Oligonucleotide probes containing pyrimidine analogs reveal diminished hydrogen bonding capacity of the DNA adduct O6-methyl-G in DNA duplexes

Oligonucleotide hybridization probes containing nucleoside analogs offer a potential strategy for binding specific DNA sequences that bear pro-mutagenic O⁶-G alkylation adducts. To optimize O⁶-Me-G-targeting probes, an understanding of how base pairs with O⁶-Me-G are stabilized is needed. In this study, we compared the ability of O⁶-Me-G and G to hydrogen bond with three pyrimidine-like nucleobases (Z, 4-thio-U, and 3-deaza-C) bearing varied hydrogen bond donor and acceptor groups. We found that duplexes containing the pyrimidine analog nucleoside:G pairs were more thermodynamically stable than those containing pyrimidine analog nucleoside:O⁶-alkyl-G pairs. Thus, hydrogen bonding alone was not sufficient to impart selectivity to probes that target O⁶-G alkylation adducts in DNA.     

Cytosine Methylation Effects on the Repair of O6-Methylguanines within CG Dinucleotides

O⁶-Alkyldeoxyguanine adducts induced by tobacco-specific nitrosamines are repaired by O⁶-alkylguanine DNA alkyltransferase (AGT), which transfers the O⁶-alkyl group from the damaged base to a cysteine residue within the protein. In the present study, a mass spectrometry-based approach was used to analyze the effects of cytosine methylation on the kinetics of AGT repair of O⁶-methyldeoxyguanosine (O⁶-Me-dG) adducts placed within frequently mutated 5′-CG-3′ dinucleotides of the p53 tumor suppressor gene. O⁶-Me-dG-containing DNA duplexes were incubated with human recombinant AGT protein, followed by rapid quenching, acid hydrolysis, and isotope dilution high pressure liquid chromatography-electrospray ionization tandem mass spectrometry analysis of unrepaired O⁶-methylguanine. Second-order rate constants were calculated in the absence or presence of the C-5 methyl group at neighboring cytosine residues. We found that the kinetics of AGT-mediated repair of O⁶-Me-dG were affected by neighboring 5-methylcytosine (^MeC) in a sequence-dependent manner. AGT repair of O⁶-Me-dG adducts placed within 5′-CG-3′ dinucleotides of p53 codons 245 and 248 was hindered when ^MeC was present in both DNA strands. In contrast, cytosine methylation within p53 codon 158 slightly increased the rate of O⁶-Me-dG repair by AGT. The effects of ^MeC located immediately 5′ and in the base paired position to O⁶-Me-dG were not additive as revealed by experiments with hypomethylated sequences. Furthermore, differences in dealkylation rates did not correlate with AGT protein affinity for cytosine-methylated and unmethylated DNA duplexes or with the rates of AGT-mediated nucleotide flipping, suggesting that ^MeC influences other kinetic steps involved in repair, e.g. the rate of alkyl transfer from DNA to AGT.Metabolic activation of the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)³ produces methyl- and pyridyloxobutyldiazonium ions that can react with DNA to give O⁶-methyldeoxyguanosine (O⁶-Me-dG) and O⁶- pyridyloxobutyl deoxyguanosine (O⁶-POB-dG) lesions (1). Both adducts are strongly mutagenic because DNA polymerases preferentially misinsert thymine opposite O⁶-alkylguanines, resulting in G → A transitions (2). Studies in laboratory animals have provided evidence for a direct involvement of O⁶-Me-dG in NNK-mediated carcinogenesis (3).A specialized repair protein, O⁶-alkylguanine DNA alkyltransferase (AGT), removes the alkyl group from the O⁶ position of modified guanine bases, such as O⁶-Me-dG and O⁶-POB-dG, restoring normal guanine bases and preventing mutagenesis. AGT preferentially binds double-stranded DNA through a helix-turn-helix motif (4). In the resulting AGT-DNA complex, one recognition helix of the protein is found within the minor groove of the DNA, whereas the other one interacts with the phosphodiester backbone (4). The adducted nucleotide is flipped into a binding pocket within the protein, whereas Arg-128 takes its place in the double helix (4). A hydrogen bonding network around the active site involving His-148, Glu-172, and a water molecule promotes the deprotonation of the active site cysteine (Cys-145) (4, 5). The resulting thiolate anion acts as a nucleophile, displacing the O⁶ substituent of O⁶-alk-G