Recognition of guanine-guanine mismatches by the dimeric form of 2-amino-1,8-naphthyridine.

Dimeric 2-amino-1,8-naphthyridine selectively binds to a G-G mismatch with high affinity (K(d) = 53 nM). We have investigated a binding mechanism of naphthyridine dimer 2 to a G-G mismatch by spectroscopic studies, thermodynamic analysis, and structure-activity studies for the thermal stabilization of the mismatch. 1H NMR spectra of a complex of 2 with 9-mer duplex d(CATCGGATG)2 containing a G-G mismatch showed that all hydrogens in two naphthyridine rings of 2 were observed upfield compared to those of 2 in a free state. The 2D-NOESY experiments showed that each naphthyridine of 2 binds to a guanine in the G-G mismatch within the pi-stack. In CD spectra, a large conformational change of the G-G mismatch-containing duplex was observed upon complex formation with 2. Isothermal calorimetry titration of 2 binding to the G-G mismatch showed that the stoichiometry for the binding is about 1:1 and that the binding is enthalpy-controlled. It is clarified by structure-activity studies that show (i) the linker connecting two naphthyridine rings was essential for the stabilization of the G-G mismatch, (ii) the binding efficiency was very sensitive to the linker structure, and (iii) the binding of two naphthyridines to each one of two Gs in the G-G mismatch is essential for a strong stabilization. These results strongly supported the intercalation of both naphthyridine rings of 2 into DNA base pairs and the formation of a hydrogen bonded complex with the G-G mismatch.     

Fluorescence and electrochemical detection of pyrimidine/purine transversion by a ferrocenyl aminonaphthyridine derivative

A novel hydrogen bond-forming ligand for pyrimidine/purine transversion, which contains both a fluorescent naphthyridine moiety and a ferrocenyl group as an electrochemical indicator, is described. Hydrogen bond-mediated recognition for a target nucleobase at an abasic site in a DNA duplex is confirmed by both fluorescence and electrochemical measurements. The analysis by fluorescence titration reveals that the ligand shows significant fluorescent quenching upon formation of a 1 : 1 complex with the target nucleobase opposite the abasic site, and the selectivity is in the order of cytosine > thymine > adenine, guanine, reflecting the stability of the hydrogen bond formation.     

荧光小分子2-氨基-5,6,7-三甲基-1,8-萘啶识别dsDNA中胞嘧啶凸出

双链DNA（dsDNA）中单碱基凸出结构（bulge structure）具有重要生物学意义,这种结构也是DNA靶向药物的目标部位之一.荧光小分子2-氨基-5,6,7-三甲基-1,8-萘啶（ATMND）能够通过氢键识别胞嘧啶（cytosine）,因而对dsDNA中凸出的胞嘧啶表现出明显的特异性结合.与其余三种凸出的碱基相比,ATMND与凸出部位胞嘧啶的结合伴随着ATMND荧光的明显猝灭,因而可以用于胞嘧啶凸出结构的识别.利用解旋温度测量、荧光、圆二色光谱对ATMND和存在胞嘧啶凸出结构的dsDNA相互作用进行了研究.荧光滴定结果表明ATMND和dsDNA中凸出部位未配对的胞嘧啶的结合常数K11=4.8×105M？1.通过对含胞嘧啶凸出结构的dsDNA与ATMND结合前后的解旋温度曲线进行解析,发现胞嘧啶凸出结构相邻碱基对凸出的胞嘧啶与ATMND的结合有较大的影响.荧光测量结果也表明ATMND荧光的猝灭效率与凸出结构相邻碱基的类型有关,当相邻碱基为鸟嘌呤（guanine,G）时,荧光猝灭效率最高.基于dsDNA中凸出的碱基对ATMND荧光猝灭效率存在明显差异这一现象,设计了探针DNA实现了乳腺癌相关基因（PGR gene rs3740753）中单核苷酸多态性（G/C变异）的荧光分型.     

