Long‐Wavelength Fluorescence from 2‐Aminopurine‐Nucleobase Dimers in DNA

When 2‐aminopurine (2AP) is substituted for adenine in DNA, it is widely accepted that its fluorescence spectrum is essentially unchanged from that of the free fluorophore. We show that 2AP in DNA exhibits long‐wavelength emission and excitation bands, in addition to the familiar short‐wavelength spectra, as a result of formation of a ground‐state heterodimer with an adjacent, π‐stacked, natural base. The observation of dual emission from 2AP in a variety of oligodeoxynucleotide duplexes and single strands demonstrates the generality of this phenomenon. The photophysical and conformational properties of the long‐wavelength‐emitting 2AP‐nucleobase dimer are examined. Analogous long‐wavelength fluorescence is seen when 2AP π‐stacks with aromatic amino acid sidechains in the active sites of methyltransferase enzymes during DNA nucleotide flipping.     

Electrochemical Detection of Abasic Site‐Containing DNA

A simple and rapid approach for detecting apurinic (AP) sites in DNA, based on direct stripping chronopotentiometric measurements of the adenine and guanine nucleobases at a graphite electrode is described. Tetrahydrofuranyl residues, lacking a nucleobase moiety, were utilized for designing the AP sites and were incorporated in 19‐mer oligonucleotides. The change of adenine‐to‐guanine response ratio (A/G) in one‐, two‐ or three‐substituted adenosine residues for stable analogs of AP sites was exploited for electrochemical measurements of the adenine loss. The resulting A/G response ratio decreases linearly upon increasing the number of AP sites in the oligonucleotides; the values of A/G electrochemical signals were slightly enhanced when compared to the actual purine content. HPLC analysis of the released nucleobases confirmed that the sulfuric acid‐induced oligonucleotide cleavage provides complete apurination and dissolution of the released nucleobases in aqueous solution. Additional experiments with mixtures of free nucleobases and purine nucleosides reveal that the larger A/G ratio observed in the electrochemical analysis of AP‐site‐containing oligomers is attributed to the influence of the acid and/or thermal decomposition products (particularly the sugar fragments). This study represents the first step in developing a simple and direct electrochemical assay of AP sites in single‐stranded DNA.     

Thermodynamic Impact of Abasic Sites on Simulated Translesion DNA Synthesis

Loss of a base in DNA and the creation of an abasic (apurinic/apyrimidinic, AP) site is a frequent lesion that may occur spontaneously, or as a consequence of the action of DNA‐damaging agents. The AP lesion is mutagenic or lethal if not repaired. We report a systematic thermodynamic investigation by differential scanning calorimetry on the evolution, during primer extension, of a model AP site in chemically simulated DNA translesion synthesis. Incorporation of dAMP (deoxyadenosine monophosphate), as well as dTMP (deoxythymidine monophosphate), opposite an AP site is enthalpically unfavorable, although incorporation of dTMP is more enthalpically unfavorable than that of dAMP. This finding is in a good agreement with experimental data showing that AP sites block various DNA polymerases of eukaryotic and prokaryotic origin and that, if bypassed, dAMP is preferentially inserted, whereas insertion of dTMP is less likely. The results emphasize the importance of thermodynamic contributions to the insertion of nucleotides opposite an AP site by DNA polymerases.     

An Isosteric and Fluorescent DNA Base Pair Consisting of 4‐aminophthalimide and 2,4‐diaminopyrimidine as C‐Nucleosides

A 13mer DNA duplex containing the artificial 4‐aminophthalimide:2,4‐diaminopyrimidine (4AP:DAP) base pair in the central position was characterized by optical and NMR spectroscopy. The fluorescence of 4AP in the duplex has a large Stokes shift of Δλ =124 nm and a quantum yield of Φ _F=24 %. The NMR structure shows that two interstrand hydrogen bonds are formed and confirms the artificial base pairing. In contrast, the 4‐N ,N ‐dimethylaminophthalimide moiety prefers the syn conformation in DNA. The fluorescence intensity of this chromophore in DNA is very low and the NMR structure shows no significant interaction with DAP. Primer‐extension experiments with DNA polymerases showed that not only is the 4AP C nucleotide incorporated at the desired position opposite DAP in the template, but also that the polymerase is able to progress past this position to give the full‐length product. The observed selectivity supports the NMR results.     

