BASE-SPECIFIC DAMAGE INDUCED BY 4-THIOURIDINE PHOTOSENSITIZATION WITH 334-nm RADIATION IN M13 PHAGE DNA

Abstract— Site-specific DNA damage caused by 334-nm radiation in the presence of the rare Escherichia coli base 4-thiouridine was investigated in vitro by detecting the sites of the termination of DNA synthesis with irradiated M13 phage DNA used as a template. Single-strand breakage was also examined. The results indicate that 334-nm radiation at very low fluences in the presence of 4-thiouridine induces termination of strand synthesis at thymine base sites and at the base immediately prior to thymine. Termination at these sites was diminished by treatment with hot piperidine. Strand cleavage by piperidine treatments was observed preferentially at the guanine site, but only after irradiation at much larger fluences. It is hypothesized that at low fluences 4-thiouridine forms photoadducts with thymine that block DNA synthesis, while at high fluences the guanine site is damaged via oxygen species.     

Effects of a high-affinity antibody fragment on DNA polymerase reactions near a (6-4) photoproduct site

Pyrimidine (6-4) pyrimidone photodimers are major photoproducts that have mutagenic and carcinogenic consequences. One major reason for these biological effects of (6-4) photoproducts may be base mispairing/DNA replication errors due to hydrogen bonding to bases opposite these damaged sites. We synthesized a modified 41-mer DNA containing a (6-4) photoproduct using a preformed building block, then employed it as a template for primer extension reactions catalyzed by Klenow fragment and DNA polymerases alpha, beta and delta (pol alpha, pol beta and pol delta). None of these DNA polymerases were able to bypass the (6-4) photoproduct and elongation terminated at or near the 3'-pyrimidone of the photoproduct, depending on the dNTP concentration. When a single-chain Fv (scFv) with high affinity for the (6-4) photoproduct was included in the polymerization reaction, DNA synthesis was inhibited at base positions four, six, eight or eight nucleotides prior to the 3'-pyrimidone by Klenow fragment, pol alpha, pol beta or pol delta, respectively. These results suggest that the scFv can bind to the template DNA containing a (6-4) photoproduct and inhibit extension reactions by polymerases.     

A 4-[(3R,4R)-dihydroxypyrrolidino]pyrimidin-2-one nucleobase for a CG base pair in triplex DNA

In order to expand target sequences in triplex DNA formation, the development of a nucleobase that recognizes a CG base pair in dsDNA was attempted. A 4-[(3R,4R)-dihydroxypyrrolidino]pyrimidin-2-one nucleobase was found to recognize a CG base pair with high sequence-selectivity.     

Discovery and synthesis of new UV-induced intrastrand C(4-8)G and G(8-4)C photolesions

UV irradiation of cellular DNA leads to the formation of a number of defined mutagenic DNA lesions. Here we report the discovery of new intrastrand C(4-8)G and G(8-4)C cross-link lesions in which the C(4) amino group of the cytosine base is covalently linked to the C(8) position of an adjacent dG base. The structure of the novel lesions was clarified by HPLC-MS/MS data for UV-irradiated DNA in combination with chemical synthesis and direct comparison of the synthetic material with irradiated DNA. We also report the ability to generate the lesions directly in DNA with the help of a photoactive precursor that was site-specifically incorporated into DNA. This should enable detailed chemical and biochemical investigations of these lesions.     

