations surrounding DNA and RNA helices, and this model predicts that Na(+) would outcompete Cs(+) by 1.8-2.1-fold; i.e., with Cs(+) in 2-fold excess of Na(+) the ion atmosphere would contain an equal number of each cation (Nucleic Acids Res. 2015, 43, 8405). However, our ion counting experiments indicate that there is no significant preference for Na(+) over Cs(+). There is an ∼25% preferential occupancy of Li(+) over larger cations in the ion atmosphere but, counter to general expectations from existing models, no size dependence for the other alkali metal ions. Further, we followed the folding of the P4-P6 RNA and showed that differences in folding with different alkali metal ions observed at high concentration arise from cation-anion interactions and not cation size effects. Overall, our results provide a critical test of a computational prediction, fundamental information about ion atmosphere properties, and parameters that will aid in the development of next-generation nucleic acid computational models.

Gemini surfactants are good candidates to bind, protect, and deliver nucleic acids. Herein, the concept of amino acids (namely glycine) as counter ions of gemini surfactants for gene therapy application was explored. This study was conducted on DNA and RNA oligomers and two quaternary bis-imidazolium salts, having 2,5-dioxahexane and 2,8-dioxanonane spacer groups. The toxicity level of surfactants was assessed by an MTT assay, and their ability to bind nucleic acids was tested through electrophoresis. The nucleic acid conformation was established based on circular dichroism and infrared spectroscopic analyses. The structures of the formed complexes were characterized by small-angle scattering of synchrotron radiation. Both studied surfactants appear to be suitable for gene therapy; however, although they vary by only three methylene groups in the spacer, they differ in binding ability and toxicity. The tested oligonucleotides maintained their native conformations upon surfactant addition and the studied lipoplexes formed a variety of structures. In systems based on a 2,5-dioxahexane spacer, a hexagonal phase was observed for DNA-surfactant complexes and a micellar phase was dominant with RNA. For the surfactant with a 2,8-dioxanonane spacer group, the primitive cubic phase prevailed., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The expansion of CAG repeats in the human genome causes the neurological disorder Huntington's disease. The small-molecule naphthyridine-azaquinolone NA we reported earlier bound to the CAG/CAG motif in the hairpin structure of the CAG repeat DNA. In order to investigate and improve NA-binding to the CAG repeat DNA and RNA, we conducted systematic structure-binding studies of NA to CAG repeats. Among the five new NA derivatives we synthesized, surface plasmon resonance (SPR) assay showed that all of the derivatives modified from amide linkages in NA to a carbamate linkage failed to bind to CAG repeat DNA and RNA. One derivative, NBzA, modified by incorporating an additional ring to the azaquinolone was found to bind to both d(CAG)9 and r(CAG)9 . NBzA binding to d(CAG)9 was similar to NA binding in terms of large changes in the SPR assay and circular dichroism (CD) as well as pairwise binding, as assessed by electron spray ionization time-of-flight (ESI-TOF) mass spectrometry. For the binding to r(CAG)9 , both NA and NBzA showed stepwise binding in ESI-TOF MS, and NBzA-binding to r(CAG)9 induced more extensive conformational change than NA-binding. The tricyclic system in NBzA did not show significant effects on the binding, selectivity, and translation, but provides a large chemical space for further modification to gain higher affinity and selectivity. These studies revealed that the linker structure in NA and NBzA was suitable for the binding to CAG DNA and RNA, and that the tricyclic benzoazaquinolone did not interfere with the binding., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

i-Motifs are four-stranded DNA structures consisting of two parallel DNA duplexes held together by hemi-protonated and intercalated cytosine base pairs (C:CH(+)). They have attracted considerable research interest for their potential role in gene regulation and their use as pH responsive switches and building blocks in macromolecular assemblies. At neutral and basic pH values, the cytosine bases deprotonate and the structure unfolds into single strands. To avoid this limitation and expand the range of environmental conditions supporting i-motif folding, we replaced the sugar in DNA by 2-deoxy-2-fluoroarabinose. We demonstrate that such a modification significantly stabilizes i-motif formation over a wide pH range, including pH 7. Nuclear magnetic resonance experiments reveal that 2-deoxy-2-fluoroarabinose adopts a C2'-endo conformation, instead of the C3'-endo conformation usually found in unmodified i-motifs. Nevertheless, this substitution does not alter the overall i-motif structure. This conformational change, together with the changes in charge distribution in the sugar caused by the electronegative fluorine atoms, leads to a number of favorable sequential and inter-strand electrostatic interactions. The availability of folded i-motifs at neutral pH will aid investigations into the biological function of i-motifs in vitro, and will expand i-motif applications in nanotechnology., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The stimulatory effect of the aqueous extract of G. lucidum, a basidiomycetes class fungus in the APE1-enzyme-mediated processing of solitary and bistranded clustered abasic sites DNA damages is presented. Abasic sites are considered the most common type of DNA damage lesions. Our study shows enhanced activity of APE1 in the processing of abasic sites in the presence of the polysaccharides fraction of G. lucidum. Remarkable increase in the amount of single-strand breaks (SSBs) and double-strand breaks (DSBs) from solitary and bistranded clustered abasic sites respectively with APE1 in the presence of the extract was found. This trend is maintained when abasic sites in DNA oligomers are exposed to fibroblast cell extracts in the presence of the extract. While DNA conformational alteration is negligible, APE1 enzyme shows characteristic changes in the alpha helix and beta strand ratio after incubation with G. lucidum extract. The enhanced reactivity of APE1 at the molecular level in the presence of G. lucidium is attributed to this effect. This study potentially amplifies the scope of the use of G. lucidum, which was earlier shown to have only reactive oxygen species (ROS) scavenging properties with regards to DNA damage inhibition.