and regenerating normal guanine (Fig. 1) (4, 5). The alkylated protein is inactive and is rapidly degraded by the ubiquitin proteolytic pathway (6–8).Open in a separate windowFIGURE 1.Direct repair of O⁶-alkyl-guanine adducts by AGT.AGT-mediated repair of O⁶-Me-dG lesions includes multiple kinetic steps (9). First, the AGT protein must bind adducted DNA in a reactive conformation. The alkylated nucleotide is flipped out of the DNA base stack to enter the hydrophobic pocket within AGT, and the methyl group is transferred from DNA to the protein. Finally, alkylated AGT protein dissociates from the repaired DNA. Zang et al. (9) reported that the chemical step of alkyl transfer is rate-limiting in the case of O⁶-Me-dG, but not O⁶-benzyl-dG. Furthermore, previous studies have shown that the repair of O⁶-Me-dG by mammalian AGT is influenced by the nature of the O⁶-alkyl group, the length of oligonucleotide duplex, the placement of the adduct, and the identities of neighboring nucleotides (10–14).Removing the alkyl group from O⁶-Me-dG by AGT regenerates normal guanine and protects the genome from G → A transition mutations. For example, Wolf et al. (15) examined the relationship between the inactivation of the AGT gene by promoter hypermethylation and the mutational spectrum of the p53 tumor suppressor gene in non-small cell lung cancer. These authors found that only 8% of lung tumors had G → A transition mutations in the p53 gene when the promoter region of the gene coding for AGT was not methylated, thereby allowing protein expression (15). In contrast, 33% of tumors with a methylated AGT promoter had G → A mutations within the p53 gene (15). The p53 gene is mutated in over 50% of non-small cell lung cancer tumors (16).All CpG sites within the coding sequence of the p53 gene are endogenously methylated (17). Importantly, the same sites are among the major p53 mutational “hotspots” in smoking-induced lung cancer, e.g. codons 157, 158, 245, 248, 249, and 273 (18). Of all p53 mutations, G → A transitions account for 18–24% of genetic changes observed in lung cancer, including 35% of mutations at the CG dinucleotides (15, 19). Given the established role of NNK-induced O⁶-alkylguanine lesions in tobacco carcinogenesis and mutagenesis (20), they are likely to be involved in the induction of smoking-associated G → A transitions in the p53 gene.The presence of ^MeC residues may hinder the repair of O⁶-Me-dG lesions within endogenously methylated CG dinucleotides (14). For example, Bentivenga and Bresnick (14) showed that the repair of O⁶-Me-dG by recombinant AGT in the context of codon 248 of the p53 gene was reduced by 75% when ^MeC was placed immediately 5′ to the O⁶-Me-dG lesion. However, the effects of cytosine methylation on AGT repair of O⁶-Me-dG in other sequence contexts have not been previously investigated, and it is not known which individual steps in the removal of O⁶-methyl group are affected by neighboring ^MeC.Cytosine methylation leads to small structural changes of DNA duplex, including an increase in the base pair rise, roll, and local curvature angles, narrowing of the DNA minor groove, and decreased depth of the major groove (21–23). These structural alterations may influence the affinity of the AGT protein for alkylated DNA. Furthermore, ^MeC enhances base stacking (24) and stabilizes the DNA duplex by increasing the molecular polarizability of the cytosine base (25), which can have an effect on the rate of AGT-mediated nucleotide flipping. The alkyl transfer step itself may be mediated by the presence of ^MeC through its effects on transition state geometry.The goal of the present study was to systematically examine the effects of cytosine methylation on AGT-mediated repair of O⁶-Me-dG lesions placed within 5′-CG-3′ dinucleotides representing major p53 mutational hotspots observed in lung cancer. The kinetics of alkyl transfer were analyzed using rapid-quench methods coupled with quantitative analyses of O⁶-Me-dG by isotope dilution-high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) (26). Furthermore, we examined the effects of cytosine methylation on AGT binding to O⁶-Me-dG-containing DNA and its influence on the rate of O⁶-Me-dG nucleotide flipping in the presence of AGT protein.     

