Diketopyrrolopyrrole J‐Aggregates Formed by Self‐Organization with DNA

Bis(2‐thienyl)diketopyrrolopyrrole with two Zn^II‐cyclens (ZnCyc‐DPP) was designed and synthesized to evaluate the selective binding of Zn^II‐cyclen with thymine base in single‐strand DNA as a tool for the construction of a highly ordered multichromophore system on DNAs. Through UV/Vis titrations, gel filtration chromatography, and circular dichroism spectroscopy, ZnCyc‐DPP formed J‐type DPP aggregates with oligo‐dT_n DNAs. The DPP aggregates absorbed on a gold electrode exhibited good photocurrent responses. The present results show that binding Zn^II‐cyclen–chromophore conjugates and thymine bases together is a powerful tool for preparing DNA‐templated multichromophoric systems with specific functions.     

基于T-T碱基错配的荧光传感器特异性检测汞离子

在本文中,我们研制了一种基于T-T碱基错配特异性键合汞离子的荧光传感器用于汞离子的检测。该传感器由两条分别标记了荧光基团（F）和淬灭基团（Q）的DNA探针组成,并且含有两对用于结合汞离子的T-T错配碱基。当汞离子存在时,两条探针之间形成T-Hg2+-T结构,作用力增强,从而拉近了荧光基团与淬灭基团之间的距离,发生能量转移,使荧光信号在一定程度上被淬灭。在优化的条件下,我们使用该传感器对汞离子进行检测,动力学响应范围为50nM到1000nM,线性相关方程为y= 5281.13 - 1650.56 lg[Hg2+] ( R2 = 0.985),检测下限为79nM。此外,我们还考察了该传感器的选择性,当用其它干扰离子（浓度都为1.0µM）代替待测离子进行实验时,没有发生明显的荧光淬灭,说明该传感器具有较高的选择性。该传感器的构建为汞离子的检测提供了一条快速、简便的新途径。     

Base pairing patterns of DNA base lesion spiroiminodihydantoin: A DFT study

The DNA base lesion spiroiminodihydantoin (Sp) is produced in biological systems endogenously and can cause mutation and cancer. It is considered to be more mutagenic and deleterious than 8‐oxoguanine and other oxidized guanine products such as guanidinohydantoin (Gh) and imidazolone. In this work, the base pairing patterns of Sp with each of the normal nucleic acid bases of DNA have been investigated thoroughly using the B3LYP, M06‐2X, and wB97X‐D functionals of density functional theory in conjunction with the aug‐cc‐pVDZ basis set. It is found that the magnitudes of interaction energies between the bases and Sp follow the order: Sp‐guanine >> Sp‐cytosine > Sp‐adenine > Sp‐thymine. The strong Sp‐guanine abnormal base pairing may be the main cause of the observed mutagenicity of Sp. © 2013 Wiley Periodicals, Inc.     

The Radical Cationic Repair Pathway of Cyclobutane Pyrimidine Dimer: The Effect of Sugar‐Phosphate Backbone

Radical cationic repair process of cis–syn thymine dimer has been investigated when (1) sugar‐phosphate backbones were substituted by hydrogen atoms, (2) phosphate group was substituted by two hydrogen atoms each on a sugar ring and (3) sugar‐phosphate backbone was taken into account. The effect of the interactions between N1 and N1′ lone pairs and the C6‐C6′ antibonding orbital are the most important evidences for the cleavage of the C6‐C6′ bond in the first step of radical cationic repair mechanism in the absence of the sugar‐phosphate backbone. The impact of the N1 and N1′ lone pairs on the C6‐C6′ bond cleavage decreases and the energy barrier of the cleavage of that bond significantly increases in the presence of the deoxynucleoside sugars and the sugar‐phosphate backbone.     