The high mobility group box 1 protein has been identified as a key player in chromatin homeostasis including transcription regulation, recombination, repair, and chromatin remodeling. Emerging findings indicate HMGB1 protein over expression in nearly all types of human cancers and inflammatory disorders. Thus it is considered as a potential therapeutic target for treating various malignancies. We screened the promoter region of hmgb1 gene and selected a positive regulatory element of 25 base pair duplex (25RY) (-165 to -183) as a potential target for chemotherapeutic intervention. The molecular interaction of actinomycin (ACT) with the regulatory region of hmgb1 gene was characterized by spectroscopic, calorimetric and molecular docking studies. The hypochromic and bathochromic shift in the absorption spectrum, stabilization of 25RY duplex against thermal denaturation, perturbation of CD spectrum of duplex and enhancement of fluorescence intensity of actinomycin indicate strong binding of actinomycin to the hmgb1 promoter region (25RY).The energetics was characterized to be endothermic and entropy driven. All these results are in good agreement with in silico investigation that suggest minor groove binding with effective intercalation at GC bases of actinomycin to 25RY. This study identifies hmgb1 gene promoter region a potential target for the anticancer therapautiucs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Accumulating evidence suggests that G-quadruplexes play vital roles in gene expression, DNA replication, and recombination. Three distinct promoters (PI, PII, and PIII) regulate human acetyl-CoA carboxylase 1 (ACC1) gene expression. In this study, we asked whether the G-rich sequences within the human ACC1 (PI and PII) promoters can form G-quadruplex structures and regulate normal DNA transactions. Using multiple complementary methods, we show that G-rich sequences of PI and PII promoters form intramolecular G-quadruplex structures and then establish unambiguously the topologies of these structures. Importantly, G-quadruplex formation in ACC1 gene promoter region blocks DNA replication and suppresses transcription, and this effect was further augmented by G-quadruplex stabilizing ligands. Altogether, these results are consistent with the notion that G-quadruplex structures exist within the human ACC1 gene promoter region, whose activity can be suppressed by G-quadruplex stabilizing ligands, thereby revealing a novel regulatory mechanism of ACC1 gene expression and as a possible therapeutic target.

Saporin L3 from the leaves of the common soapwort is a catalyst for hydrolytic depurination of adenine from RNA. Saporin L3 is a type 1 ribosome inactivating protein (RIP) composed only of a catalytic domain. Other RIPs have been used in immunotoxin cancer therapy, but off-target effects have limited their development. In the current study, we use transition state theory to understand the chemical mechanism and transition state structure of saporin L3. In favorable cases, transition state structures guide the design of transition state analogues as inhibitors. Kinetic isotope effects (KIEs) were determined for an A14C mutant of saporin L3. To permit KIE measurements, small stem-loop RNAs that contain an AGGG tetraloop structure were enzymatically synthesized with the single adenylate bearing specific isotopic substitutions. KIEs were measured and corrected for forward commitment to obtain intrinsic values. A model of the transition state structure for depurination of stem-loop RNA (5'-GGGAGGGCCC-3') by saporin L3 was determined by matching KIE values predicted via quantum chemical calculations to a family of intrinsic KIEs. This model indicates saporin L3 displays a late transition state with the N-ribosidic bond to the adenine nearly cleaved, and the attacking water nucleophile weakly bonded to the ribosyl anomeric carbon. The transition state retains partial ribocation character, a feature common to most N-ribosyl transferases. However, the transition state geometry for saporin L3 is distinct from ricin A-chain, the only other RIP whose transition state is known.