Repair of O6-methylguanine adducts in human telomeric G-quadruplex DNA by O6-alkylguanine-DNA alkyltransferase

O⁶-alkylguanine-DNA alkyltransferase (AGT) is a single-cycle DNA repair enzyme that removes pro-mutagenic O⁶-alkylguanine adducts from DNA. Its functions with short single-stranded and duplex substrates have been characterized, but its ability to act on other DNA structures remains poorly understood. Here, we examine the functions of this enzyme on O⁶-methylguanine (6mG) adducts in the four-stranded structure of the human telomeric G-quadruplex. On a folded 22-nt G-quadruplex substrate, binding saturated at 2 AGT:DNA, significantly less than the ∼5 AGT:DNA found with linear single-stranded DNAs of similar length, and less than the value found with the telomere sequence under conditions that inhibit quadruplex formation (4 AGT:DNA). Despite these differences, AGT repaired 6mG adducts located within folded G-quadruplexes, at rates that were comparable to those found for a duplex DNA substrate under analogous conditions. Repair was kinetically biphasic with the amplitudes of rapid and slow phases dependent on the position of the adduct within the G-quadruplex: in general, adducts located in the top or bottom tetrads of a quadruplex stack exhibited more rapid-phase repair than did adducts located in the inner tetrad. This distinction may reflect differences in the conformational dynamics of 6mG residues in G-quadruplex DNAs.     

Effect O6‐guanine alkylation on DNA flexibility studied by comparative molecular dynamics simulations

Alkylation of guanine at the O6 atom is a highly mutagenic DNA lesion because it alters the coding specificity of the base causing G:C to A:T transversion mutations. Specific DNA repair enzymes, e.g. O⁶‐alkylguanin‐DNA‐Transferases (AGT), recognize and repair such damage after looping out the damaged base to transfer it into the enzyme active site. The exact mechanism how the repair enzyme identifies a damaged site within a large surplus of undamaged DNA is not fully understood. The O⁶‐alkylation of guanine may change the deformability of DNA which may facilitate the initial binding of a repair enzyme at the damaged site. In order to characterize the effect of O⁶‐methyl‐guanine (O⁶‐MeG) containing base pairs on the DNA deformability extensive comparative molecular dynamics (MD) simulations on duplex DNA with central G:C, O⁶‐MeG:C or O⁶‐MeG:T base pairs were performed. The simulations indicate significant differences in the helical deformability due to the presence of O⁶‐MeG compared to regular undamaged DNA. This includes enhanced base pair opening, shear and stagger motions and alterations in the backbone fine structure caused in part by transient rupture of the base pairing at the damaged site and transient insertion of water molecules. It is likely that the increased opening motions of O⁶‐MeG:C or O⁶‐MeG:T base pairs play a decisive role for the induced fit recognition or for the looping out of the damaged base by repair enzymes. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 23–32, 2015.     

Kinetic analysis of steps in the repair of damaged DNA by human O6-alkylguanine-DNA alkyltransferase

Rates of individual steps in the removal of alkyl groups from O6-methyl (Me) and -benzyl (Bz) guanine in oligonucleotides by human O6-alkylguanine DNA alkyltransferase (AGT) were estimated using rapid reaction kinetic methods. The overall reaction yields hyperbolic plots of rate versus AGT concentration for O6-MeG but linear plots for the O6-BzG reaction, which is approximately 100-fold faster. The binding of AGT and DNA (double-stranded 30-mer/36-mer complex) appears to be diffusion-limited. The rate of dissociation of the complex is approximately 25-fold slower (approximately 1 s(-1)) for DNA containing O6-MeG or O6-BzG than unmodified DNA. The fluorescent dC-analog 6-methylpyrrolo[2,3-d]pyrimidine-2(3H) one deoxyribonucleoside (pyrrolo dC), which pairs with G, was positioned opposite G, O6-MeG, or O6-BzG and used as a probe of the rate of base flipping. A rapid increase of fluorescence (k approximately 200 s(-1)) was observed with O6-MeG and O6-BzG and AGT but not with a Gly mutation at Arg128, which has been implicated in base flipping with crystal structures. Only weak and slower fluorescence changes were observed with G:pyrrolo dC or T:2-aminopurine pairs. These rate estimates were used in a kinetic model in which AGT binds and scans DNA rapidly, flips O6-alkylG residues, transfers the alkyl group in a chemical step that is rate-limiting in the case of O6-MeG but not O6-BzG, and releases the dealkylated DNA. The results explain the overall patterns of rates of alkyl group removal versus AGT concentration and the effects of the mutations, as well as the greater affinity of AGT for DNA with O6-alkylG lesions.     

