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Papers by Manjori Ganguly

The incorporation of 7-deazapurine modifications into DNA is frequently used in biochemical studi... more The incorporation of 7-deazapurine modifications into DNA is frequently used in biochemical studies, especially to investigate the effect of the modifications on the DNA duplex stability. It is assumed that 7-deaza-2'-deoxyadenosine (7-deaza-dA) forms a normal Watson-Crick base pair with thymine. In this work, the high resolution crystallographic structure of the 7-deaza-dA modified Dickerson-Drew Dodecamer (DDD) is reported. The incorporation of the 7-deaza-2'-deoxyadenosine modification has minimal effect on the structure; a cation-binding site adjacent to the lesion is maintained. NMR melting studies as well as UV thermal melting experiments show a destabilization of a 7-deaza-2'-deoxyadenosine modified DNA duplex relative to an unmodified duplex.

The Journal of Physical Chemistry B, 2011

Nucleic Acids Research, 2011

The oxidation of DNA resulting from reactive oxygen species generated during aerobic respiration ... more The oxidation of DNA resulting from reactive oxygen species generated during aerobic respiration is a major cause of genetic damage that, if not repaired, can lead to mutations and potentially an increase in the incidence of cancer and aging. A major oxidation product generated in cells is 8-oxoguanine (oxoG), which is removed from the nucleotide pool by the enzymatic hydrolysis of 8-oxo-2 0-deoxyguanosine triphosphate and from genomic DNA by 8-oxoguanine-DNA glycosylase. Finding and repairing oxoG in the midst of a large excess of unmodified DNA requires a combination of rapid scanning of the DNA for the lesion followed by specific excision of the damaged base. The repair of oxoG involves flipping the lesion out of the DNA stack and into the active site of the 8-oxoguanine-DNA glycosylase. This would suggest that thermodynamic stability, in terms of the rate for local denaturation, could play a role in lesion recognition. While prior X-ray crystal and NMR structures show that DNA with oxoG lesions appears virtually identical to the corresponding unmodified duplex, thermodynamic studies indicate that oxoG has a destabilizing influence. Our studies show that oxoG destabilizes DNA (""G of 2-8 kcal mol À1 over a 16-116 mM NaCl range) due to a significant reduction in the enthalpy term. The presence of oxoG has a profound effect on the level and nature of DNA hydration indicating that the environment around an oxoGC is fundamentally different than that found at GC. The temperature-dependent imino proton NMR spectrum of oxoG modified DNA confirms the destabilization of the oxoGC pairing and those base pairs that are 5 0 of the lesion. The instability of the oxoG modification is attributed to changes in the hydrophilicity of the base and its impact on major groove cation binding.

Nucleic Acids Research, 2007

The incorporation of 7-deazaguanine modifications into DNA is frequently used to probe protein re... more The incorporation of 7-deazaguanine modifications into DNA is frequently used to probe protein recognition of H-bonding information in the major groove of DNA. While it is generally assumed that 7-deazaguanine forms a normal Watson-Crick base pair with cytosine, detailed thermodynamic and structural analyses of this modification have not been reported. The replacement of the 7-N atom on guanine with a C-H, alters the electronic properties of the heterocycle and eliminates a major groove cation-binding site that could affect the organization of salts and water in the major groove. We report herein the characterization of synthetic DNA oligomers containing 7-deazaguanine using a variety of complementary approaches: UV thermal melting, differential scanning calorimetry (DSC), circular dichroism (CD), chemical probing and NMR. The results indicate that the incorporation of a 7-deazaguanine modification has a significant effect on the dynamic structure of the DNA at the flanking residue. This appears to be mediated by changes in hydration and cation organization.

Journal of the American Chemical Society, 2009

The replacement of the 7-N atom on guanine (G) with a C-H to give 7-deazaguanine (c 7 G) alters t... more The replacement of the 7-N atom on guanine (G) with a C-H to give 7-deazaguanine (c 7 G) alters the electronic properties of the heterocyclic base and eliminates a potential major groove cation binding site, which affects the organization of salts and water in the major groove. This has a destabilizing effect on DNA. We report herein the characterization of DNA oligomers containing 7-(aminomethyl)-7-deazaguanine (1) residues using a variety of spectroscopic and thermodynamic approaches. 1 is an intramolecular model for the major groove binding of cations and basic amino acid residues to G. In contrast to c 7 G, the tethering of a cation in the major groove using 1 affords

Biophysical Journal, 2010

G6-53 unfolds at~250 pN. Using their characteristic unfolding forces as a reporter, we were able ... more G6-53 unfolds at~250 pN. Using their characteristic unfolding forces as a reporter, we were able to directly quantify the partitioning of G6-53 between the apo and Ni2þ bound states at different Ni2þ concentration and measure the binding affinity of Ni2þ to G6-53. The distinct unfolding forces of apo and holo forms of G6-53 also allow us to discriminate different species in the process of folding and Ni2þ binding and measure their kinetic evolution. We unfolded G6-53 by force and waited to allow it to fold and bind with Ni2þ. We found that the unfolded G6-53 folds to apo form before incorporating Ni2þ. The folding rate of G6-53 is independent of Ni2þ concentration, while the binding rate of Ni2þ to apo form of G6-53 is directly proportional to the Ni2þ concentration. Our kinetic data can be fully described using a ''folding before binding'' model. We anticipate that this novel assay will find unique applications in the study of various protein-ligand interactions.