Photochemistry and Photobiology of the Spore Photoproduct: A 50‐Year Journey

Fifty years ago, a new thymine dimer was discovered as the dominant DNA photolesion in UV‐irradiated bacterial spores [Donnellan, J. E. & Setlow R. B. (1965) Science, 149, 308–310], which was later named the spore photoproduct (SP). Formation of SP is due to the unique environment in the spore core that features low hydration levels favoring an A‐DNA conformation, high levels of calcium dipicolinate that acts as a photosensitizer, and DNA saturation with small, acid‐soluble proteins that alters DNA structure and reduces side reactions. In vitro studies reveal that any of these factors alone can promote SP formation; however, SP formation is usually accompanied by the production of other DNA photolesions. Therefore, the nearly exclusive SP formation in spores is due to the combined effects of these three factors. Spore photoproduct photoreaction is proved to occur via a unique H‐atom transfer mechanism between the two involved thymine residues. Successful incorporation of SP into an oligonucleotide has been achieved via organic synthesis, which enables structural studies that reveal minor conformational changes in the SP‐containing DNA. Here, we review the progress on SP photochemistry and photobiology in the past 50 years, which indicates a very rich SP photobiology that may exist beyond endospores.     

A doorway state leads to photostability or triplet photodamage in thymine DNA

Ultraviolet irradiation of DNA produces electronic excited states that predominantly eliminate the excitation energy by returning to the ground state (photostability) or following minor pathways into mutagenic photoproducts (photodamage). The cyclobutane pyrimidine dimer (CPD) formed from photodimerization of thymines in DNA is the most common form of photodamage. The underlying molecular processes governing photostability and photodamage of thymine-constituted DNA remain unclear. Here, a combined femtosecond broadband time-resolved fluorescence and transient absorption spectroscopies were employed to study a monomer thymidine and a single-stranded thymine oligonucleotide. We show that the protecting deactivation of a thymine multimer is due to an ultrafast single-base localized stepwise mechanism where the initial excited state decays via a doorway state to the ground state or proceeds via the doorway state to a triplet state identified as a major precursor for CPD photodamage. These results provide new mechanistic characterization of and a dynamic link between the photoexcitation of DNA and DNA photostability and photodamage.     

Distinguishing Individual DNA Bases in a Network by Non‐Resonant Tip‐Enhanced Raman Scattering

The importance of identifying DNA bases at the single‐molecule level is well recognized for many biological applications. Although such identification can be achieved by electrical measurements using special setups, it is still not possible to identify single bases in real space by optical means owing to the diffraction limit. Herein, we demonstrate the outstanding ability of scanning tunneling microscope (STM)‐controlled non‐resonant tip‐enhanced Raman scattering (TERS) to unambiguously distinguish two individual complementary DNA bases (adenine and thymine) with a spatial resolution down to 0.9 nm. The distinct Raman fingerprints identified for the two molecules allow to differentiate in real space individual DNA bases in coupled base pairs. The demonstrated ability of non‐resonant Raman scattering with super‐high spatial resolution will significantly extend the applicability of TERS, opening up new routes for single‐molecule DNA sequencing.     

Functional monomers and polymers. XCIV. Photochemical reactions on the synthetic polymers containing thymine bases in the presence of adenine derivatives

Photodimerization reactions of polyacrylate and polymethacrylate derivatives and the dimer model compound containing thymine bases were studied in the presence of adenine derivatives in dimethyl sulfoxide; N,N-dimethylformamide; and dimethyl sulfoxide–ethylene glycol solutions. The photodimerization of thymine bases both in the polymers and in the dimer model compound was found to be quenched by the addition of adenine derivatives. Base-base interaction in the ground state was also studied by ultraviolet (UV) spectroscopy in the three solvents. The quenching of the photodimerizationof thymine bases in the presence of adenine derivatives was discussed in terms of the specific interaction between adenine and thymine bases both in ground and excited states.     