Repair of oligodeoxyribonucleotides by O(6)-alkylguanine-DNA alkyltransferase

Activity of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) is an important source of tumor cell resistance to alkylating agents. AGT inhibitors may prove useful in enhancing chemotherapy. AGT is inactivated by reacting stoichiometrically with O(6)-benzylguanine (b(6)G), which is currently in clinical trials for this purpose. Short oligodeoxyribonucleotides containing a central b(6)G are more potent inactivators of AGT than b(6)G. We examined whether human AGT could react with oligodeoxyribonucleotides containing multiple b(6)G residues. The single-stranded 7-mer 5'-d[T(b(6)G)(5)G]-3' was an excellent AGT substrate with all five b(6)G adducts repaired although one adduct was repaired much more slowly. The highly b(6)G-resistant Y158H and P140K AGT mutants were also inactivated by 5'-d[T(b(6)G)(5)G]-3'. Studies with 7-mers containing a single b(6)G adduct showed that 5'-d[TGGGG(b(6)G)G]-3' was more poorly repaired by wild-type AGT than 5'-d[T(b(6)G)GGGGG]-3' and 5'-d[TGG(b(6)G)GGG]-3' and was even less repairable by mutants Y158H and P140K. This positional effect was unaffected by interchanging the terminal 5'- or 3'-nucleotides and was also observed with single-stranded 16-mer oligodeoxyribonucleotides containing O(6)-methylguanine, where a minimum of four nucleotides 3' to the lesion was required for the most efficient repair. Annealing with the reverse complementary strands to produce double-stranded substrates increased the ability of AGT to repair adducts at all positions except at positions 2 and 15. Our results suggest that AGT recognizes the polarity of single-stranded DNA, with the best substrates having an adduct adjacent to the 5'-terminal residue. These findings will aid in designing novel AGT inhibitors that incorporate O(6)-alkylguanine adducts in oligodeoxyribonucleotide contexts.     

Stable isotope labeling-mass spectrometry analysis of methyl- and pyridyloxobutyl-guanine adducts of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in p53-derived DNA sequences

The p53 tumor suppressor gene is a primary target in smoking-induced lung cancer. Interestingly, p53 mutations observed in lung tumors of smokers are concentrated at guanine bases within endogenously methylated (Me)CG dinucleotides, e.g., codons 157, 158, 245, 248, and 273 ((Me)C = 5-methylcytosine). One possible mechanism for the increased mutagenesis at these sites involves targeted binding of metabolically activated tobacco carcinogens to (Me)CG sequences. In the present work, a stable isotope labeling HPLC-ESI(+)-MS/MS approach was employed to analyze the formation of guanine lesions induced by the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) within DNA duplexes representing p53 mutational "hot spots" and surrounding sequences. Synthetic DNA duplexes containing p53 codons 153-159, 243-250, and 269-275 were prepared, where (Me)C was incorporated at all physiologically methylated CG sites. In each duplex, one of the guanine bases was replaced with [1,7,NH(2)-(15)N(3)-2-(13)C]-guanine, which served as an isotope "tag" to enable specific quantification of guanine lesions originating from that position. After incubation with NNK diazohydroxides, HPLC-ESI(+)-MS/MS analysis was used to determine the yields of NNK adducts at the isotopically labeled guanine and at unlabeled guanine bases elsewhere in the sequence. We found that N7-methyl-2'-deoxyguanosine and N7-[4-oxo-4-(3-pyridyl)but-1-yl]guanine lesions were overproduced at the 3'-guanine bases within polypurine runs, while the formation of O(6)-methyl-2'-deoxyguanosine and O(6)-[4-oxo-4-(3-pyridyl)but-1-yl]-2'-deoxyguanosine adducts was specifically preferred at the 3'-guanine base of 5'-GG and 5'-GGG sequences. In contrast, the presence of 5'-neighboring (Me)C inhibited O(6)-guanine adduct formation. These results indicate that the N7- and O(6)-guanine adducts of NNK are not overproduced at the endogenously methylated CG dinucleotides within the p53 tumor suppressor gene, suggesting that factors other than NNK adduct formation are responsible for mutagenesis at these sites.     