Bulged Guanine is Uniquely Sensitive to Damage Caused by Visible‐Light Irradiation of Ethidium Bound to DNA: A Possible Role in Mutagenesis

The interaction of ethidium bromide (=3,8‐diamino‐5‐ethyl‐6‐phenylphenanthridinium bromide; EB) with a series of duplex DNA oligomers having single‐base bulges and single‐base mis‐pairs was investigated (Fig. 1). Physical and spectroscopic analysis reveals no definitive evidence for selective binding of EB at the bulge or mis‐pair. However, irradiation of the bound EB with VIS light leads to lesions in the DNA selectively in the sequence having a bulged guanine. This reaction is attributed to the formation of an exciplex between the lowest excited singlet state of the EB and the bulged guanine. The exciplex is trapped by H₂O, which initiates a sequence of reactions that lead to piperidine‐requiring strand cleavage at this site. Significantly, the damaged bulged guanine is not recognized by E. coli formamidopyrimidine glycosylase (Fpg), which is part of a base‐excision repair system for oxidative damage to DNA. Thus, DNA containing a bulged guanine and having a bound intercalator may be irreparably damaged by exposure to VIS light, even though normal duplex DNA is relatively inert under these conditions.     

Structural basis for bifunctional zinc(II) macrocyclic complex recognition of thymine bulges in DNA

The zinc(II) complex of 1-(4-quinoylyl)methyl-1,4,7,10-tetraazacyclododecane (cy4q) binds selectively to thymine bulges in DNA and to a uracil bulge in RNA. Binding constants are in the low-micromolar range for thymine bulges in the stems of hairpins, for a thymine bulge in a DNA duplex, and for a uracil bulge in an RNA hairpin. Binding studies of Zn(cy4q) to a series of hairpins containing thymine bulges with different flanking bases showed that the complex had a moderate selectivity for thymine bulges with neighboring purines. The dissociation constants of the most strongly bound Zn(cy4q)-DNA thymine bulge adducts were 100-fold tighter than similar sequences with fully complementary stems or than bulges containing cytosine, guanine, or adenine. In order to probe the role of the pendent group, three additional zinc(II) complexes containing 1,4,7,10-tetraazacyclododecane (cyclen) with aromatic pendent groups were studied for binding to DNA including 1-(2-quinolyl)methyl-1,4,7,10-tetraazacyclododecane (cy2q), 1-(4-biphenyl)methyl-1,4,7,10-tetraazacyclododecane (cybp), and 5-(1,4,7,10-tetraazacyclododecan-1-ylsulfonyl)-N,N-dimethylnaphthalen-1-amine (dsc). The Zn(cybp) complex binds with moderate affinity but little selectivity to DNA hairpins with thymine bulges and to DNA lacking bulges. Similarly, Zn(dsc) binds weakly both to thymine bulges and hairpins with fully complementary stems. The zinc(II) complex of cy2q has the 2-quinolyl moiety bound to the Zn(II) center, as shown by (1)H NMR spectroscopy and pH-potentiometric titrations. As a consequence, only weak (500 μM) binding is observed to DNA with no appreciable selectivity. An NMR structure of a thymine-bulge-containing hairpin shows that the thymine is extrahelical but rotated toward the major groove. NMR data for Zn(cy4q) bound to DNA containing a thymine bulge is consistent with binding of the zinc(II) complex to the thymine N3(-) and stacking of the quinoline on top of the thymine. The thymine-bulge bound zinc(II) complex is pointed into the major groove, and there are interactions with the guanine positioned 5' to the thymine bulge.     

