The formation of DNA adducts by the attack of intermediates derived from toxic substances at C8 o... more The formation of DNA adducts by the attack of intermediates derived from toxic substances at C8 of 2'-deoxyguanosine (dG) is a common damaging event. Although the majority of studies on C8-dG adducts have focused on lesions containing a C8-N-C tether between the bulky moiety and the nucleobase, the formation of O-linked lesions with a similar tether topology and C-linked adducts involving direct C8-C connectivity have also been uncovered. Several studies have been done to try to better understand the structural impact and mutagenicity of O-linked and C-linked aryl C8-dG adducts, including lesions arising from unsubstituted and chloro-substituted phenols and the food mutagen ochratoxin A (OTA). Information about the structural preferences of the adducts in duplexes containing the NarI sequence has been gained from optical spectroscopy (UV-vis, CD, and fluorescence), 19F NMR spectroscopy, and computational chemistry (density functional theory calculations at the nucleobase, nucleoside, and nucleotide levels and molecular dynamics simulations of adducted duplexes). The replication of select adducts has also been investigated using primer-elongation assays, and model high-fidelity and Y-family polymerases. Although the (unsubstituted) O-linked phenoxy-dG adduct preferentially induces a single duplex conformation and is replicated as per natural dG, chloro substitution blocks DNA replication. In contrast, the unsubstituted C-linked phenyl-dG adduct induces mismatches, while the C-linked ortho- and para-phenoxy-dG lesions lead to conformational heterogeneity of adducted DNA indicative of strong mutagenic potential. Finally, the C-linked OTA-derived lesion exhibits the greatest conformational flexibility in duplexes, which provides structural explanations for observed outcomes in OTA-exposed cells. Overall, the variation in the conformational preferences of DNA containing O-linked and C-linked aryl-dG adducts highlights the fact that the type of C8 linkage, the presence and location of functional groups in the bulky moiety, the adduct ionization state, and the sequence context can have profound effects on the conformational outcomes of adducted DNA, which directly dictate the activity of the original toxin.
Utility of internal fluorescent guanine replacements for effective signaling of divalent metal io... more Utility of internal fluorescent guanine replacements for effective signaling of divalent metal ion binding.
DNA base compositional analysis is something which is rarely undertaken today, but it is still a ... more DNA base compositional analysis is something which is rarely undertaken today, but it is still a useful criterion for phage taxonomy. A variety of techniques are described including hydrolysis of the DNA to the level of bases or nucleosides and separation by paper chromatography or HPLC. Spectroscopic and spectrofluorometric procedures are also outlined.
Publisher Summary This chapter examines the structural and biological impact of radical addition ... more Publisher Summary This chapter examines the structural and biological impact of radical addition reactions with DNA nucleobases. The basic hypothesis of chemical carcinogenesis is that formation of a covalent bond between a chemical and DNA to form a DNA adduct represents the first essential step in the tumor initiation process. The nucleobases, especially 2′-deoxyguanosine (dG), are preferential targets of electrophilic attachment in which a single electrophile can react with all four DNA bases and at multiple sites. The hydroxyl radical can also abstract a single electron from dG to generate the base radical cation (G• + ). In duplex DNA, the G• + will be stabilized by its delocalization into adjacent bases. Both calculations and kinetic measurements indicate that GG sequences have a lower oxidation potential than an isolated G. The first step involves the synthesis of an oligonucleotide containing a single well-defined modification at an individual site. Four major approaches that can be used to prepare the adducted DNA include—synthesis of the modified phosphoramidite for solid-phase DNA synthesis and postsynthetic modification of DNA bearing a convertible nucleoside. The fungal carcinogen, OTA, has also been shown by the P-postlabeling method to form a C8-dG adduct in vivo through the intermediacy of radical species.
SYPRO OrangeTM is a commercial dye commonly employed in a thermofluorimetric analysis (TFA) to st... more SYPRO OrangeTM is a commercial dye commonly employed in a thermofluorimetric analysis (TFA) to stain thermally-denatured proteins and obtain their melting temperature (Tm values). Herein, the ability of DNA-aptamer-based ligands...