A comparison of DNA conformational changes in a solution containing the poly-l-lysine with the number of monomers z=3, 5, 17, 20, 270, 325 and polyamines (spermine and spermidine) was carried out in 1M and 5mM NaCl solutions. It was shown that despite the identical results of DNA condensation induced by compounds, their influence on the DNA conformation prior to packaging depends on whether they belong to a long polycations or short oligomers. DNA secondary and tertiary structures were examined using Circular Dichroism, UV-vis Spectrophotometry, Dynamic Light Scattering, Low Gradient Viscometry, Flow Birefringence, and AFM. The phase diagrams for systems of DNA-polycations, DNA-oligomers, DNA-polyamines were drawn., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The condensing effect of aminoglycoside antibiotics on the structure of double-stranded DNA was examined. The selective condensation of DNA by small molecules is an interesting approach in biotechnology. Here, we present the interaction between calf thymus DNA and three types of antibiotic molecules: tobramycin, kanamycin, and neomycin. Several techniques were applied to study this effect. Atomic force microscopy, transmission electron microscopy images, and nuclear magnetic resonance spectra showed that the interaction of tobramycin with double-stranded DNA caused the rod, toroid, and sphere formation and very strong condensation of DNA strands, which was not observed in the case of other aminoglycosides used in the experiment. Studies on the mechanisms by which small molecules interact with DNA are important in understanding their functioning in cells, in designing new and efficient drugs, or in minimizing their adverse side effects. Specific interactions between tobramycin and DNA double helix was modeled using molecular dynamics simulations. Simulation study shows the aminoglycoside specificity to bend DNA double helix, shedding light on the origins of toroid formation. This phenomenon may lighten the ototoxicity or nephrotoxicity issues, but also other adverse reactions of aminoglycoside antibiotics in the human body.

Previous studies have shown that promoter regions of many proto-oncogenes can fold into G-quadruplexes, which are potentially involved in the regulation of genes. Bioinformatics analysis suggested that there was a G-rich sequence within -48 to -26 region of the human MET promoter (named Pu23WT). In this study, we proved that Pu23WT adopted an intramolecular parallel G-quadruplex structure under physiological conditions in vitro, and the cationic porphyrin TMPyP4 enhanced the stability of the Pu23WT G-quadruplex. To better understand the functions of Pu23WT in the MET expression, we performed a series of analysis on several cancer cells. Experimental data revealed that TMPyP4 treatment attenuated the expression of MET in HepG2, BGC823, and U87MG cells, resulting in the cellular proliferation inhibition, G1 phase cell cycle arrest and cell migration retardation. ChIP assay results indicated that TMPyP4 probably prohibited the Pu23WT G-quadruplex from binding to the activator Sp1, which could be one of the mechanisms that led to the transcription inhibition of MET gene. It is the first study on the G-quadruplex structure in the human MET promoter and its functions in cancer cells. We believe that this structure is a potential target for anticancer treatment., (© 2015 Wiley Periodicals, Inc.)

Stable RNA triplexes play key roles in many biological processes. However, due to Hoogsteen base pairing, triplexes are thermodynamically less stable than the corresponding duplexes. To understand the factors effecting the stabilization of RNA triplexes by octahedral ruthenium(ii) complexes, two Ru(ii) complexes, [Ru(bpy)2(7-CH3-dppz)](2+) (Ru) and [Ru(phen)2(7-CH3-dppz)](2+) (Ru), have been synthesized and characterized in this work. The interactions of the two Ru(ii) complexes with the poly(U)˙poly(A)*poly(U) triplex are investigated by spectrophotometry, spectrofluorometry, circular dichroism as well as viscometry. The results demonstrate that the two complexes are able to enhance the stability of the RNA triplex and serve as molecular "light switches" for the triplex. However, Ru and Ru affecting the stabilization of the third strand are significantly weaker than that of the Watson-Crick base-paired duplex, suggesting that the binding of the two complexes with the triplex is favored by the Watson-Crick base-paired duplex to a large extent. In addition, considering the nature of Ru and Ru, we presume that their binding differences may be due to different ancillary ligand effects. This study further advances our knowledge on the interaction of RNA triple-stranded structures with metal complexes, particularly with Ru(ii) complexes.

Proflavine, a known intercalator of DNA and RNA, promotes duplex formation by nucleic acids with natural and non-natural backbones that otherwise form duplexes with low thermal stability, and even some that show no sign of duplex formation in the absence of proflavine. These findings demonstrate the potential for intercalators to be used as cofactors for the assembly of rationally designed nucleic acid structures, and could provide fundamental insights regarding intercalation of natural nucleic acid duplexes.

Four uracil-containing intrastrand cross-links have been detected in human cells upon UV irradiation of 5-bromouracil-containing DNA, namely 5'-G[8-5]U-3', 5'-U[5-8]G-3', 5'-A[8-5]U-3', and 5'-A[2-5]U-3'. These lesions feature unique composition and connectivity compared with other intrastrand cross-links reported in the literature. For the first time, structural information obtained using molecular dynamics (MD) simulations reveal that all four lesions distort the DNA helix, which can involve an extrahelical location of the cross-link, changes in the helical interactions of the complementary nucleotides, or disruption of hydrogen bonding in the flanking base pairs up to two positions from the cross-linked site; however, the degree of distortion varies between the cross-links, being affected by the sequence, nucleobase-nucleobase connectivity, and the purine involved. Most importantly, the relative distortion of the damaged DNA provides the first structural explanation for the observed abundances of the four uracil-containing cross-links. Furthermore, the highly distorted conformations suggest that these lesions will likely have severe implications for DNA replication and repair processes in cells.