DNA脱碱基位点存在下非氢键配体的增强结合研究

众所周知,插入剂的DNA特性结合位点位于DNA碱基对之间,然而这种非共价相互作用对于含脱碱基（AP）位点的DNA来讲还没有引起足够的重视,虽然在生物细胞中总是存在着DNA脱碱基位点。本文以原黄素（proflavine,PF）为例研究了插入剂对DNA中AP位点的结合特性。实验结果表明,相对于插入位点而言,AP位点是PF的优先结合位点,AP位点的本征结合常数比插入结合常数高一个数量级以上。此外,PF的结合使含脱碱基位点DNA的热稳定性明显提高,表明PF在脱碱基位点的结合构像明显不同于插入结合时的分子定向。本文结果将有助于判断小分子的DNA结合方式所决定的药物的生物化学及生物物理效用。     

Competitive Assay for Theophylline Based on an Abasic Site‐Containing DNA Duplex Aptamer and a Fluorescent Ligand

A fluorescence assay for theophylline, one of the common drugs for acute and chronic asthmatic conditions, has been developed based on an abasic site‐containing DNA duplex aptamer (AP aptamer) in combination with an abasic site‐binding fluorescent ligand, riboflavin. The assay is based on the competitive binding of theophylline and riboflavin at the abasic (AP) site of the AP aptamer. In the absence of theophylline, riboflavin binds to the receptor nucleotide opposite the AP site, which leads to fluorescence quenching of the riboflavin. Upon addition of theophylline, competitive binding occurs between theophylline and riboflavin, which results in an effective fluorescence restoration due to release of riboflavin from the AP site. From an examination of the optimization of the AP aptamers, the complex of riboflavin with a 23‐mer AP aptamer (5′‐TCT GCG TCC AGX GCA ACG CAC AC‐3′/5′‐GTG TGC GTT GCC CTG GAC GCA GA‐3′; X : the AP site (Spacer C3, a propylene residue)) possessing cytosine as a receptor nucleotide was found to show a selective and effective fluorescence response to theophylline; the limit of detection for theophylline was 1.1 μM . Furthermore, fluorescence detection of theophylline was successfully demonstrated with high selectivity in serum samples by using the optimized AP aptamer and riboflavin.     

O6‐Alkylguanine DNA Alkyltransferase Repair Activity Towards Intrastrand Cross‐Linked DNA is Influenced by the Internucleotide Linkage

Oligonucleotides containing an alkylene intrastrand cross‐link (IaCL) between the O⁶‐atoms of two consecutive 2′‐deoxyguanosines (dG) were prepared by solid‐phase synthesis. UV thermal denaturation studies of duplexes containing butylene and heptylene IaCL revealed a 20 °C reduction in stability compared to the unmodified duplexes. Circular dichroism profiles of these IaCL DNA duplexes exhibited signatures consistent with B‐form DNA. Human O⁶‐alkylguanine DNA alkyltransferase (hAGT) was capable of repairing both IaCL containing duplexes with slightly greater efficiency towards the heptylene analog. Interestingly, repair efficiencies of hAGT towards these IaCL were lower compared to O⁶‐alkylene linked IaCL lacking the 5′‐3′‐phosphodiester linkage between the connected 2′‐deoxyguanosine residues. These results demonstrate that the proficiency of hAGT activity towards IaCL at the O⁶‐atom of dG is influenced by the backbone phosphodiester linkage between the cross‐linked residues.     