Ochratoxin A (OTA, 1: X = Cl) is a fungal carcinogen that facilitates single-strand DNA cleavage ... more Ochratoxin A (OTA, 1: X = Cl) is a fungal carcinogen that facilitates single-strand DNA cleavage and DNA adduction when metabolically activated. To determine if redox-active transition metals induce OTA-mediated DNA damage, we have examined the toxin's ability to bind Cu(II) and Fe(III) in aqueous media and facilitate DNA cleavage in their presence using agarose gel electrophoresis and supercoiled plasmid DNA. Using fluorescence spectroscopy, 1 was found to bind Cu(II) readily at physiological pH, while acidic conditions (pH 2.6) were employed to study Fe(III) binding due to the formation of Fe-oxide precipitates at higher pH values. Structure-activity relationships employing synthetic derivatives of 1 implied that 1 binds both Cu(II) and Fe(III) by its phenolic oxygen, while the carboxylic acid of its phenylalanine moiety binds Cu(II), but does not appear to play a role in Fe(III) coordination at pH 2.6. In terms of metal-mediated DNA cleavage, no role for 1 could be detected in Fe-induced DNA strand scission. With Cu(II), DNA cleavage by the 1:1 copper-bound complex of 1 could only be initiated by addition of a suitable reducing agent (sodium ascorbate). However, 1 was found to facilitate DNA cleavage by the Cu(II) complex of 1,10-phenanthroline (Cu(OP)2); a prototypical Cu-mediated nuclease system that cleaves DNA upon activation by an external reducing agent. Structure-activity relationships employing analogs lacking the chlorine atom, ochratoxin B (2: X = H), and the lactone (12), indicated that the chlorine atom is essential for activity of the OTA in potentiating DNA cleavage by Cu(OP)2. The implications of our findings to the genotoxic properties of 1 are discussed.Key words: ochratoxin, DNA cleavage, copper, iron, 1,10-phenanthroline.
Phenolic toxins and mutagenic diazoquinones generate C-linked adducts at the C8 site of 2&amp... more Phenolic toxins and mutagenic diazoquinones generate C-linked adducts at the C8 site of 2'-deoxyguanosine (dG) through the intermediacy of radical species. We have previously reported the site-specific incorporation of these adducts into oligonucleotides using a postsynthetic palladium-catalyzed cross-coupling strategy [Omumi (2011 ) J. Am. Chem. Soc. 133 , 42 - 50 ]. We report here the structural impact of these lesions within two decanucleotide sequences containing either 5'- and 3'-flanking pyrimidines or purines. In the complementary strands, the base opposite (N) the C-linked adduct was varied to determine the possibility of mismatch stabilization by the modified nucleobases. The resulting adducted duplex structures were characterized using UV thermal denaturation studies, circular dichroism, fluorescence spectroscopy, and molecular dynamics (MD) simulations. The experimental data showed the C-linked adducts to destabilize the duplex when base paired with its normal partner C but to increase duplex stability within a G:G mismatch. The stabilization within the G:G mismatch was sequence dependent, with flanking purine bases playing a key role in the stabilizing influence of the adduct. MD simulations showed no large structural changes to the B form double helix, regardless of the (anti/syn) adduct preference. Consideration of H-bonding and stacking interactions derived from the MD simulations together with the thermal melting data and changes in fluorescent emission of the adducts upon hybridization to the complementary strands implied that the C-linked phenolic adducts preferentially adopt the syn-conformation within both duplexes regardless of the opposite base N. Given that biological outcome in terms of mutagenicity appears to be strongly correlated to the conformational preference of the corresponding N-linked C8-dG adducts, the potential biological implications of phenolic C-linked adducts are discussed.
Insertion of an indanone handle into DNA oligonucleotides permits base-catalyzed aldol to create ... more Insertion of an indanone handle into DNA oligonucleotides permits base-catalyzed aldol to create fluorescent molecular rotor (FMR) chalcones with turn-on fluorescence and brightness suitable for biosensing applications.
Fluorescent molecular rotors (FMRs) are critical tools for monitoring nucleic acid structure and ... more Fluorescent molecular rotors (FMRs) are critical tools for monitoring nucleic acid structure and function. Many valuable probes are capable of sitespecific insertion into oligonucleotides, although the methods of doing so can be arduous and lack modularity. Development of modular and more user amenable insertion methods is thus crucial to expand the biotechnological applications of oligonucleotides in what is a rapidly expanding field. Herein we report the utility of 6-hydroxy-indanone (6HI) with a glycol backbone for on-strand aldehyde capture as a modular aldol approach with synthetic ease for site-specific insertion of internal FMRs. The 6HI phosphoramidite is produced in two steps and inserted into DNA using solid-phase synthesis. On-strand aldol with aromatic aldehydes containing N-donors proceeds in quantitative yield via sodium hydroxide to create a highly functional and unique library of FMR chalcones with emissions that span the visible region (em 490- 680 nm). The FMRs pr...