Direct labeling of 5-methylcytosine and its applications

Cytosine methylation is one of the most important epigenetic events, and much effort has been directed to develop a simple reaction for methylcytosine detection. In this paper, we describe the design of tag-attachable ligands for direct methylcytosine labeling and their application to fluorescent and electrochemical assays. The effect of the location of bipyridine substituents on the efficiency of osmium complexation at methylcytosine was initially investigated. As a result, a bipyridine derivative with a substituent at the C4 position showed efficient complexation at the methylcytosine residue of single-stranded DNA in a reaction mixture containing potassium osmate and potassium hexacyanoferrate(III). On the basis of this result, a bipyridine derivative with a tag-attachable amino linker at the C4 position was synthesized. The efficiency of metal complex formation in the presence of the osmate and the synthetic ligand was clearly changed by the presence/absence of a methyl group at the C5 position of cytosine. The succinimidyl esters of functional labeling units were then attached to the bipyridine ligand fixed on the methylcytosine. These labels attached to methylcytosine enabled us to detect the target methylcytosine in DNA both fluorometrically and electrochemically. For example, we were able to fluorometrically obtain information on the methylation status at a specific site by means of fluorescence resonance energy transfer from a hybridized fluorescent DNA probe to a fluorescent label on methylcytosine. In addition, by the combination of electrochemically labeled methylcytosine and an electrode modified by probe DNAs, a methylcytosine-selective characteristic current signal was observed. This direct labeling of methylcytosine is a conceptually new methylation detection assay with many merits different from conventional assays.     

A transient product of DNA alkylation can be stabilized by binding localization

A 9-aminoacridine conjugate of a silyl-protected bis(acetoxymethyl)phenol (bisQMP) was synthesized and evaluated as an inducible cross-linking agent of DNA to test our ability to harness the chemistry of reactive quinone methide intermediates (QM). The acridine component was chosen for its ability to delivery an appendage to the major groove of DNA, and the silyl-protected component was chosen for its ability to generate two quinone methide equivalents in tandem upon addition of fluoride. This design created competition between reaction of (1) the 2-amino group of guanine that reacts irreversibly to form a stable QM adduct and (2) the more nucleophilic N7 group of guanine that reacts more efficiently but reversibly to form a labile QM adduct. This lability was apparently compensated by co-localization of the N7 group and QM in the major groove since the N7 adduct appeared to dominate the profile of products formed by duplex DNA. The controlling influence of acridine was also expressed in the sensitivity of the conjugate to ionic strength. High salt concentration inhibited covalent reaction just as it inhibits intercalation of the cationic acridine. As expected for QM formation, the presence of fluoride was indeed necessary for initiating reaction, and no direct benzylic substitution was observed. The conjugate also cross-linked DNA with high efficiency, forming one cross-link for every four alkylation events. Both alkylation and cross-linking products formed by duplex DNA were labile to hot piperidine treatment which led to approximately 40% strand scission and approximately 50% reversion to a material with an electrophoretic mobility equivalent to the parent DNA. All guanines exhibited at least some reactivity including those which were recalcitrant to cross-linking by an oligonucleotide-bisQMP conjugate designed for triplex formation [Zhou, G.; Pande, P.; Johnson, A. E.; Rokita, S. E. Bioorg. Med. Chem. 2001, 9, 2347-2354].     

Sequence Dependence of Fluorescent Quenching of Chromophores Covalently Bonded to Oligo-DNAs before and after Duplex Formation

The labeling of oligo- and polynucleotides with fluorescent probes is an important technique for the analysis of DNAs and RNAs. The effect of duplex formation with complementary oligo-DNA on the quenching behavior of two fluorescent chromophores (eosin, Eo and tetramethylrhodamine, TMR) attached to the 5′-terminal of various 10mer oligo-DNAs was investigated and the dependence of the quenching on DNA base sequence is discussed. We found that guanine residues played a major role in the quenching of the fluorescence of the chromophores. Guanine residues on the complementary DNA near the chromophores, in particular, had a significant influence on the quenching.     

Reactivity of ferrocenylcarbodiimide with DNA duplex containing single-mismatched base pairs.

Ferrocenylcarbodiimide (1), which is known to react with a guanine (G) or thymine (T) base of single stranded DNA, was allowed to react with DNA duplex having a single mismatched base pair of G-T, T-T, or T-cytosine (C). Electrophoreograms of the reaction mixture showed that 1 could react with G or T base of the mismatched sites on the DNA duplex. However, 1 also reacted with the G base of the terminal site on the DNA duplex. This showed that 1 can react with an unpaired base or unstable base pair such as a terminal or mismatched base on the DNA duplex. Electrochemical mismatch detection could be achieved after hybridization of the ferrocenylated mismatched DNA duplex with a selected DNA probe-immobilized electrode. These results revealed that 1 has a potentiality of serving as a labeling reagent of mismatched bases on the DNA duplex, which is important in the search for heterozygous single nucleotide polymorphisms (SNPs).     