New butyltin complexes with 2-sulfobenzoic acid: [Sn(C4H9)2{O3SC6H4COO-2}(H2O)]·(C2H5OH) (DBTsbz), [Sn(C4H9)3{O3SC6H4COOH-2}] (TBTsbz) and [Sn2(C4H9)6{μ-O3SC6H4COO-2}] (DTBTsbz) are very effective cytotoxic agents against tumor cells. The molecular interaction of these complexes with lipid membranes and DNA has been investigated. The IR spectra and changes of (1)H, (13)C chemical shifts suggest that SO3 and COO groups of 2-sulfobenzoato ligand interact with O atom of glycerin fragment of DPPC. Moreover, the compounds form Sn-OP bonds with phosphate groups of DPPC, which was shown by the lower frequency shift of the νs(PO2(-)) and νas(PO2(-)) band, by change of (31)P NMR signals and by DFT calculation. Another possibility is the interaction of the phosphate group of DPPC owing to formation of hydrogen bond O-H…O-P between water molecule coordinated to Sn and oxygen atom from the phosphate group. Using TCSPC-FCS we characterized DNA supramolecular assemblies' formation upon increasing TBTsbz, DTBTsbz and DBTsbz concentration. Diffusion time, lifetime and particle number changes are altered systematically with increasing Ccomp/CDNAbp ratio in following effectiveness order DBTsbz > TBTsbz > DTBTsbz. From those parameters we can conclude that all these compounds lead to a change of DNA winding, strand but not to DNA compaction. Investigated compounds show very high cytotoxic activity against cancer cell lines. All compounds exhibit efficient in vitro antitumor activity toward Jurkat (T-cell leukemia), CL-1 (T-lymphoblastoid cell line), GL-1 (B cell lymphoma cell line) and D-17 (canine osteosarcoma). The DBTsbz is more effective then carboplatin against canine osteosarcoma., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The increasing awareness of RNA's central role in biology calls for a new understanding of how RNAs, like proteins, recognize biological partners. Because RNA is inherently flexible, it assumes a variety of conformations. This conformational flexibility can be a critical aspect of how RNA attracts and binds molecular partners. Structurally, RNA consists of rigid basepaired duplexes, separated by flexible non-basepaired regions. Here, using an RNA system consisting of two short helices, connected by a single-stranded (non-basepaired) junction, we explore the role of helix length and junction sequence in determining the range of conformations available to a model RNA. Single-molecule Förster resonance energy transfer reports on the RNA conformation as a function of either mono- or divalent ion concentration. Electrostatic repulsion between helices dominates at low salt concentration, whereas junction sequence effects determine the conformations at high salt concentration. Near physiological salt concentrations, RNA conformation is sensitive to both helix length and junction sequence, suggesting a means for sensitively tuning RNA conformations., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

A full understanding of RNA-mediated biology would require the knowledge of three-dimensional (3D) structures, structural flexibility, and stability of RNAs. To predict RNA 3D structures and stability, we have previously proposed a three-bead coarse-grained predictive model with implicit salt/solvent potentials. In this study, we further develop the model by improving the implicit-salt electrostatic potential and including a sequence-dependent coaxial stacking potential to enable the model to simulate RNA 3D structure folding in divalent/monovalent ion solutions. The model presented here can predict 3D structures of RNA hairpins with bulges/internal loops (<77 nucleotides) from their sequences at the corresponding experimental ion conditions with an overall improved accuracy compared to the experimental data; the model also makes reliable predictions for the flexibility of RNA hairpins with bulge loops of different lengths at several divalent/monovalent ion conditions. In addition, the model successfully predicts the stability of RNA hairpins with various loops/stems in divalent/monovalent ion solutions., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Bacterial riboswitches couple small-molecule ligand binding to RNA conformational changes that widely regulate gene expression, rendering them potential targets for antibiotic intervention. Despite structural insights, the ligand-mediated folding mechanisms of riboswitches are still poorly understood. Using single-molecule fluorescence resonance energy transfer (smFRET), we have investigated the folding mechanism of an H-type pseudoknotted preQ1 riboswitch in dependence of Mg(2+) and three ligands of distinct affinities. We show that, in the absence of Mg(2+), both weakly and strongly bound ligands promote pseudoknot docking through an induced-fit mechanism. By contrast, addition of as low as 10 μM Mg(2+) generally shifts docking toward conformational selection by stabilizing a folded-like conformation prior to ligand binding. Supporting evidence from transition-state analysis further highlights the particular importance of stacking interactions during induced-fit and of specific hydrogen bonds during conformational selection. Our mechanistic dissection provides unprecedented insights into the intricate synergy between ligand- and Mg(2+)-mediated RNA folding.