Multispectroscopic DNA interaction and Docking studies

Two new water soluble oxovanadium(IV) complexes with formulae Na[VO(his)(met)SO₄] (1) and Na[VO(gly)(met)SO₄] (2), (gly=glycine his=histidine, and met=metformin) were synthesized and characterized by LCMS, UV‐Visible absorption, infrared spectra, magnetic moment, elemental analysis, thermal analysis and electronic spectral studies. The metal center was found in an octahedral geometry. DNA binding interaction of these complexes with CT DNA has been explored by UV‐Visible absorption, fluorescence, viscosity measurements and cleavage studies. Finally the binding of the complexes with CT‐DNA could be surface binding, mainly in the groove binding. The complexes were docked in to B‐DNA sequence, 5’(D*AP*CP*CP*GP*AP*CP*GP*TP*CP*GP*GP*T)‐3’ retrieved from protein data bank (PDB ID: 423D), using Discovery Studio 2.1 software.     

Surface‐Assisted Large‐Scale Ordering of DNA Origami Tiles

The arrangement of DNA‐based nanostructures into extended higher order assemblies is an important step towards their utilization as functional molecular materials. We herein demonstrate that by electrostatically controlling the adhesion and mobility of DNA origami structures on mica surfaces by the simple addition of monovalent cations, large ordered 2D arrays of origami tiles can be generated. The lattices can be formed either by close‐packing of symmetric, non‐interacting DNA origami structures, or by utilizing blunt‐end stacking interactions between the origami units. The resulting crystalline lattices can be readily utilized as templates for the ordered arrangement of proteins.     

Chromium‐metformin ternary complexes: Thermal,DNA interaction and Docking studies

Three chromium ternary complexes with metformin (met) as a primary ligand and bipyridine (bipy) or ortho‐phenylenediamine (opda) or ortho‐phenanthroline (phen) as secondary ligand were synthesized. These complexes [Cr (Cl)₂(Hmet)(bipy)]‐( 1 ), [Cr (Cl)₂(Hmet)(opda)]‐( 2 ) and [Cr (Cl)₂(Hmet)(phen)]‐( 3 ) were characterized by LC–MS, elemental analysis, molar conductance, thermal analysis, infrared spectroscopy, electronic spectroscopy. The geometrical structures have been found to be octahedral. Degradation pattern of the compounds is shown by thermal studies. The Kinetic parameters‐ energy of activation (E_a), enthalpy (ΔH), entropy (ΔS) and free energy changes (ΔG) have been determined by thermogravimetric data. Coats‐Redfern integration method with thirteen kinetic models was used to calculate the kinetic and thermodynamic parameters for the degradation of all the complexes. The stabilities of the complexes were obtained from their molecular orbital structures from which the quantum chemical parameters were calculated using the HOMO‐LUMO energies. UV–Visible absorption, fluorescence, and viscosity measurements have been conducted to assess the interaction of the complexes with CT DNA. The complexes showed absorption hyperchromism in its UV–Vis spectrum with DNA. The binding constants K_b from UV–Vis absorption studies were 3.1x10⁴, 4.4x10⁴, 5x10⁴ M^?1 for 1, 2, 3 respectively and Stern–Volmer quenching constants (K_sq) from fluorescence studies were 0.137, 0.532, 0.631 for 1, 2, 3 respectively. Finally, viscosity measurements revealed that the binding of the complexes with CT‐DNA could be surface binding, mainly due to groove binding. The activity of complexes towards DNA cleavage decrease in the order of 3 > 2 > 1.The light switching properties of the complexes were also evaluated. The complexes were docked in to B‐DNA sequence, 5′(D*AP*CP*CP*GP*AP*CP*GP*TP*CP*GP*GP*T)‐3′ retrieved from protein data bank (PDB ID: 423D), using Discovery Studio 2.1 software.     

Os2–Os4 Switch Controls DNA Knotting and Anticancer Activity

Dinuclear trihydroxido‐bridged osmium–arene complexes are inert and biologically inactive, but we show here that linking dihydroxido‐bridged Os^II–arene fragments by a bridging di‐imine to form a metallacycle framework results in strong antiproliferative activity towards cancer cells and distinctive knotting of DNA. The shortened spacer length reduces biological activity and stability in solution towards decomposition to biologically inactive dimers. Significant differences in behavior toward plasmid DNA condensation are correlated with biological activity.     