Synthesis and characterization of DNA duplexes containing an N(4)C-ethyl-N(4)C interstrand cross-link

Short DNA duplexes containing an N(4)C-ethyl-N(4)C interstrand cross-link, C-C, were synthesized on controlled pore glass supports. Duplexes having two, three, or four A/T base pairs on either side of the C-C cross-link and terminating with a C(4) overhang at their 5'-ends were prepared. The cross-link was introduced using a convertible nucleoside approach. Thus, an oligonucleotide terminating at its 5'-end with O(4)-triazoyl-2'-deoxyuridine was first prepared on the support. The triazole group of support-bound oligomer was displaced by the aminoethyl group of 5'-dimethoxytrityl-3'-O-tert-butyldimethylsilyl-N(4)-(2-aminoethyl)deoxycytidine to give the cross-link. The dimethoxytrityl group was removed, and the upper and lower strands of the duplex were extended from two 5'-hydroxyl groups of the cross-link using protected nucleoside 3'-phosphoramidites. The tert-butyldimethylsilyl group of the resulting partial duplex was then removed, and the chain was extended in the 3'-direction from the resulting 3'-hydroxyl of the cross-link using protected nucleoside 5'-phosphoramidites. The cross-linked duplexes were purified by HPLC and characterized by enzymatic digestion and MALDI-TOF mass spectrometry. Duplexes with three or four A/T base pairs on either side of the C-C cross-link gave sigmoidal shaped A(260) profiles when heated, a behavior consistent with cooperative denaturation of the A/T base pairs. Each cross-linked duplex could be ligated to an acceptor duplex using T4 DNA ligase, a result that suggests that the C-C cross-link does not interfere with the ligation reaction, even when it is located only two base pairs from the site of ligation. The ability to synthesize duplexes with a defined interstrand cross-link and to incorporate these duplexes into longer pieces of DNA should enable preparation of substrates that can be used for a variety of biophysical and biochemical experiments, including studies of DNA repair.     

Fluorescence Quenching by Intercalation of a Pyrene Group Tethered to an N4‐modified Cytosine in Duplex DNA

A series of duplex DNA oligomers was prepared that contain a pyrene chromophore linked by a trimethylene chain (‐(CH₂)₃‐) to N⁴ of a cytosine. The pyrene group stabilizes the DNA as evidenced by an increase in melting temperature. The absorption spectrum of the linked pyrene chromophore shows a temperature‐dependent shift and there is also a strong induced circular dichroism spectrum attributed to the pyrene group. The fluorescence of the pyrene chromophore is strongly quenched at room temperature by linkage to the DNA, but it increases above the melting temperature. We attribute these observations to intramolecular intercalation of the pyrene group at a base pair adjacent to its linkage site at cytosine.     

Metal-mediated DNA base pairing of easily prepared 2-oxo-imidazole-4-carboxylate nucleotides

Metal-mediated DNA base pairs, which consist of two ligand-type artificial nucleobases and a bridging metal ion, have attracted increasing attention in recent years as a different base pairing mode from natural base pairing. Metal-mediated base pairing has been extensively studied, not only for metal-dependent thermal stabilisation of duplexes, but also for metal assembly by DNA templates and construction of functional DNAs that can be controlled by metals. Here, we report the metal-mediated base paring properties of a novel 2-oxo-imidazole-4-carboxylate (Im^OC) nucleobase and a previously reported 2-oxo-imidazole-4-carboxamide (Im^OA) nucleobase, both of which can be easily derived from a commercially available uridine analogue. The Im^OC nucleobases were found to form stable Im^OC–Cu^II–Im^OC and Im^OC–Hg^II–Im^OC base pairs in the presence of the corresponding metal ions, leading to an increase in the duplex melting temperature by +20 °C and +11 °C, respectively. The Im^OC bases did not react with other divalent metal ions and showed superior metal selectivity compared to similar nucleobase design reported so far. The Im^OC–Cu^II–Im^OC base pair was much more stable than mismatch pairs with other natural nucleobases, confirming the base pair specificity in the presence of Cu^II. Furthermore, we demonstrated the quantitative assembly of three Cu^II ions inside a DNA duplex with three consecutive Im^OC–Im^OC pairs, showing great potential of DNA-template based Cu^II nanoarray construction. The study of easily-prepared Im^OC base pairs will provide a new design strategy for metal-responsive DNA materials.