We previously identified EMICORON as a novel G-quadruplex (G4) ligand showing high selectivity for G4 structures over the duplex DNA, causing telomere damage and inhibition of cell proliferation in transformed and tumor cells. Here, we evaluated the antitumoral effect of EMICORON on advanced models of human colon cancer that could adequately predict human clinical outcomes. Our results showed that EMICORON was well tolerated in mice, as no adverse effects were reported, and a low ratio of sensitivity across human and mouse bone marrow cells was observed, indicating a good potential for reaching similar blood levels in humans. Moreover, EMICORON showed a marked therapeutic efficacy, as it inhibited the growth of patient-derived xenografts (PDX) and orthotopic colon cancer and strongly reduced the dissemination of tumor cells to lymph nodes, intestine, stomach, and liver. Finally, activation of DNA damage and impairment of proliferation and angiogenesis are proved to be key determinants of EMICORON antitumoral activity. Altogether, our results, performed on advanced experimental models of human colon cancer that bridge the translational gap between preclinical and clinical studies, demonstrated that EMICORON had an unprecedented antitumor activity warranting further studies of EMICORON-based combination treatments., (©2015 American Association for Cancer Research.)

Human nuclear type IB topoisomerase (Top1) inhibitors are widely used and powerful anticancer agents. In this study, we introduce and validate a single-molecule supercoil relaxation assay as a molecular pharmacology tool for characterizing therapeutically relevant Top1 inhibitors. Using this assay, we determined the effects on Top1 supercoil relaxation activity of four Top1 inhibitors; three clinically relevant: camptothecin, LMP-400, LMP-776 (both indenoisoquinoline derivatives), and one natural product in preclinical development, lamellarin-D. Our results demonstrate that Top1 inhibitors have two distinct effects on Top1 activity: a decrease in supercoil relaxation rate and an increase in religation inhibition. The type and magnitude of the inhibition mode depend both on the specific inhibitor and on the topology of the DNA substrate. In general, the efficacy of inhibition is significantly higher with supercoiled than with relaxed DNA substrates. Comparing single-molecule inhibition with cell growth inhibition (IC50) measurements showed a correlation between the binding time of the Top1 inhibitors and their cytotoxic efficacy, independent of the mode of inhibition. This study demonstrates that the single-molecule supercoil relaxation assay is a sensitive method to elucidate the detailed mechanisms of Top1 inhibitors and is relevant for the cellular efficacy of Top1 inhibitors., (©2015 American Association for Cancer Research.)

With the aim to attain an alkylating agent with enhanced DNA-affinity, we have successfully synthesised lysine-linked bis-3-chloropiperidine 1 bearing an anthraquinone moiety known to bind double-stranded DNA. Consistent with our expectations, compound 1 appears to intercalate into the DNA double helix, which can be observed by conformational changes of plasmid DNA suggesting alkylation and intercalation-induced DNA unwinding. The results of this work can provide a meaningful starting point for investigating the molecular mechanism of action of this novel DNA alkylating conjugate 1 with improved affinity for DNA., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The porphyrin compound, TMPyP4 (5,10,15,20-Tetrakis-(N-methyl-4-pyridyl)porphine), is widely used as a photosensitizer and a modulator of nucleic acid secondary structure stability. Our group recently showed in cultured cells and forebrain slice cultures that this compound can also down regulate expression of Tyrosine hydroxylase (Th), which encodes the rate-limiting enzyme in catecholamine biosynthesis, by stabilizing DNA secondary structures in the Th proximal promoter. The current study sought to establish whether treatment with TMPyP4 could modify mouse Th expression levels in vivo. Intraperitoneal administration of low TMPyP4 doses (10mg/kg), similar to those used for photosensitization, did not significantly reduce Th transcript levels in several catecholaminergic regions. Administration of a high dose (40 mg/kg), similar to those used for tumor xenograph reduction, unexpectedly induced flaccid paralysis in an age and sex-dependent manner. In vitro analyses revealed that TMPyP4, but not putative metabolites, inhibited Acetylcholinesterase activity and pre-treatment of TMPyP4 with Hemeoxygenase-2 (HO-2) rescued Acetylcholinesterase function. Age-dependent differences in HO-2 expression levels may account for some of the variable in vivo effects of high TMPyP4 doses. Together, these studies indicate that only low doses of TMPyP4, such as those typically used for photosensitization, are well tolerated in vivo. Thus, despite its widespread use in vitro, TMPyP4 is not ideal for modifying neuronal gene expression in vivo by manipulating nucleic acid secondary structure stability, which highlights the need to identify more clinically suitable compounds that can modulate nucleic acid secondary structure and gene expression.