Mutagenicity associated with O6-methylguanine-DNA damage and mechanism of nucleotide flipping by AGT during repair

Methylated guanine damage at O6 position (i.e. O6MG) is dangerous due to its mutagenic and carcinogenic character that often gives rise to G:C-A:T mutation. However, the reason for this mutagenicity is not known precisely and has been a matter of controversy. Further, although it is known that O6-alkylguanine-DNA alkyltransferase (AGT) repairs O6MG paired with cytosine in DNA, the complete mechanism of target recognition and repair is not known completely. All these aspects of DNA damage and repair have been addressed here by employing high level density functional theory in gas phase and aqueous medium. It is found that the actual cause of O6MG mediated mutation may arise due to the fact that DNA polymerases incorporate thymine opposite to O6MG, misreading the resulting O6MG:T complex as an A:T base pair due to their analogous binding energies and structural alignments. It is further revealed that AGT mediated nucleotide flipping occurs in two successive steps. The intercalation of the finger residue Arg128 into the DNA double helix and its interaction with the O6MG:C base pair followed by rotation of the O6MG nucleotide are found to be crucial for the damage recognition and nucleotide flipping.     

Inhibition of repair of O-methyldeoxyguanosine and enhanced mutagenesis in rat-liver epithelial cells

The pro-mutagenicity of chemically-induced methylation of DNA at the O⁶ position of dexoyguanosine was studied in cultured adult rat liver epithelial cells. To modify the level of O⁶-methyldeoxyguanosine (O⁶-medGuo) resulting from exposure to an alkylating agent, partial depletion of the O⁶-alkylguanine-DNA alkyltransferase (AGT) repair system was produced by pretreatment of ARL 18 cells with a non-toxic dose of exogenous O⁶-methylguanine (O⁶-meG). Exposure of cells to 0.6 mM O⁶-meG for 4 h depleted AGT activity by about 40%. Intact and pretreated cells were exposed to a range of doses of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), and mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus was quantified by measurement of 6-thioguanine-resistant mutants. The mutagenicity of MNNG was dose dependent and was greater in O⁶-meG pretreated cultures than in intact cultures. Immunoslot blot measurement of O⁶-medGuo employing a mouse monoclonal antibody demonstrated that MNNG produced O[su6-medGuo and that the intact liver cells were efficient in eliminating this lesion from their DNA. Since depletion of AGT would be expected to affect the rate of elimination of only O⁶-medGuo, it is concluded that this lesion is highly pro-mutagenic.     

6-Carboxyfluorescein and structurally similar molecules inhibit DNA binding and repair by O⁶-alkylguanine DNA alkyltransferase

Human O⁶-alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O⁶-alkylguanine and O⁴-alkylthymine adducts in single-stranded and duplex DNAs. These activities protect normal cells and tumor cells against drugs that alkylate DNA; drugs that inactivate AGT are under test as chemotherapeutic enhancers. In studies using 6-carboxyfluorescein (FAM)-labeled DNAs, AGT reduced the fluorescence intensity by ∼40% at binding saturation, whether the FAM was located at the 5′ or the 3′ end of the DNA. AGT protected residual fluorescence from quenching, indicating a solute-inaccessible binding site for FAM. Sedimentation equilibrium analyses showed that saturating AGT-stoichiometries were higher with FAM-labeled DNAs than with unlabeled DNAs, suggesting that the FAM provides a protein binding site that is not present in unlabeled DNAs. Additional fluorescence and sedimentation measurements showed that AGT forms a 1:1 complex with free FAM. Active site benzylation experiments and docking calculations support models in which the primary binding site is located in or near the active site of the enzyme. Electrophoretic analyses show that FAM inhibits DNA binding (IC₅₀ ∼ 76 μM) and repair of DNA containing an O⁶-methylguanine residue (IC₅₀ ∼ 63 μM). Similar results were obtained with other polycyclic aromatic compounds. These observations demonstrate the existence of a new class of non-covalent AGT-inhibitors. After optimization for binding-affinity, members of this class might be useful in cancer chemotherapy.     