Design of a new fluorescent cofactor for DNA methyltransferases and sequence-specific labeling of DNA

Sequence-specific labeling of DNA is of immense interest for analytical and functional studies of DNA. We present a novel approach for sequence-specific labeling of DNA using a newly designed fluorescent cofactor for the DNA methyltransferase from Thermus aquaticus (M.TaqI). Naturally, M.TaqI catalyzes the nucleophilic attack of the exocyclic amino group of adenine within the double-stranded 5'-TCGA-3' DNA sequence onto the methyl group of the cofactor S-adenosyl-L-methionine (AdoMet) leading to methyl group transfer. The design of a new fluorescent cofactor for covalent labeling of DNA was based on three criteria: (1) Replacement of the methionine side chain of the natural cofactor AdoMet by an aziridinyl residue leads to M.TaqI-catalyzed nucleophilic ring opening and coupling of the whole nucleoside to DNA. (2) The adenosyl moiety is the molecular anchor for cofactor binding. (3) Attachment of a fluorophore via a flexible linker to the 8-position of the adenosyl moiety does not block cofactor binding. According to these criteria the new fluorescent cofactor 8-amino[1'-(N'-dansyl)-4'-aminobutyl]-5'-(1-aziridinyl)-5'-deoxyadenosine (3) was synthesized. 3 binds about 4-fold better than the natural cofactor AdoMet to M.TaqI and is coupled with a short duplex oligodeoxynucleotide by M.TaqI. The identity of the expected modified nucleoside was verified by electrospray ionization mass spectrometry after enzymatic fragmentation of the product duplex. In addition, the new cofactor 3 was used to sequence-specifically label plasmid DNA in a M.TaqI-catalyzed reaction.     

Observation of a single-stranded DNA/pyrrolobenzodiazepine adduct

Pyrrolobenzodiazepine (PBD) antitumor agents have, to date, only been observed to bind to duplex DNA, apparently requiring a minor groove environment for covalent bond formation between their C11-position and the C2-NH(2) functionality of a guanine base. Using an HPLC/MS assay we have now observed and isolated for the first time PBD adducts with single-stranded DNA fragments. Surprisingly, these adducts could only be formed through dissociation of duplex DNA adducts and not by direct interaction of PBDs with single-stranded DNA. They were sufficiently stable for characterization by MALDI-TOF-MS and remained intact after storing at -20 °C for at least 20 days, although the PBD became detached from the DNA within 7 days if stored at room temperature. Furthermore, addition of a complementary strand allowed the duplex adduct to reform. The relative stability of single-stranded PBD/DNA adducts despite a complete loss of minor groove structure was further confirmed by CD spectroscopic analysis. The CD signal induced by the presence of a PBD molecule in the single-stranded adducts remained prominent despite heating for 2 h at 50-60 °C, thus indicating their relatively robust nature.     

Structural studies of an oligodeoxynucleotide containing a trimethylene interstrand cross-link in a 5'-(CpG) motif: model of a malondialdehyde cross-link.

Malondialdehyde (MDA), a known mutagen and suspected carcinogen, is a product of lipid peroxidation and byproduct of eicosanoid biosynthesis. MDA can react with DNA to generate potentially mutagenic adducts on adenine, cytosine, and particularly guanine. In addition, repair-dependent frame shift mutations in a GCGCGC region of Salmonella typhimurium hisD3052 have been attributed to formation of interstrand cross-links (Mukai, F. H. and Goldstein, B. D. Science 1976, 191, 868--869). The cross-linked species is unstable and has never been characterized but has been postulated to be a bis-imino linkage between N(2) positions of guanines. An analogous linkage has now been investigated as a stable surrogate using the self-complementary oligodeoxynucleotide sequence 5'-d(AGGCG*CCT)(2,) in which G* represents guanines linked via a trimethylene chain between N(2) positions. The solution structure, obtained by NMR spectroscopy and molecular dynamics using a simulated annealing protocol, revealed the cross-link only minimally distorts duplex structure in the region of the cross-link. The tether is accommodated by partially unwinding the duplex at the lesion site to produce a bulge and tipping the guanine residues; the two guanines and the tether attain a nearly planar conformation. This distortion did not result in significant bending of the DNA, a result which was confirmed by gel electrophoresis studies of multimers of a 21-mer duplex containing the cross-link.     