The Electrostatic Origin of Low‐Hydration Polymorphism in DNA

In recent years, significant progress has been made towards uncovering the physical mechanisms of low‐hydration polymorphism in double‐helical DNA. The effect appears to be mechanistically similar in different biological systems, and it is due to the ability of water to form spanning H‐bonded networks around biomacromolecules via a quasi‐two‐dimensional percolation transition. In the case of DNA, disintegration of the spanning H‐bonded network leads to electrostatic condensation of DNA strands because, below the percolation threshold, water loses its high dielectric permittivity, whereas the concentration of neutralizing counterions becomes high. In this Concept article arguments propose that this simple electrostatic mechanism represents the universal origin of low‐hydration polymorphism in DNA.     

Synthesis,Characterization, and Repair of a Flexible O6‐2′‐Deoxyguanosine‐alkylene‐O6‐2′‐deoxyguanosine Intrastrand Cross‐Link

Oligonucleotides tethered by an alkylene linkage between the O⁶‐atoms of two consecutive 2′‐deoxyguanosines, which lack a phosphodiester linkage between these residues, have been synthesized as a model system of intrastrand cross‐linked (IaCL) DNA. UV thermal denaturation studies of duplexes formed between these butylene‐ and heptylene‐linked oligonucleotides with their complementary DNA sequences revealed about 20 °C reduction in stability relative to the unmodified duplex. Circular dichroism spectra of the model IaCL duplexes displayed a signature characteristic of B‐form DNA, suggesting minimal global perturbations are induced by the lesion. The model IaCL containing duplexes were investigated as substrates of O⁶‐alkylguanine DNA alkyltransferase (AGT) proteins from human and E. coli (Ada‐C and OGT). Human AGT was found to repair both model IaCL duplexes with greater efficiency towards the heptylene versus butylene analog adding to our knowledge of substrates this protein can repair.     

A Tri‐copper(II) Complex Displaying DNA‐Cleaving Properties and Antiproliferative Activity against Cancer Cells

A new disubstituted terpyridine ligand and the corresponding tri‐copper(II) complex have been prepared and characterised. The binding affinity and binding mode of this tri‐copper complex (as well as the previously reported mono‐ and di‐copper analogues) towards duplex DNA were determined by using UV/Vis spectroscopic titrations and fluorescent indicator displacement (FID) assays. These studies showed the three complexes to bind moderately (in the order of 10⁴ M ^?1) to duplex DNA (ct‐DNA and a 26‐mer sequence). Furthermore, the number of copper centres and the nature of the substituents were found to play a significant role in defining the binding mode (intercalative or groove binding). The nuclease potential of the three complexes was investigated by using circular plasmid DNA as a substrate and analysing the products by agarose‐gel electrophoresis. The cleaving activity was found to be dependent on the number of copper centres present (cleaving potency was in the order: tri‐copper>di‐copper>mono‐copper). Interestingly, the tri‐copper complex was able to cleave DNA without the need of external co‐reductants. As this complex displayed the most promising nuclease properties, cell‐based studies were carried out to establish if there was a direct link between DNA cleavage and cellular toxicity. The tri‐copper complex displayed high cytotoxicity against four cancer cell lines. Of particular interest was that it displayed high cytotoxicity against the cisplatin‐resistant MOLT‐4 leukaemia cell line. Cellular uptake studies showed that the tri‐copper complex was able to enter the cell and more importantly localise in the nucleus. Immunoblotting analysis (used to monitor changes in protein levels related to the DNA damage response pathway) and DNA‐flow cytometric studies suggested that this tri‐copper(II) complex is able to induce cellular DNA damage.     

Fabrication of Defined Polydopamine Nanostructures by DNA Origami‐Templated Polymerization

A versatile, bottom‐up approach allows the controlled fabrication of polydopamine (PD) nanostructures on DNA origami. PD is a biosynthetic polymer that has been investigated as an adhesive and promising surface coating material. However, the control of dopamine polymerization is challenged by the multistage‐mediated reaction mechanism and diverse chemical structures in PD. DNA origami decorated with multiple horseradish peroxidase‐mimicking DNAzyme motifs was used to control the shape and size of PD formation with nanometer resolution. These fabricated PD nanostructures can serve as “supramolecular glue” for controlling DNA origami conformations. Facile liberation of the PD nanostructures from the DNA origami templates has been achieved in acidic medium. This presented DNA origami‐controlled polymerization of a highly crosslinked polymer provides a unique access towards anisotropic PD architectures with distinct shapes that were retained even in the absence of the DNA origami template.     