The formation of DNA adducts by the attack of intermediates derived from toxic substances at C8 o... more The formation of DNA adducts by the attack of intermediates derived from toxic substances at C8 of 2'-deoxyguanosine (dG) is a common damaging event. Although the majority of studies on C8-dG adducts have focused on lesions containing a C8-N-C tether between the bulky moiety and the nucleobase, the formation of O-linked lesions with a similar tether topology and C-linked adducts involving direct C8-C connectivity have also been uncovered. Several studies have been done to try to better understand the structural impact and mutagenicity of O-linked and C-linked aryl C8-dG adducts, including lesions arising from unsubstituted and chloro-substituted phenols and the food mutagen ochratoxin A (OTA). Information about the structural preferences of the adducts in duplexes containing the NarI sequence has been gained from optical spectroscopy (UV-vis, CD, and fluorescence), 19F NMR spectroscopy, and computational chemistry (density functional theory calculations at the nucleobase, nucleoside, and nucleotide levels and molecular dynamics simulations of adducted duplexes). The replication of select adducts has also been investigated using primer-elongation assays, and model high-fidelity and Y-family polymerases. Although the (unsubstituted) O-linked phenoxy-dG adduct preferentially induces a single duplex conformation and is replicated as per natural dG, chloro substitution blocks DNA replication. In contrast, the unsubstituted C-linked phenyl-dG adduct induces mismatches, while the C-linked ortho- and para-phenoxy-dG lesions lead to conformational heterogeneity of adducted DNA indicative of strong mutagenic potential. Finally, the C-linked OTA-derived lesion exhibits the greatest conformational flexibility in duplexes, which provides structural explanations for observed outcomes in OTA-exposed cells. Overall, the variation in the conformational preferences of DNA containing O-linked and C-linked aryl-dG adducts highlights the fact that the type of C8 linkage, the presence and location of functional groups in the bulky moiety, the adduct ionization state, and the sequence context can have profound effects on the conformational outcomes of adducted DNA, which directly dictate the activity of the original toxin.
Utility of internal fluorescent guanine replacements for effective signaling of divalent metal io... more Utility of internal fluorescent guanine replacements for effective signaling of divalent metal ion binding.
DNA base compositional analysis is something which is rarely undertaken today, but it is still a ... more DNA base compositional analysis is something which is rarely undertaken today, but it is still a useful criterion for phage taxonomy. A variety of techniques are described including hydrolysis of the DNA to the level of bases or nucleosides and separation by paper chromatography or HPLC. Spectroscopic and spectrofluorometric procedures are also outlined.
Publisher Summary This chapter examines the structural and biological impact of radical addition ... more Publisher Summary This chapter examines the structural and biological impact of radical addition reactions with DNA nucleobases. The basic hypothesis of chemical carcinogenesis is that formation of a covalent bond between a chemical and DNA to form a DNA adduct represents the first essential step in the tumor initiation process. The nucleobases, especially 2′-deoxyguanosine (dG), are preferential targets of electrophilic attachment in which a single electrophile can react with all four DNA bases and at multiple sites. The hydroxyl radical can also abstract a single electron from dG to generate the base radical cation (G• + ). In duplex DNA, the G• + will be stabilized by its delocalization into adjacent bases. Both calculations and kinetic measurements indicate that GG sequences have a lower oxidation potential than an isolated G. The first step involves the synthesis of an oligonucleotide containing a single well-defined modification at an individual site. Four major approaches that can be used to prepare the adducted DNA include—synthesis of the modified phosphoramidite for solid-phase DNA synthesis and postsynthetic modification of DNA bearing a convertible nucleoside. The fungal carcinogen, OTA, has also been shown by the P-postlabeling method to form a C8-dG adduct in vivo through the intermediacy of radical species.
SYPRO OrangeTM is a commercial dye commonly employed in a thermofluorimetric analysis (TFA) to st... more SYPRO OrangeTM is a commercial dye commonly employed in a thermofluorimetric analysis (TFA) to stain thermally-denatured proteins and obtain their melting temperature (Tm values). Herein, the ability of DNA-aptamer-based ligands...