A role for mismatch repair in production of chromosome aberrations by methylating agents in human cells

We have shown previously that certain alkylation products, or alkylation derived lesions, which induce chromosome aberrations (abs) persist for at least two cell cycles in Chinese hamster ovary cells. The increase in abs in the second cycle after treatment contrasts with the classical observation of reduction in ab yield with successive mitoses following ionizing radiation. Here we present evidence that processing of lesions by mismatch repair is a mechanism for ab induction by methylating agents.Our previous studies implicated O⁶-methylguanine (O⁶MeG) as an important lesion in induction of abs, particularly in the second cell cycle after treatment. In the absence of repair of O⁶MeG by alkylguanine DNA alkyltransferase (AGT), new abs were induced in the second cycle after treatment with e.g. methylnitronitrosoguanidine (MNNG) and methylnitrosourea (MNU). Thus, we hypothesized that abs were produced not by O⁶MeG or its repair in the first S phase, but by subsequent processing of the lesions. We suggested that after replication proceeded past the O⁶MeG lesion in the first S phase, inserting an incorrect base on the newly synthesized strand, recognition and repair by mismatch repair in the second S phase led to a chromosome ab. Here we used MT1 cells, a human lymphoblastoid cell line that has a defect in strand-specific mismatch repair. MT1 cells are alkylation tolerant and have a mutator phenotype, compared with their parent line, TK6; both MT1 and TK6 cells lack AGT so do not remove the methyl group from O⁶MeG. While the initial levels of abs at the first metaphase were similar in MT1 and TK6 cells, ab levels in MT1 cells were greatly reduced in the second and third cell cycles following treatment with MNNG, dimethylnitrosamine and MNU, in contrast with the parent TK6 cells, which had more abs in the second cell cycle than in the first. This supports the hypothesis that repair of mismatched base pairs involving O⁶MeG is one mechanism for induction of chromosome abs. In contrast to the difference in response to methylating agents between TK6 cells and mismatch repair-deficient MT1 cells, the profile of ab induction by an ethylating agent, ethylnitronitrosourea, was similar in MT1 cells to those for TK6 cells and CHO cells.     

Synthesis and antitumor activity evaluation of a novel combi-nitrosourea prodrug: Designed to release a DNA cross-linking agent and an inhibitor of O6-alkylguanine-DNA alkyltransferase

The drug resistance of CENUs induced by O⁶-alkylguanine-DNA alkyltransferase (AGT), which repairs the O⁶-alkylated guanine and subsequently inhibits the formation of dG–dC cross-links, hinders the application of CENU chemotherapies. Therefore, the discovery of CENU analogs with AGT inhibiting activity is a promising approach leading to novel CENU chemotherapies with high therapeutic index. In this study, a new combi-nitrosourea prodrug 3-(3-(((2-amino-9H-purin-6-yl)oxy)methyl)benzyl)-1-(2-chloroethyl)-1-nitrosourea (6), designed to release a DNA cross-linking agent and an inhibitor of AGT, was synthesized and evaluated for its antitumor activity and ability to induce DNA interstrand cross-links (ICLs). The results indicated that 6 exhibited higher cytotoxicity against mer⁺ glioma cells compared with ACNU, BCNU, and their respective combinations with O⁶-benzylguanine (O⁶-BG). Quantifications of dG–dC cross-links induced by 6 were performed using HPLC–ESI-MS/MS. Higher levels of dG–dC cross-link were observed in 6-treated human glioma SF763 cells (mer⁺), whereas lower levels of dG–dC cross-link were observed in 6-treated calf thymus DNA, when compared with the groups treated with BCNU and ACNU. The results suggested that the superiority of 6 might result from the AGT inhibitory moiety, which specifically functions in cells with AGT activity. Molecular docking studies indicated that five hydrogen bonds were formed between the O⁶-BG analogs released from 6 and the five residues in the active pocket of AGT, which provided a reasonable explanation for the higher AGT-inhibitory activity of 6 than O⁶-BG.     