A novel 2-oxo-imidazole-4-carboxylate (Im^OC) nucleobase, which can be easily derived from a commercially available uridine analogue, was found to form stable Cu^II- and Hg^II-mediated base pairs in DNA duplexes.     

A new method for the synthesis of oligodeoxyribonucleotides containing 4-N-alkoxycarbonyldeoxycytidine derivatives and their hybridization properties.

Oligodeoxyribonucleotides incorporating 4-N-alkoxycarbonyldeoxycytidine derivatives were synthesized on polystyrene-type ArgoPore resins having a new benzyloxy(diisopropyl)silyl linker, by use of ZnBr(2) as the detritylating agent. The first 3'-terminal thymidine could be attached to the resin by successive in situ reactions of 5'-O-DMTr-thymidine with diisopropylsilanediyl ditriflate and an ArgoPore resin containing hydroxyl groups. The use of this new silanediyl-type linker allowed release of the DNA chain from the resin by treatment with TBAF under neutral conditions. The T(m) experiments apparently showed that incorporation of 4-N-alkoxycarbonyldeoxycytidines into DNA strands resulted in higher hybridization affinity with the complementary DNA strands than that of 4-N-acyldeoxycytidines. In addition, comparable T(m) studies using oligodeoxyribonucleotides incorporating acyl (RC(O)-) groups and alkoxyacyl (RO(CH(2))(n)C(O)-) groups having the same chain length show that the latter tend to exhibit higher T(m) values than the former. It turned out that 4-N-alkoxycarbonyldeoxycytidines can form base pairs not only with deoxyguanosine but also with deoxyadenosine. Based on the ab initio calculations of the hydrogen bond energies of the possible base pairs formed between 4-N-methoxycarbonyl-1-methylcytosine and 9-methyladenine and the NMR analysis of the base-pairs of (15)N-labeled 4-N-alkoxycarbonyldeoxycytidines with deoxyadenosine derivatives, we conclude that the base pair involves two unique hydrogen bonds between the cytosyl 4-NH group and the adenyl N(1) atom and between the O atom of the ester group and the adenyl 6-NH group.     

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.     

Nick sealing by T4 DNA ligase on a modified DNA template: tethering a functional molecule on D-threoninol

Efficient DNA nick sealing catalyzed by T4 DNA ligase was carried out on a modified DNA template in which an intercalator such as azobenzene had been introduced. The intercalator was attached to a D-threoninol linker inserted into the DNA backbone. Although the structure of the template at the point of ligation was completely different from that of native DNA, two ODNs could be connected with yields higher than 90% in most cases. A systematic study of sequence dependence demonstrated that the ligation efficiency varied greatly with the base pairs adjacent to the azobenzene moiety. Interestingly, when the introduced azobenzene was photoisomerized to the cis form on subjection to UV light (320-380 nm), the rates of ligation were greatly accelerated for all sequences investigated. These unexpected ligations might provide a new approach for the introduction of functional molecules into long DNA strands in cases in which direct PCR cannot be used because of blockage of DNA synthesis by the introduced functional molecule. The biological significance of this unexpected enzymatic action is also discussed on the basis of kinetic analysis.     

Synthesis and properties of a new fluorescent bicyclic 4-N-carbamoyldeoxycytidine derivative

[reaction: see text] A bicyclic 4-N-carbamoyldeoxycytidine derivative (1, dC(hpp)) geometrically locked was synthesized as a new fluorescent nucleobase. The hybridization properties of oligodeoxynucleotides containing dC(hpp) were investigated by use of T(m) analysis. It was found that dC(hpp) forms stable base pairs not only with the complementary guanine base, but also with the adenine base. Interestingly, the fluorescence of dC(hpp) was suppressed only when a dC(hpp)-dG base pair was formed.     