The Photochemistry of Thymine in Frozen Aqueous Solution: Trimeric and Minor Dimeric Products

Early work identified three compounds, namely the c,s cyclobutane dimer, the so‐called (6‐4) photoproduct (5‐hydroxy‐6‐4′‐(5‐methylpyrimidin‐2′‐one)‐5,6‐dihydrothymine) and a trimer hydrate, as products formed upon UV irradiation of thymine in frozen aqueous solution. More recent work has shown that an (α‐4) product, namely α‐4′‐(5′‐methylpyrimidine‐2′‐one)‐thymine, is a likely product formed under these reaction conditions. During a thorough reinvestigation of the photochemistry of Thy in ice at ?78.5°C, we found that a variety of other products could be detected. In addition to the c,s dimer, the other three known cyclobutane dimers, namely the c,a, t,s and t,a forms, are produced, although in considerably smaller amounts. The so‐called “spore product” of thymine (5,6‐dihydro‐5‐(α‐thyminyl)thymine) is likewise formed. Two other dimers have been identified as minor products; one of these has been determined to be 5‐(thymin‐3‐yl)‐5,6‐dihydrothymine and the other has been tentatively assigned to be a (5‐4) adduct (6‐hydroxy‐5‐4′‐(5‐methylpyrimidin‐2′‐one)‐5,6‐dihydrothymine). Compounds with the behavior expected of true trimeric compounds have been isolated via HPLC and characterized by mass spectrometry and photochemical behavior. One of these materials, putatively containing an oxetane ring, decomposes thermally to a secondary trimeric product that is then converted into the known trimer hydrate.     

Formation of a Thymine‐HgII‐Thymine Metal‐Mediated DNA Base Pair: Proposal and Theoretical Calculation of the Reaction Pathway

A reaction mechanism that describes the substitution of two imino protons in a thymine:thymine (T:T) mismatched DNA base pair with a Hg^II ion, which results in the formation of a (T)N3‐Hg^II‐N3(T) metal‐mediated base pair was proposed and calculated. The mechanism assumes two key steps: The formation of the first Hg^II? N3(T) bond is triggered by deprotonation of the imino N3 atom in thymine with a hydroxo ligand on the Hg^II ion. The formation of the second Hg^II? N3(T) bond proceeds through water‐assisted tautomerization of the remaining, metal‐nonbonded thymine base or through thymine deprotonation with a hydroxo ligand of the Hg^II ion already coordinated to the thymine base. The thermodynamic parameters ΔG_R=?9.5 kcal mol^?1, ΔH_R=?4.7 kcal mol^?1, and ΔS_R=16.0 cal mol^?1 K^?1 calculated with the ONIOM (B3LYP:BP86) method for the reaction agreed well with the isothermal titration calorimetric (ITC) measurements by Torigoe et al. [H. Torigoe, A. Ono, T. Kozasa, Chem. Eur. J. 2010 , 16, 13218–13225]. The peculiar positive reaction entropy measured previously was due to both dehydration of the metal and the change in chemical bonding. The mercury reactant in the theoretical model contained one hydroxo ligand in accord with the experimental pK_a value of 3.6 known for an aqua ligand of a Hg^II center. The chemical modification of T:T mismatched to the T‐Hg^II‐T metal‐mediated base pair was modeled for the middle base pair within a trinucleotide B‐DNA duplex, which ensured complete dehydration of the Hg^II ion during the reaction.     

Interaction Between DNA and Some Salicylic Acid Derivatives and Characterization of Their DNA Targets

A nanofiber polypyrrole (PPy) film was electrochemically deposited on a Pt electrode and used for immobilization of single‐stranded DNA (ssDNA) and investigation of hybridization events. Then, the interaction of DNA with four salicylic acid (SA) derivatives was studied with electrochemical methods. The oxidation peak of guanine was decreased by increasing the concentrations of salicylic acid derivatives. The binding constants of these compounds with four different sequences of DNA including different percentages of guanine‐cytosine and adenine‐thymine bases were calculated and it was clarified that sequences with higher percentage of adenine‐thymine bases have a higher binding constant in their interaction with SA derivatives.     