Bulged structures have been identified in nucleic acids and have been shown to be linked to biomolecular processes involved in numerous diseases. Thus, chemical agents with affinity for bulged nucleic acids are of general biological significance. Herein, the mechanism of specific recognition and stabilization of bulged DNA and RNA by helical bimetallic species was established through detailed molecular biophysics and biochemistry assays. These agents, known as 'flexicates', are potential mimetics of α-helical peptides in cancer treatment, exhibiting antimicrobial and antitumor effects. The flexicates have positive impacts on the thermal stability of DNA duplexes containing bulges, which means that the flexicates interact with the duplexes containing bulges, and that these interactions stabilize the secondary structures of these duplexes. Notably, the stabilising effect of the flexicates increases with the size of the bulge, the maximal stabilization is observed for the duplexes containing a bulge composed of at least three bases. The flexicates bind most preferentially to the bulges composed of pyrimidines flanked on both sides also by pyrimidines. It is suggested that it is so because these bulges exhibit greatest conformational variability in comparison with other combinations of bases in the bulge loop and bases flanking the bulge. Finally, the results indicate that there is only one dominant binding site for the flexicates on the DNA and RNA bulges and that the flexicates bind directly to the bulge or in its close proximity. It is also shown that the flexicates effectively bind to RNA duplexes containing the bulged region of HIV-1 TAR RNA.

Interaction of oleuropein, the major bio-phenol in olive leaf and fruit, with salmon sperm double-stranded DNA was investigated by employing electronic absorption titrations, fluorescence quenching spectroscopy, competitive fluorescence spectroscopy, thermal denaturation and voltammetric studies. Titration of oleuropein with the DNA caused a hypochromism accompanied with a red shift indicating an intercalative mode of interaction. Binding constant of 1.4×10(4) M(-1) was obtained for this interaction. From the curves of fluorescence titration of oleuropein with the DNA, binding constant and binding sites were calculated to be 8.61×10(3) M(-1) and 1.05, respectively. Competitive studies with ethidium bromide (a well-known DNA intercalator) showed that the bio-phenol could take the place of ethidium bromide in the DNA intercalation sites. The interaction of oleuropein with DNA was also studied electrochemically. In the presence of the DNA, the anodic and cathodic peak currents of oleuropein decreased accompanied with increases in peak-to-peak potential separation and formal potential, indicating the intercalation of oleuropein into the DNA double helix. Moreover, melting temperature of the DNA was found to increase in the presence of oleuropein, indicating the stabilization of the DNA double helix due to an intercalative interaction., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Oxaliplatin is one of the most important anticancer drugs at present. However, the mechanism of action of oxaliplatin is still controversial. In this study, the interactions between oxaliplatin and a plasmid DNA have been studied so as to reveal the structural basis of its activity. The structural characteristic of pUC19 DNA (2ng/μL) incubated with 100μmol/L and 1000μmol/L of oxaliplatin for the different time on a freshly cleaved highly ordered pyrolytic graphite (HOPG) surface was investigated by atomic force microscopy (AFM). High resolution AFM observation indicated that oxaliplatin can induce pUC19 DNA molecules change from the extended conformation to the entangled structures with many nodes, and finally to the compact particles. The present AFM results provide structural evidence about the interactions between oxaliplatin and circular duplex DNA containing multiple targets., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Transcription termination for genes encoding polyadenylated mRNAs requires a functional poly(A) signal (PAS) in the nascent pre-mRNA. Often called PAS-dependent termination, or PADT, it is widely assumed that the PAS requirement reflects an obligatory poly(A) site cleavage requirement for termination. Cleavage is thought to provide entry for a 5'-to-3' exonuclease that targets RNA polymerase II via the nascent transcript-i.e., the torpedo model. To assess the role of cleavage in PADT, we developed a PADT assay using HeLa nuclear extract. Here we examine the basal mechanism of PADT and show that cleavage at the poly(A) site is not required for PADT. Isolated elongation complexes undergo termination in a PAS-dependent manner when incubated in buffer, in the absence of extract, nucleotides, or cleavage at the poly(A) site. Thus, PADT-proficient complexes undergo a conformational change that triggers termination. PADT is inhibited by α-amanitin, which presumably blocks the required conformational change., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A new electrochemical biosensor was developed to demonstrate the effect of Acridine Orange (AO) on DNA damage. Then, the biosensor was used to check the inhibitors effect of three flavonols antioxidants (myricetin, fisetin and kaempferol) on DNA damage. Acridine Orange (AO) was used as a damaging agent because it shows a high affinity to nucleic acid and stretch of the double helical structure of DNA. Decreasing on the oxidation signals of adenine and guanine (in the DNA) in the presence of AO were used as probes to study the antioxidants power, using DNA-modified screen printed graphene electrode (DNA/SPGE). The results of our study showed that the DNA-biosensor could be suitable biosensor to investigate the inhibitors ability of the flavonols antioxidants on the DNA damage. The linear dependency was detected in the two regions in the ranges of 1.0-15.0 and 15.0-500.0 pmol L(-1). The detection limit was found 0.5 pmol L(-1) and 0.6 pmol L(-1) for guanine and adenine, respectively. To confirm the electrochemical results, Uv-Vis and fluorescence spectroscopic methods were used too. Finally molecular dynamic (MD) simulation was performed on the structure of DNA in a water box to study any interaction between the antioxidant, AO and DNA., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Chemical modification interference is a powerful method for surveying an entire RNA molecule to identify functionally important chemical groups. The basic idea is to generate a pool of end-labeled RNAs wherein each RNA molecule is chemically modified (e.g., by diethyl pyrocarbonate [DEPC], hydrazine, dimethyl sulfate, CMCT, or kethoxal) at a different position. The pool of RNAs is then allowed to participate in the reaction of interest. The functionally important RNA molecules (e.g., those bound by protein or that successfully participate in a processing reaction) are then separated from the nonfunctional RNA molecules (e.g., those not bound by protein or unable to participate in a processing reaction). This is often achieved by straightforward gel electrophoretic analysis. In the case of protein binding, it is necessary to be able to separate bound RNA from unbound RNA, which can be accomplished using electrophoretic mobility shift assays, filter binding, or affinity approaches (e.g., by immunoprecipitation or the use of tagged proteins). None of these techniques requires that a large fraction of RNA be bound or reacted, and, as a result, they are quite sensitive. Here we describe one example of a chemical modification interference assay in which RNA is modified with DEPC or hydrazine before binding to a protein. This technique can be readily adapted for use with other chemicals., (© 2015 Cold Spring Harbor Laboratory Press.)