Structure of cis-[Pt(NH_3)(2-picoline)]~(2+) and DNA adduct and its bonding characteristics

Several methods including molecular mechanics, molecular dynamics, ONIOM that combines quantum chemistry with molecular mechanics and standard quantum chemistry are used to study the configuration and electron structures of an adduct of the DMA segment d(ATACATG*G*TACATA)-d(TATGTACCATGTAT) with cis-[Pt(NH3)(2-Picoline)]2+. The investigation shows that the configuration optimized by ONIOM is similar to that determined by NMR. Strong chemical bonds between Pt of the complex and two N7s of neighboring guanines in the DNA duplex and hydrogen bond between the NH3of the complex and O6 of a nearby guanine have a large impact on the configuration of the adduct. Chemical bonds, the aforementioned hydrogen bond, and the interaction between a methyl of the complex and a methyl of the base in close proximity are critical for the complex to specifically recognize DNA.     

Induction of a remarkable conformational change in a human telomeric sequence by the binding of naphthyridine dimer: inhibition of the elongation of a telomeric repeat by telomerase

The binding of a dimeric form of the 2-amino-1,8-naphthyridine derivative (naphthyridine dimer) to a human telomeric sequence, TTAGGG, was investigated by UV melting, CD spectra, and CSI-MS measurements. Both the 9-mer d(TTAGGGTTA) and the 15-mer d(TTAGGGTTAGGGTTA) showed apparent melting temperatures (T(m)) of 45.6 and 63.6 degrees C, respectively, in the presence of naphthyridine dimer (30 microM) in sodium cacodylate buffer (50 mM, pH 7.0) containing 100 mM NaCl. The CD spectra at 235 and 255 nm of the 9-mer increased in intensity accompanied with strong induced CDs at 285 and 340 nm upon complex formation with naphthyridine dimer. UV titration of the binding of naphthyridine dimer to the 9-mer at 320 nm showed a hypochromism of the spectra. A Scatchard plot of the data showed the presence of multiple binding sites with different association constants. Cold spray ionization mass spectrometry of the complex between naphthyridine dimer and the 9-mer clearly showed that one to three molecules of the ligand bound to the dimer duplex of the 9-mer. Telomeric repeat elongation assay showed that the binding of naphthyridine dimer to the telomeric sequence inhibits the elongation of the sequence by telomerase.     

Red/NIR G‐Quadruplex Sensing,Harvesting Blue Light by a Coumarin–Naphthalene Diimide Dyad

A conceptually new light‐up nucleic acid fluorescent probe resulting from the conjugation of a coumarin to a naphthalene diimide exhibits a single wavelength emission at 498 nm when free in solution and an additional red/NIR emission when bound to G‐quadruplex DNA. The light‐up response centred at 666 nm is highly specific for quadruplex DNA when compared to duplex DNA or to RNA quadruplexes.     

Sequence specificity of alkali-labile DNA damage photosensitized by suprofen

On irradiation at UVB wavelengths, in aerated neutral aqueous solution, the anti-inflammatory drug suprofen (SP) photosensitizes the production of alkali-labile cleavage sites in DNA much more efficiently than direct strand breaks. It is active at submillimolar concentrations despite having no significant binding affinity for DNA. Gel sequencing studies utilizing 32P-end-labeled oligonucleotides have revealed that piperidine-sensitive lesions are formed predominantly at the positions of guanine (G) bases, with the extent of modification being UV dose- and SP concentration-dependent. Quite distinct patterns of G-specific damage are observed in single-stranded and duplex DNA molecules. The uniform attack at all G residues in single-stranded DNA, which is enhanced in D2O, is compatible with a Type-II mechanism. SP is a known generator of singlet oxygen whose participation in the reaction is supported by the effects of quenchers and scavengers. In duplex DNA, piperidine-induced cleavage occurs with high selectivity at the 5'-G of GG and (less prominently) GA doublets. This behavior is characteristic of a Type-I process involving electron transfer from DNA to photoexcited SP molecules. The ability of SP to sensitize the formation of Type-I and Type-II photo-oxidation products from 2'-deoxyguanosine attests to the feasibility of competing mechanisms in DNA.     