Hydrogen-abstracted adenine-thymine radicals with interesting transferable properties

The formation of radicals on DNA bases through various pathways can lead to harmful structural alterations. Such processes are of interest for preventing alteration of healthy DNA and, conversely, to develop more refined methods for inhibiting the replication of unwanted mutagenic DNA. In the present work, we explore theoretically the energetic and structural properties of the nine possible neutral radicals formed via hydrogen abstraction from the adenine-thymine base pair. The lowest energy radical is formed by loss of a hydrogen atom from the methyl group of thymine. The next lowest energy radicals, lying 8 and 9 kcal mol-1 higher than the global minimum, are those in which hydrogens are removed from the two nitrogens that would join the base pair to 2-deoxyribose in double-stranded DNA. The other six radicals lie between 16 and 32 kcal mol-1 higher in energy. Unlike the guanine-cytosine base pair, adenine-thymine (A-T) exhibits only minor structural changes upon hydrogen abstraction, with all A-T derived radicals maintaining planarity. Moreover, the energetic ordering for the radicals of the two isolated bases (adenine and thymine) is preserved upon formation of the base pair, though with a wider spread of energies. Even more significantly, the energetic interleaving of the (A-H)*-T and A-(T-H)* radicals is correctly predicted from the X-H bond dissociation energies of the isolated adenine and thymine. This suggests that the addition of the hydrogen-bonded complement base only marginally affects the bond energies.     

Sequence, structure and energy transfer in DNA

Excitation energy transfer in DNA has similarities to charge transfer, but the transport is of an excited state, not of mass or charge. Use of the fluorescent, modified adenine base 2‐aminopurine (2AP) as an energy trap in short (3‐ to 20‐base) single‐ and double‐stranded DNA oligomers is reviewed. Variation of 2AP’s neighboring sequence shows (1) relatively efficient transfer from adenine compared to that from cytosine and thymine, (2) efficient transfer from guanine, but only when 2AP is at the 3′ end, (3) approximate equality of efficiencies for 3′ to 5′ and 5′ to 3′ directional transfer in adenine tracks. The overall, average transfer distance at room temperature is about four adenine bases or less before de‐excitation. The transfer fluorescence excitation spectral shape is similar to that of the absorption spectrum of the neighboring normal bases, confirming that initial excitation of the normal bases, followed by emission from 2AP (i.e. energy transfer), is occurring. Transfer apparently may take place both along one strand and cross‐strand, depending on the oligomer sequence. Efficiency increases when the temperature is decreased, rising above 50% (overall efficiency) in decamers of adenine below ?60°C (frozen media). Modeling of the efficiencies of transfer from the nearest several adenine neighbors of 2AP in these oligomers suggests that the nearest two neighbors transfer with near 100% efficiency. As bases in B DNA, as well as in single‐stranded DNA, are separated by less than 5 Å (less than the size of a base), standard Förster transfer theory should not apply. Indeed, while both theory and experiment show efficiency decreasing with donor–acceptor distance, the experimental dependence clearly disagrees with Förster 1/r⁶ dependence. It is not yet clear what the best theoretical approach is, but any calculation must deal accurately with the excited states of bases, including strong base–base interactions and structural fluctuations, and should reflect the increase of efficiency with temperature decrease and the relative insensitivity to strandedness (single, double). Attempts to use DNA as a molecular “fiber optic” face three primary challenges. First, reasonable efficiency over more than a base or two occurs only in adenine stretches at temperatures well below freezing. Second, transfer in these adenine tracks is efficient in both directions. Third, absorption of UV light occurs randomly, making excitation at a specific site on this “fiber optic” a challenge.     

Construction of 5‐amino‐1,10‐phenanthroline‐Fe(II) Nanostructures on Glassy Carbon Electrode: Simultaneous and Selective Determination of Purine and Pyrimidine DNA Bases

5‐amino‐1,10‐phenanthroline‐Fe(II) complex is immobilized onto GC electrode and used for determination of DNA bases. Modifications are traced by electrochemical methods. All DNA bases are electroactive on the modified electrode. The I_ps increased linearly with increase of DNA bases concentration. A wide response range was observed for each base (～4 orders for guanine (GA) and adenine (A); and ～2.5 orders for thymine (T) and cytosine (C)) with DLs of 0.15, 4.44, 133.0 and 230.0 nM, respectively. The electrode was applied for determination of calf‐thymus DNA bases. The value obtained for [(GA+C)/(A+T)], 0.78, is in good agreement with standard value, 0.77.     