Covalent capture of a human O(6)-alkylguanine alkyltransferase-DNA complex using N(1),O(6)-ethanoxanthosine, a mechanism-based crosslinker

The DNA repair protein O⁶-alkylguanine alkyltransferase (AGT) is responsible for removing promutagenic alkyl lesions from exocyclic oxygens located in the major groove of DNA, i.e. the O⁶ and O⁴ positions of guanine and thymine. The protein carries out this repair reaction by transferring the alkyl group to an active site cysteine and in doing so directly repairs the premutagenic lesion in a reaction that inactivates the protein. In order to trap a covalent AGT–DNA complex, oligodeoxyribonucleotides containing the novel nucleoside N¹,O⁶-ethanoxanthosine (^eX) have been prepared. The ^eX nucleoside was prepared by deamination of 3′,5′-protected O⁶-hydroxyethyl-2′-deoxyguanosine followed by cyclization to produce 3′,5′-protected N¹,O⁶-ethano-2′-deoxyxanthosine, which was converted to the nucleoside phosphoramidite and used in the preparation of oligodeoxyribonucleotides. Incubation of human AGT with a DNA duplex containing ^eX resulted in the formation of a covalent protein–DNA complex. Formation of this complex was dependent on both active human AGT and ^eX and could be prevented by chemical inactivation of the AGT with O⁶-benzylguanine. The crosslinking of AGT to DNA using ^eX occurs with high yield and the resulting complex appears to be well suited for further biochemical and biophysical characterization.     

Specifically alkylated DNA fragments. Synthesis and physical characterization of d[CGC(O6Me)GCG] and d[CGT(O6Me)GCG]

Two hexamer DNA fragments containing a carcinogenic modified base, O⁶-methyl guanine, have been synthesized by a solid-phase phosphotriester method, in which the unmodified guanine residues present were O⁶ protected with the 4-nitrophenylethyl group. These two alkylated oligonucleotides were found to have similar T_m's about 40° lower than the unmodified parent compund, d(CG)₃. Moreover, the presence of the (O⁶Me)G appears to inhibit the B→Z transition, as determined by CD spectroscopy.     

DNA nicking by HinP1I endonuclease: bending, base flipping and minor groove expansion

HinP1I recognizes and cleaves the palindromic tetranucleotide sequence G↓CGC in DNA. We report three structures of HinP1I–DNA complexes: in the presence of Ca²⁺ (pre-reactive complex), in the absence of metal ion (binary complex) and in the presence of Mg²⁺ (post-reactive complex). HinP1I forms a back-to-back dimer with two active sites and two DNA duplexes bound on the outer surfaces of the dimer facing away from each other. The 10 bp DNA duplexes undergo protein-induced distortions exhibiting features of A-, B- and Z-conformations: bending on one side (by intercalation of a phenylalanine side chain into the major groove), base flipping on the other side of the recognition site (by expanding the step rise distance of the local base pair to Z-form) and a local A-form conformation between the two central C:G base pairs of the recognition site (by binding of the N-terminal helix in the minor groove). In the pre- and post-reactive complexes, two metals (Ca²⁺ or Mg²⁺) are found in the active site. The enzyme appears to cleave DNA sequentially, hydrolyzing first one DNA strand, as seen in the post-reactive complex in the crystalline state, and then the other, as supported by the observation that, in solution, a nicked DNA intermediate accumulates before linearization.     