DNA cross-linking technology is an attractive tool for the detection, regulation, and manipulation of genes. In this study, a series of photolabile 4-oxo-enal-modified oligonucleotides functionalized with photosensitive ο-nitrobenzyl derivatives were rationally designed as a new kind of photocaged cross-linking agents. A comprehensive evaluation of cross-linking reactions for different nucleobases in complementary strands under different conditions suggested that the modified DNA oligonucleotides tended to form interstrand cross-linking to nucleobases with the potential of thymidine > guanosine » cytidine ~ adenosine. Different from previous literature reports that cytidine and adenosine were preferential cross-linked nucleobases with 4-oxo-enal moieties, our study represents the first example of DNA cross-linking for T and G selectivity using 4-oxo-enal moiety. The cross-linked adducts were identified and their cross-linking mechanism was also illustrated. This greatly expands the applications of 4-oxo-enal derivatives in the studies of DNA damage and RNA structure.

Four novel photoactivated binitroimidazole prodrugs were synthesized. These agents produced DNA interstrand cross-links (ICLs) and direct strand breaks (DSB) upon UV irradiation, whereas no or very few DNA ICLs and DSBs were observed without UV treatment. Although these four molecules (1-4) contain the same binitroimidazole moiety, they bear four different leaving groups, which resulted in their producing different yields of DNA damage. Compound 4, with nitrogen mustard as a leaving group, showed the highest ICL yield. Surprisingly, compounds 1-3, without any alkylating functional group, also induced DNA ICL formation, although they did so with lower yields, which suggested that the binitroimidazole moiety released from UV irradiation of 1-3 is capable of cross-linking DNA. The DNA cross-linked products induced by these compounds were completely destroyed upon 1.0 M piperidine treatment at 90 °C (leading to cleavage at dG sites), which revealed that DNA cross-linking mainly occurred via alkylation of dGs. We proposed a possible mechanism by which alkylating agents were released from these compounds. HRMS and NMR analysis confirmed that free nitrogen mustards were generated by UV irradiation of 4. Suppression of DNA ICL and DSB formation by a radical trap, TEMPO, indicated the involvement of free radicals in the photo reactions of 3 and 4 with DNA. On the basis of these data, we propose that UV irradiation of compounds 1-4 generated a binitroimidazole intermediate that cross-links DNA. The higher ICL yield observed with 4 resulted from the amine effector nitrogen mustard released from UV irradiation.