The Role of One‐Electron Reduction of Lipid Hydroperoxides in Causing DNA Damage

The in vivo metabolism of plasma lipids generates lipid hydroperoxides that, upon one‐electron reduction, give rise to a wide spectrum of genotoxic unsaturated aldehydes and epoxides. These metabolites react with cellular DNA to form a variety of pre‐mutagenic DNA lesions. The mechanisms of action of the radical precursors of these genotoxic electrophiles are poorly understood. In this work we investigated the nature of DNA products formed by a one‐electron reduction of (13S)‐hydroperoxy‐(9Z,11E)‐octadecadienoic acid (13S‐HPODE), a typical lipid molecule, and the reactions of the free radicals thus generated with neutral guanine radicals, G(?H)^.. A novel approach was devised to generate these intermediates in solution. The two‐photon‐induced ionization of 2‐aminopurine (2AP) within the 2′‐deoxyoligonucleotide 5′‐d(CC[2AP]TCGCTACC) by intense nanosecond 308 nm excimer laser pulses was employed to simultaneously generate hydrated electrons and radical cations 2AP^.+. The latter radicals either in cationic or neutral forms, rapidly oxidize the nearby G base to form G(?H)^.. In deoxygenated buffer solutions (pH 7.5), the hydrated electrons rapidly reduce 13S‐HPODE and the highly unstable alkoxyl radicals formed undergo a prompt β‐scission to pentyl radicals that readily combine with G(?H)^.. Two novel guanine products in these oligonucleotides, 8‐pentyl‐ and N²‐pentylguanine, were identified. It is shown that the DNA secondary structure significantly affects the ratio of 8‐pentyl‐ and N²‐pentylguanine lesions that changes from 0.9:1 in single‐stranded, to 1:0.2 in double‐stranded oligonucleotides. The alkylation of guanine by alkyl radicals derived from lipid hydroperoxides might contribute to the genotoxic modification of cellular DNA under hypoxic conditions. Thus, further research is warranted on the detection of pentylguanine lesions and other alkylguanines in vivo.     

Single‐Walled Carbon Nanotubes Modified Gold Electrodes as an Impedimetric DNA Sensor

A gold surface modified with a self‐assembled monolayer of 11‐amino‐1‐undecanethiol (AUT) was used for the covalent immobilization of oxidized single‐walled carbon nanotubes (SWNTs). The as‐described SWNTs‐modified substrate was subsequently used to attach single‐stranded deoxyribonucleic acid (ssDNA) used as a substrate for DNA hybridization. Electrochemical impedance spectroscopy measurements were performed to follow the DNA hybridization process by using the redox couple [Fe(CN)₆]^3−/4− as a marker ion. Specifically, changes in charge transfer resistance obtained from the Nyquist plots were used as the sensing parameter of DNA hybridization. The substrate sensitivity towards changes in target DNA concentration, its selectivity toward different DNA sequences and its reusability are successfully demonstrated in this report.     

Designer Extracellular Matrix Based on DNA–Peptide Networks Generated by Polymerase Chain Reaction

Cell proliferation and differentiation in multicellular organisms are partially regulated by signaling from the extracellular matrix. The ability to mimic an extracellular matrix would allow particular cell types to be specifically recognized, which is central to tissue engineering. We present a new functional DNA‐based material with cell‐adhesion properties. It is generated by using covalently branched DNA as primers in PCR. These primers were functionalized by click chemistry with the cyclic peptide c(RGDfK), a peptide that is known to predominantly bind to αvβ3 integrins, which are found on endothelial cells and fibroblasts, for example. As a covalent coating of surfaces, this DNA‐based material shows cell‐repellent properties in its unfunctionalized state and gains adhesiveness towards specific target cells when functionalized with c(RGDfK). These cells remain viable and can be released under mild conditions by DNase I treatment.