Synthesis and Hybridization Properties of Oligonucleotides Containing (2 S ,3 R )-9-(2,3,4-Trihydroxybutyl)Adenine

The synthesis and properties of oligonucleotides (ONs) containing 9-(2,3,4-trihydroxybutyl)adenine, A ^C2 and A ^C3, are described. The ON containing A ^C2 involves the 3′ → 4′ and 3′ → 5′ phosphodiester linkages in the strand, whereas that containing A ^C3 possesses the 3′ → 4′ and 2′ → 5′ phosphodiester linkages. It was found that incorporation of the analogs, A ^C2 or A ^C3, into ONs significantly reduces the thermal and thermodynamic stabilities of the ON/DNA duplexes, but does not largely decrease the thermal and thermodynamic stabilities of the ON/RNA duplexes as compared with the case of the ON/DNA duplexes. It was revealed that the base recognition ability of A ^C2 is greater than that of A ^C3 in the ON/RNA duplexes.     

Highly effective colorimetric and visual detection of ATP by a DNAzyme-aptamer sensor

A new and simple method was developed to detect adenosine triphosphate (ATP) by using a DNAzyme aptamer sensor. The DNAzyme used was a single‐stranded DNA that could combine with hemin. The aptamer, a single, short nucleic acid sequence that can specifically bind with many targets, was an anti‐ATP aptamer. Two DNA sequences were designed: i) a functional chain (Chain A) consisting of two parts, i.e., the anti‐ATP aptamer (recognition part) and the DNAzyme (signal transduction part) and ii) a blocker chain (Chain B), which could partially hybridize with Chain A. The hybridized chains A and B were unfolded by the addition of ATP and hemin, and the blocker chain and the complex of the functional chain with ATP and hemin were in solution. The DNAzyme in the functional chain formed a G‐quadruplex with hemin and then catalyzed the oxidation by H₂O₂ of 2,2′‐azinobis(3‐ethylbenzthiazoline‐6‐sulfonic acid) (ABTS²⁻) to the colored ABTS^.− radical. The color change caused by this reaction could be clearly observed by naked eye, and the absorbance was recorded at 414 nm. The detection limit was 1×10⁻⁶ M .     

Oligonucleotide N3′→P5′ Phosphoramidates and Thio‐Phoshoramidates as Potential Therapeutic Agents

Nucleic acids analogues, i.e., oligonucleotide N3′→P5′ phosphoramidates and N3′→P5′ thio‐phosphoramidates, containing 3′‐amino‐3′‐deoxy nucleosides with various 2′‐substituents were synthesized and extensively studied. These compounds resist nuclease hydrolysis and form stable duplexes with complementary native phosphodiester DNA and, particularly, RNA strands. An increase in duplexes' melting temperature, ΔT_m, relative to their phosphodiester counterparts, reaches 2.2–4.0° per modified nucleoside. 2′‐OH‐ (RNA‐like), 2′‐O‐Me‐, and 2′‐ribo‐F‐nucleoside substitutions result in the highest degree of duplex stabilization. Moreover, under close to physiological salt and pH conditions, the 2′‐deoxy‐ and 2′‐fluoro‐phosphoramidate compounds form extremely stable triple‐stranded complexes with either single‐ or double‐stranded phosphodiester DNA oligonucleotides. Melting temperature, T_m, of these triplexes exceeds T_m values for the isosequential phosphodiester counterparts by up to 35°. 2′‐Deoxy‐N3′→P5′ phosphoramidates adopt RNA‐like C3′‐endo or N‐type nucleoside sugar‐ring conformations and hence can be used as stable RNA mimetics. Duplexes formed by 2′‐deoxy phosphoramidates with complementary RNA strands are not substrates for RNase H‐mediated cleavage in vitro. Oligonucleotide phosphoramidates and especially thio‐phosphoramidates conjugated with lipid groups are cell‐permeable and demonstrate high biological target specific activity in vitro. In vivo, these compounds show good bioavailability and efficient biodistribution to all major organs, while exerting acceptable toxicity at therapeutically relevant doses. Short oligonucleotide N3′→P5′ thio‐phosphoramidate conjugated to 5′‐palmitoyl group, designated as GRN163L (Imetelstat), was recently introduced as a potent human telomerase inhibitor. GRN163L is not an antisense agent; it is a direct competitive inhibitor of human telomerase, which directly binds to the active site of the enzyme and thus inhibits its activity. This compound is currently in multiple Phase‐I and Phase‐I/II clinical trials as potential broad‐spectrum anticancer agent.