A computational foray into the mechanism and catalysis of the adduct formation reaction of guanine with crotonaldehyde

Crotonaldehyde, a common environmental pollutant and product of endogenous lipid peroxidation, reacts with guanine to form DNA adducts with pronounced genotoxicity and mutagenicity. Here, we explore the molecular mechanism of this adduct formation using double-hybrid density functional theory methods. The reaction can be envisaged to occur in a two-step fashion via an aza-Michael addition leading to an intermediate ring-open adduct followed by a cyclization reaction giving the mutagenic ring-closed adduct. We find that (i) a 1,2-type addition is favored over a 1,4-type addition for the aza-Michael addition, and (ii) an initial tautomerization of the guanine moiety in the resulting ring-open adduct significantly reduces the barrier toward cyclization compared to the direct cyclization of the ring-open adduct in its keto-form. Overall, the aza-Michael addition is found to be rate-determining. We further find that participation of a catalytic water molecule significantly reduces the energy barriers of both the addition and cyclization reaction. © 2018 Wiley Periodicals, Inc.     

Structure of <Emphasis Type="Italic">cis</Emphasis>-[Pt(NH<Subscript>3</Subscript>)(2-picoline)]<Superscript>2+</Superscript> and DNA adduct and its bonding characteristics

Several methods including molecular mechanics, molecular dynamics, ONIOM that combines quantum chemistry with molecular mechanics and standard quantum chemistry are used to study the configuration and electron structures of an adduct of the DNA segment d(ATACATG*G*TACATA)d(TATGTACCATGTAT) with cis-[Pt(NH₃)(2-Picoline)]²⁺. The investigation shows that the configuration optimized by ONIOM is similar to that determined by NMR. Strong chemical bonds between Pt of the complex and two N7s of neighboring guanines in the DNA duplex and hydrogen bond between the NH₃of the complex and O6 of a nearby guanine have a large impact on the configuration of the adduct. Chemical bonds, the aforementioned hydrogen bond, and the interaction between a methyl of the complex and a methyl of the base in close proximity are critical for the complex to specifically recognize DNA.     

Direct chemical evidence for charge transfer between photoexcited 2-aminopurine and guanine in duplex DNA

Photoexcited 2-aminopurine (Ap*) is extensively exploited as a fluorescent base analogue in the study of DNA structure and dynamics. Quenching of Ap* in DNA is often attributed to stacking interactions between Ap* and DNA bases, despite compelling evidence indicating that charge transfer (CT) between Ap* and DNA bases contributes to quenching. Here we present direct chemical evidence that Ap* undergoes CT with guanine residues in duplex DNA, generating oxidative damage at a distance. Irradiation of Ap in DNA containing the modified guanine, cyclopropylguanosine (CPG), initiates hole transfer from Ap* followed by rapid ring opening of the CPG radical cation. Ring opening accelerates hole trapping to a much shorter time regime than for guanine radicals in DNA; consequently, trapping effectively competes with back electron transfer (BET) leading to permanent CT chemistry. Significantly, BET remains competitive, even with this much faster trapping reaction, consistent with measured kinetics of DNA-mediated CT. The distance dependence of BET is sharper than that of forward CT, leading to an inverted dependence of product yield on distance; at short distances product yield is inhibited by BET, while at longer distances trapping dominates, leading to permanent products. The distance dependence of product yield is distinct from forward CT, or charge injection. As with photoinduced charge transfer in other chemical and biological systems, rapid kinetics for charge injection into DNA need not be associated with a high yield of DNA damage products.