Effect of glucocorticoids and their complexes with apolipoprotein A‐I on the secondary structure of eukaryotic DNA

The tetrahydrocortisol–apolipoprotein A‐I complex specifically interacts with eukaryotic DNA isolated from rat liver. This interaction is highly cooperative and of a saturating nature. One DNA molecule binds about 54 molecules of the complex. Small‐angle X‐ray scattering has shown that hydrogen bonds between nitrous bases are destroyed and that single‐stranded structures are formed at the interaction of the tetrahydrocortisol–apolipoprotein A‐I complex with eukaryotic DNA. The most probable site of binding the tetrahydrocortisol–apolipoprotein A‐I complex with DNA is the sequence of the CC(GCC)_n type entering the structure of many genes, among them the structure of the human apolipoprotein A‐I gene. Oligonucleotide of this type has been synthesized. The association constant (K_ass) of its complexation was shown to be 1.66 · 10⁶ M^?1. Substitution of tetrahydrocortisol for cortisol in the complex results in a considerable decrease of K_ass. IR‐spectroscopy study has shown that the interaction of tetrahydrocortisol with oligonucleotide CC(GCC)_3–5 is accompanied by the formation of hydrogen bonds via the CO‐NH, PO₂, and OH groups of desoxycytidinephosphate. The tetrahydrocortisol–apolipoprotein A‐I complex alters the DNA secondary structure formed at the interaction with the hormone, causing the structural transition “order → tangle.” It is assumed that in the GC‐pairs of the given DNA sequence, tetrahydrocortisol initiates the rupture of hydrogen bonds, while the hydrophobic interactions between nitrous bases and apoA‐I intensify this process. © 2004 Wiley Periodicals, Inc. Int J Quantum Chem, 2005     

On the Formation of Thymine Photodimers in Thymine Single Strands and Calf Thymus DNA

Solar light leads to thymine dimers that are mutagenic and primary cause of skin cancer. Here, we report absorption and synchrotron radiation circular dichroism (CD) spectra of T_n single strands with different number n of bases (n = 2–7, 10, 11) recorded after various 254 nm irradiation times. From a principal component analysis of the CD spectra, we extract fingerprint spectra of both the cyclobutane pyrimidine dimer (CPD) and the pyrimidine (6‐4) pyrimidone photoadduct (64PP). Extending the CD measurements to the vacuum ultraviolet region in combination with systematic examinations of size effects is a new approach to gain insight on the dimeric photoproducts. We find a simple linear correlation between n and average number of dimers formed after 1 h of irradiation. The probability for a thymine to engage in a dimer increases from 32% for n = 2 to 41% for n = 11, which implies limited effects of terminal thymines, i.e., the reaction does not occur preferentially at the extremities of the single strands as previously stated. It is even possible to form two dimers with only two bridging thymines. Finally, experiments conducted on calf thymus DNA provided a similar signature of the photodimer, but differences are also evident.     

Gas chromatographic-mass spectrometric method for the assessment of oxidative damage to double-stranded dna by quantification of thymine glycol residues

A technique for the measurement of thymine glycol at parts per million concentrations in double-stranded polymeric DNA is described. The procedure utilizes base to ring-open DNA-bound thymine glycol in the presence of monomeric [²H₄]thymine glycol as an internal standard, followed by reduction, solvolytic cleavage, and quantification of the characteristic methyl-2-methylglycerate released from polymeric DNA. Methyl-2-methyl-glycerate is derivatized to form the di-tert-butyldimethylsilyl [(TBDMS)₂] ether to enhance its gas chromatographic properties and electron ionization detection. This assay was tested by measuring thymine gIyco1 levels in native, undamaged DNA (not purposefully oxidized). The measured quantities of thymine glycol are proportional to the amount of DNA analyzed. Components of DNA not containing oxidizable thymine do not contribute to the measured signal from methyl-2-methylglycerate-(TBDMS)₂. These results indicate that there is approximately one thymine glycol per lo6 bases in undamaged DNA and that this value increases with storage of DNA in refrigerated aqueous solutions.     