Ochratoxin A (OTA, 1: X = Cl) is a fungal carcinogen that facilitates single-strand DNA cleavage ... more Ochratoxin A (OTA, 1: X = Cl) is a fungal carcinogen that facilitates single-strand DNA cleavage and DNA adduction when metabolically activated. To determine if redox-active transition metals induce OTA-mediated DNA damage, we have examined the toxin's ability to bind Cu(II) and Fe(III) in aqueous media and facilitate DNA cleavage in their presence using agarose gel electrophoresis and supercoiled plasmid DNA. Using fluorescence spectroscopy, 1 was found to bind Cu(II) readily at physiological pH, while acidic conditions (pH 2.6) were employed to study Fe(III) binding due to the formation of Fe-oxide precipitates at higher pH values. Structure-activity relationships employing synthetic derivatives of 1 implied that 1 binds both Cu(II) and Fe(III) by its phenolic oxygen, while the carboxylic acid of its phenylalanine moiety binds Cu(II), but does not appear to play a role in Fe(III) coordination at pH 2.6. In terms of metal-mediated DNA cleavage, no role for 1 could be detected in Fe-induced DNA strand scission. With Cu(II), DNA cleavage by the 1:1 copper-bound complex of 1 could only be initiated by addition of a suitable reducing agent (sodium ascorbate). However, 1 was found to facilitate DNA cleavage by the Cu(II) complex of 1,10-phenanthroline (Cu(OP)2); a prototypical Cu-mediated nuclease system that cleaves DNA upon activation by an external reducing agent. Structure-activity relationships employing analogs lacking the chlorine atom, ochratoxin B (2: X = H), and the lactone (12), indicated that the chlorine atom is essential for activity of the OTA in potentiating DNA cleavage by Cu(OP)2. The implications of our findings to the genotoxic properties of 1 are discussed.Key words: ochratoxin, DNA cleavage, copper, iron, 1,10-phenanthroline.
Phenolic toxins and mutagenic diazoquinones generate C-linked adducts at the C8 site of 2&amp... more Phenolic toxins and mutagenic diazoquinones generate C-linked adducts at the C8 site of 2'-deoxyguanosine (dG) through the intermediacy of radical species. We have previously reported the site-specific incorporation of these adducts into oligonucleotides using a postsynthetic palladium-catalyzed cross-coupling strategy [Omumi (2011 ) J. Am. Chem. Soc. 133 , 42 - 50 ]. We report here the structural impact of these lesions within two decanucleotide sequences containing either 5'- and 3'-flanking pyrimidines or purines. In the complementary strands, the base opposite (N) the C-linked adduct was varied to determine the possibility of mismatch stabilization by the modified nucleobases. The resulting adducted duplex structures were characterized using UV thermal denaturation studies, circular dichroism, fluorescence spectroscopy, and molecular dynamics (MD) simulations. The experimental data showed the C-linked adducts to destabilize the duplex when base paired with its normal partner C but to increase duplex stability within a G:G mismatch. The stabilization within the G:G mismatch was sequence dependent, with flanking purine bases playing a key role in the stabilizing influence of the adduct. MD simulations showed no large structural changes to the B form double helix, regardless of the (anti/syn) adduct preference. Consideration of H-bonding and stacking interactions derived from the MD simulations together with the thermal melting data and changes in fluorescent emission of the adducts upon hybridization to the complementary strands implied that the C-linked phenolic adducts preferentially adopt the syn-conformation within both duplexes regardless of the opposite base N. Given that biological outcome in terms of mutagenicity appears to be strongly correlated to the conformational preference of the corresponding N-linked C8-dG adducts, the potential biological implications of phenolic C-linked adducts are discussed.
Insertion of an indanone handle into DNA oligonucleotides permits base-catalyzed aldol to create ... more Insertion of an indanone handle into DNA oligonucleotides permits base-catalyzed aldol to create fluorescent molecular rotor (FMR) chalcones with turn-on fluorescence and brightness suitable for biosensing applications.
Fluorescent molecular rotors (FMRs) are critical tools for monitoring nucleic acid structure and ... more Fluorescent molecular rotors (FMRs) are critical tools for monitoring nucleic acid structure and function. Many valuable probes are capable of sitespecific insertion into oligonucleotides, although the methods of doing so can be arduous and lack modularity. Development of modular and more user amenable insertion methods is thus crucial to expand the biotechnological applications of oligonucleotides in what is a rapidly expanding field. Herein we report the utility of 6-hydroxy-indanone (6HI) with a glycol backbone for on-strand aldehyde capture as a modular aldol approach with synthetic ease for site-specific insertion of internal FMRs. The 6HI phosphoramidite is produced in two steps and inserted into DNA using solid-phase synthesis. On-strand aldol with aromatic aldehydes containing N-donors proceeds in quantitative yield via sodium hydroxide to create a highly functional and unique library of FMR chalcones with emissions that span the visible region (em 490- 680 nm). The FMRs pr...
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Papers by Richard Manderville