Laser desorption of DNA oligomers larger than one kilobase from cooled 4-nitrophenol

A unique matrix system consisting mostly of 4-nitrophenol has shown to be very effective for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of large DNA oligomers when a cooled sample stage was used to prevent the sublimation of this matrix under vacuum. Using this 4-nitrophenol matrix with UV laser desorption, detection of picomole quantities of DNA oligomers containing up to approximately 800 nucleotides was routinely achieved. The effectiveness of this matrix was further demonstrated by the observation of a double-stranded DNA oligomer larger than 1000 base pairs, seen as a denatured single-stranded species, with a molecular ion mass exceeding 300 000 Da. The potential applications of 4-nitrophenol as a matrix for DNA sizing are discussed.     

Phenothiazine as a redox-active DNA base substitute: comparison with phenothiazine-modified uridine

Phenothiazine can be incorporated as a redox-active probe into DNA in two conceptually different ways: the non-nucleosidic DNA base surrogate exhibits similar properties to 10-methylphenothiazine but with no preferential base-pairing properties, whereas the phenothiazine-modified uridine has different optical and electrochemical properties, but exhibits preferred Watson-Crick base pairing with adenine.     

UV INDUCED (6-4) PHOTOPRODUCTS ARE DISTRIBUTED DIFFERENTLY THAN CYCLOBUTANE DIMERS IN NUCLEOSOMES

We have compared the distributions of two stable UV photoproducts in nucleosome core DNA at the single-nucleotide level using a T4 polymerase-exonuclease mapping procedure. The distribution of pyrimidine-pyrimidone (6-4) dimers was uncovered by reversing the major UV photo-product, cis-syn cyclobutane pyrimidine dimer, with E. coli DNA photolyase and photoreactivating light. Whereas the distribution of total UV photoproducts in nucleosome core DNA forms a striking 10.3 base periodic pattern, the distribution of (6-4) dimers is much more random throughout the nucleosome core domain. Therefore, histone-DNA interactions in nucleosomes strongly modulate formation of the major class of UV-induced photoproducts, while having either a constant effect or no effect on (6-4) dimer formation.     

DNA mediated charge transport: characterization of a DNA radical localized at an artificial nucleic acid base

DNA assemblies containing 4-methylindole incorporated as an artificial base provide a chemically well-defined system in which to explore the oxidative charge transport process in DNA. Using this artificial base, we have combined transient absorption and EPR spectroscopies as well as biochemical methods to test experimentally current mechanisms for DNA charge transport. The 4-methylindole radical cation intermediate has been identified using both EPR and transient absorption spectroscopies in oxidative flash-quench studies using a dipyridophenazine complex of ruthenium as the intercalating oxidant. The 4-methylindole radical cation intermediate is particularly amenable to study given its strong absorptivity at 600 nm and EPR signal measured at 77 K with g = 2.0065. Both transient absorption and EPR spectroscopies show that the 4-methylindole is well incorporated in the duplex; the data also indicate no evidence of guanine radicals, given the low oxidation potential of 4-methylindole relative to the nucleic acid bases. Biochemical studies further support the irreversible oxidation of the indole moiety and allow the determination of yields of irreversible product formation. The construction of these assemblies containing 4-methylindole as an artificial base is also applied in examining long-range charge transport mediated by the DNA base pair stack as a function of intervening distance and sequence. The rate of formation of the indole radical cation is >/=10(7) s(-)(1) for different assemblies with the ruthenium positioned 17-37 A away from the methylindole and with intervening A-T base pairs primarily composing the bridge. In these assemblies, methylindole radical formation at a distance is essentially coincident with quenching of the ruthenium excited state to form the Ru(III) oxidant; charge transport is not rate limiting over this distance regime. The measurements here of rates of radical cation formation establish that a model of G-hopping and AT-tunneling is not sufficient to account for DNA charge transport. Instead, these data are viewed mechanistically as charge transport through the DNA duplex primarily through hopping among well stacked domains of the helix defined by DNA sequence and dynamics.     