VARIATION IN THE PHOTOCHEMICAL REACTIVITY OF THYMINE IN THE DNA OF B. SUBTILIS SPORES, VEGETATIVE CELLS AND SPORES GERMINATED IN CHLORAMPHENICOL*,†

Abstract— The chief photoproduct of thymine produced in u.v. irradiated (2537Å) vegetative cells of B. subtilis is the cyclobutane-type dimer while in spores very little of this dimer is produced (maximum yield 2·6 per cent of thymine) but a new photoproduct is produced in high yield (maximum of 28·4 per cent of thymine). This difference in photochemical response appears to be due, at least in part, to a difference in uydration of the DNA. The photochemistry of thymine in isolated DNA irradiated in solution is similar to that of DNA in irradiated vegetative cells, but differs markedly from that of isolated DNA irradiated dry. The yield of cyclobutane-type thymine dimer is much reduced in isolated DNA irradiated dry but a new photoproduct of thymine. is produced which is chromatographically similar to the spore photoproduct. The yield of this photoproduct, however, is never as great as that obtained in irradiated spores. The photochemistry of the DNA thymine of spores germinated in the presence of chloramphenicol is very similar to that of normal vegetative cells. Except for hydration, the physical state of the DNA is probably not otherwise altered by germination in the presence of chloramphenicol since DNA replication is prevented by the presence of chloramphenicol. These results are also consistent with the hypothesis that the unique photochemistry of spores is due, at least in part, to the hydration state of the DNA. The acid stability of the spore photoproduct is indicated by the fact that it is isolated from irradiated spores after hydrolysis in trifluoroacetic acid at 155°C for 60 min. It still contains the methyl group of thymine as judged by the fact that for a given dose of u.v. the same yield of photoproduct was obtained whether the spores were labeled with thymine-2–C-14 or -methyl-C-14. This photoproduct is stable to reirradiation (2537Å) in solution under condiditions where thymine dimers of the cyclobutane-type are completely converted back to monomeric thymine. On a column of molecular sieve material (Sephadex-G10), the spore photoproduct elutes in a region intermediate between the cyclobutanetype thymine dimers and monomeric thymine. Of the numerous compounds tested by paper chromatography, the spore photoproduct is most similar (but not identical) in several solvents to 5–hydroxyuracil and 5–hydroxymethyluracil. Our data do not allow us to decide if the product is a monomer or a dimer. Although the photochemistry of thymine in the DNA of spores differs markedly from that for vegetative cells, several lines of evidence make it seem doubtful that the enhanced resistance of spores to u.v. relative to that of vegetative cells can be explained solely on the basis of this difference in the photochemistry of DNA thymine.     

An ab initio MO study on the thymine dimer and its radical cation

Ab initio SCF calculations with the 6-31G basis set for the thymine dimer (cys-syn form) and the thymine dimer radical cation are reported. The fusion of the thymine bases at the C5 and C6 positions involves the formation of a cyclobutane ring with puckering. The puckering causes a notable difference in the electronic structures of the two bases of the thymine dimer. The density of the HOMO orbital of the thymine dimer is localized on the O2, N1, and C6 atoms of both thymine rings, with the higher density on one of the rings. The HOMO orbital has a bonding character on the C6(SINGLEBOND)C6 bond. In the thymine dimer radical cation, the unpaired electron is localized mainly on the lengthened C6(SINGLEBOND)C6 bond with the higher density on one of the C6 atoms and to a lesser extent on the N1 atoms of both rings. © 1996 John Wiley & Sons, Inc.     

Oxidation of DNA Bases Influenced by the Presence of Other Bases

Electrochemical detection of DNA is a highly important topic. Here we show that the electrochemical responses of one DNA base (guanine, adenine, cytosine or thymine), in terms of oxidation potential, current intensity, peak width and resolution can be highly influenced by the presence of other DNA bases at electrochemically reduced graphene oxide (ER‐GO) as well as standard glassy carbon electrode. We have observed that the effects were more significant for adenine base on ER‐GO and cytosine base on glassy carbon (GC) electrode. Differences in responses were generally low in a mixture of four different DNA bases but interestingly, deviations become significantly larger when only one or two other bases were present. Our findings are of paramount importance for future developments in DNA detection and analysis since individual DNA bases are not present in isolation in nature or in typical biosensing systems.