This study provides insights into the interactions of Pt-ttpy, that is, a metallo-organic heterocycle-comprising platinum(II) complex of terpyridine, and G-quadruplexes adopted by G-rich DNA from the transcriptional regulatory element of the c-myc gene, a well-known attractive target for artificial modulation of oncogene expression. A previously noted drug-like potential of Pt-ttpy relies on its antiproliferative activity on cancer cells and its increased selectivity for G-quadruplex binding attributed to the combination of distinct interacting modes. The predominant interaction between the herein used models of a parallel G-quadruplex exhibiting short propeller-type loops and Pt-ttpy occurs through stacking to the outer G-quartets. The presence of adenine versus thymine residue at the 5'-end overhanging region allows the coordinative binding of Pt-ttpy to the G-quadruplex structure. Interestingly, Pt-ttpy triggers the formation of the G-quadruplex even in the absence of cations. Furthermore, NMR-based characterisation revealed common structural features of Pt-ttpy-G-quadruplex complexes in the presence and absence of cations, which indicate that cations may be expelled from the cores of the corresponding structures., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The influential role of monovalent and divalent metal cations in facilitating conformational transitions in both RNA and DNA has been a target of intense biophysical research efforts. However, organic neutrally charged cosolutes can also significantly alter nucleic acid conformational transitions. For example, highly soluble small molecules such as trimethylamine N-oxide (TMAO) and urea are occasionally utilized by organisms to regulate cellular osmotic pressure. Ensemble studies have revealed that these so-called osmolytes can substantially influence the thermodynamics of nucleic acid conformational transitions. In the present work, we exploit single-molecule FRET (smFRET) techniques to measure, for first time, the kinetic origins of these osmolyte-induced changes to the folding free energy. In particular, we focus on smFRET RNA and DNA constructs designed as model systems for secondary and tertiary structure formation. These findings reveal that TMAO preferentially stabilizes both secondary and tertiary interactions by increasing kfold and decreasing kunfold, whereas urea destabilizes both conformational transitions, resulting in the exact opposite shift in kinetic rate constants (i.e., decreasing kfold and increasing kunfold). Complementary temperature-dependent smFRET experiments highlight a thermodynamic distinction between the two different mechanisms responsible for TMAO-facilitated conformational transitions, while only a single mechanism is seen for the destabilizing osmolyte urea. Finally, these results are interpreted in the context of preferential interactions between osmolytes, and the solvent accessible surface area (SASA) associated with the (i) nucleobase, (ii) sugar, and (iii) phosphate groups of nucleic acids in order to map out structural changes that occur during the conformational transitions.

Gold nanoparticles (GNPs) sensitize biomolecules to radiation in two ways: by locally increasing the radiation energy absorbed and by modifying the sensitivity of the target biomolecules to radiation. Taking DNA as the biological target, we present the first investigation of the latter chemical mechanism of radiosensitization by irradiating thin films made of GNP-DNA complexes with 10 eV electrons. Naked GNPs of 5 and 15 nm diameters were synthesized and electrostatically bound to DNA. Damage to the GNP-DNA complexes were analyzed, as a function of electron fluence, by electrophoresis. In identical 5-monolayer films, the yield of DNA damage, as well as the enhancement factor due to the presence of 5 nm positively-charged nanoparticles, increased with rising ratio of GNPs to DNA up to 1:1. In comparison, increasing the ratio of negatively-charged 15 nm GNPs to DNA did not increase damage. As verified by XPS and zeta potential measurements, the binding of plasmid DNA to the surface of the two sizes of GNPs varies owing to the characteristics of the GNP surface and electrostatic interaction. The results indicate that strong binding of GNPs to DNA could significantly influence the efficiency of the chemical radiosensitization mechanism. This mechanism appears to be an important component of the overall process of GNP radiosensitization and should be considered when modeling this phenomenon. Our results suggest that small size GNPs (diam. ≤ 5 nm) are more efficient radiosensitizers compared to larger GNPs when delivered into cancerous cells, where their action should be cell-cycle dependent.

The interaction of the widely used anticancer drug cisplatin with DNA bases was studied by EXAFS and vibrational spectroscopy (FTIR, Raman and INS), coupled with DFT/plane-wave calculations. Detailed information was obtained on the local atomic structure around the Pt(ii) centre, both in the cisplatin-purine (adenine and guanine) and cisplatin-glutathione adducts. Simultaneous neutron and Raman scattering experiments allowed us to obtain a reliable and definite picture of this cisplatin interplay with its main pharmacological target (DNA), at the molecular level. The vibrational experimental spectra were fully assigned in the light of the calculated pattern for the most favoured geometry of each drug-purine adduct, and cisplatin's preference for guanine (G) relative to adenine (A) within the DNA double helix was experimentally verified: a complete N by S substitution in the metal coordination sphere was only observed for [cDDP-A2], reflecting a somewhat weaker Pt-A binding relative to Pt-G. The role of glutathione on the drug's pharmacokinetics, as well as on the stability of platinated DNA adducts, was evaluated as this is the basis for glutathione-mediated intracellular drug scavenging and in vivo resistance to Pt-based anticancer drugs. Spectroscopic evidence of the metal's preference for glutathione's sulfur over purine's nitrogen binding sites was gathered, at least two sulfur atoms being detected in platinum's first coordination sphere.