Synthesis of two mirror image 4-helix junctions derived from glycerol nucleic acid

Structural DNA nanotechnology relies on Watson-Crick base pairing rules to assemble DNA motifs into diverse arrangements of geometric shapes and patterns. While substantial effort has been devoted to expanding the programmability of natural DNA, considerably less attention has been given to the development of nucleic acid structures based on non-natural DNA polymers. Here we describe the use of glycerol nucleic acid (GNA), a simple polymer based on an acyclic repeating unit, as an alternative genetic material for assembling nucleic acid nanostructures independent of RNA or DNA recognition. We synthesized two 4-helix junctions based entirely on GNA self-pairing and showed that GNA provides easy access to highly stable nanostructures with left- and right-handed helical configurations.     

Synthesis and DNA duplex recognition of a triplex-forming oligonucleotide with an ureido-substituted 4-phenylimidazole nucleoside

A 4-(3-n-butylureidophenyl)imidazole nucleoside was successfully incorporated into a triplex-forming oligonucleotide (TFO). Binding affinity and base pair selectivity of the TFO containing this non-natural nucleoside were studied with various duplex targets containing all four possible Watson-Crick base pairs opposite the nucleoside analog in the third strand. Triplex thermal stabilities indicate that the synthetic nucleoside acts as a universal base in binding to all four possible Watson-Crick base pairs with moderate affinity but poor selectivity. Based on an analysis of its binding thermodynamics, this can be rationalized by the absence of strong specific interactions and more favorable entropic contributions upon triplex formation.     

d(C4)相关序列形成四分子i-Motif结构的电喷雾质谱研究

设计了与富含胞嘧啶(C)的DNA序列d(C4)相关的DNA序列d(C4), d(TC4), d(AC4), d(T2C4), d(A2C4), d(C4T), d(C4A)和d(TC4T); 采用电喷雾质谱测定发现这些序列形成四分子非共价复合物离子, 根据离子的相对丰度可确定形成四链i-motif结构的数量和可能性; 同时考察了腺嘌呤(A)和胸腺嘧啶(T)在d(C4)序列的5'和3'端对其形成四分子i-motif结构的影响. 结果表明, 在d(C4)的5'端增加A碱基或T碱基更易形成四分子复合物; 5'端含T碱基比含A碱基更利于形成四分子复合物; 而在d(C4)序列中增加2个A碱基或T碱基比增加相应的单个碱基形成了更高丰度的四分子离子峰.     

AlkB recognition of a bulky DNA base adduct stabilized by chemical cross-linking

E.coli AlkB is a direct DNA/RNA repair protein that oxidatively reverses N1 alkylated purines and N3 alkylated pyrimidines to regular bases.Previous crystal structures have revealed N1-methyl adenine(1-meA) recognition by AlkB and a unique base flipping mechanism,but how the AlkB active site can accommodate bulky base adducts is largely unknown.Employing a previously developed chemical cross-linking technique,we crystallized AlkB with a duplex DNA containing a caged thymine base(cagedT).The structure reveal...     

Solution structure of xDNA: a paired genetic helix with increased diameter

We describe the structure in aqueous solution of an extended-size DNA-like duplex with base pairs that are approximately 2.4 A longer than those of DNA. Deoxy-lin-benzoadenosine (dxA) was employed as a dA analogue to form hydrogen-bonded base pairs with dT. The 10mer self-complementary extended oligodeoxynucleotide 5'-d(xATxAxATxATTxAT) forms a much more thermodynamically stable duplex than the corresponding DNA sequence, 5'-d(ATAATATTAT). NMR studies show that this extended DNA (xDNA) retains many features of natural B-form DNA, but with a few structural alterations due to its increased helical diameter. The results give insight into the structural plasticity of the natural DNA backbone and lend insight into the evolutionary origins of the natural base pairs. Finally, this structural study confirms the hypothesis that extended nucleobase analogues can form stable DNA-like structures, suggesting that alternative genetic systems might be viable for storage and transfer of genetic information.