Biochemistry, 2013

A cationic 7-aminomethyl-7-deaza-2′-deoxyguanosine (7amG) was incorporated site-specifically into... more A cationic 7-aminomethyl-7-deaza-2′-deoxyguanosine (7amG) was incorporated site-specifically into the selfcomplementary duplex d(G 1 A 2 G 3 A 4 X 5 C 6 G 7 C 8 T 9 C 10 T 11 C 12) 2 (X = 7amG). This construct placed two positively charged amines adjacent to the major groove edges of two symmetryrelated guanines, providing a model for probing how cation binding in the major groove modulates the structure and stability of DNA. Molecular dynamics calculations restrained by nuclear magnetic resonance (NMR) data revealed that the tethered cationic amines were in plane with the modified base pairs. The tethered amines did not form salt bridges to the phosphodiester backbone. There was also no indication of the amines being capable of hydrogen bonding to flanking DNA bases. NMR spectroscopy as a function of temperature revealed that the X 5 imino resonance remained sharp at 55°C. Additionally, two 5′-neighboring base pairs, A 4 :T 9 and G 3 :C 10 , were stabilized with respect to the exchange of their imino protons with solvent. The equilibrium constant for base pair opening at the A 4 :T 9 base pair determined by magnetization transfer from water in the absence and presence of added ammonia base catalyst decreased for the modified duplex compared to that of the A 4 :T 9 base pair in the unmodified duplex, which confirmed that the overall fraction of the A 4 :T 9 base pair in the open state of the modified duplex decreased. This was also observed for the G 3 :C 10 base pair, where αK op for the G 3 :C 10 base pair in the modified duplex was 3.0 × 10 6 versus 4.1 × 10 6 for the same base pair in the unmodified duplex. In contrast, equilibrium constants for base pair opening at the X 5 :C 8 and C 6 :G 7 base pairs did not change at 15°C. These results argue against the notion that electrostatic interactions with DNA are entirely entropic and suggest that major groove cations can stabilize DNA via enthalpic contributions to the free energy of duplex formation.

Biochemistry, 2008

Site-specific insertion of 5-(3-aminopropyl)-2′-deoxyuridine (Z3dU) and 7-deaza-dG into the Dicke... more Site-specific insertion of 5-(3-aminopropyl)-2′-deoxyuridine (Z3dU) and 7-deaza-dG into the Dickerson-Drew dodecamers 5′-d(C

Theory and Practice of Contemporary Pharmaceutics, 2004

The Journal of Physical Chemistry B, 2010

In many high-resolution structures of DNA there are ordered waters associated with the floor of t... more In many high-resolution structures of DNA there are ordered waters associated with the floor of the minor groove and extending outward in several layers. It is thought that this hydration structure, along with cations, reduces the Coulombic repulsion of the interstrand phosphates. In previous studies, the replacement of the 3-N atom of adenine with a C-H to afford 3-deazaadenine was shown to decrease the thermodynamic stability of DNA via a reduction in the enthalpic term. Using spectroscopic and calorimetric methods, we report herein a rigorous examination of the thermodynamics of DNA with 3-deazaadenine modifications, and report for the first time how the presence of a minor groove methyl group, i.e., 3-methyl-3-deazaadeine, affects DNA stability, hydration, and cation binding. The methylation of adenine at the N3-position to yield N3-methyladenine represents an important reaction in the toxicity of many anticancer compounds. This minor groove lesion is unstable and cannot be readily studied in terms of its effect on DNA stability or structure. Our studies show that 3-methyl-3-deazaadenine, an isostere of N3-methyladenine, significantly destabilizes DNA (DeltaDeltaG > 4 kcal x mol(-1)) due to a significant drop in the enthalpy (DeltaH) term, which is associated with a lower hydration of the duplex relative to the unfolded state.

Biochemistry, 2012

Oxidation of DNA due to exposure to reactive oxygen species is a major source of DNA damage. One ... more Oxidation of DNA due to exposure to reactive oxygen species is a major source of DNA damage. One of the oxidation lesions formed, 5-hydroxy-2′-deoxycytidine, has been shown to miscode by some replicative DNA polymerases but not by error prone polymerases capable of translesion synthesis. The 5-hydroxy-2′-deoxycytidine lesion is repaired by DNA glycosylases that require the 5-hydroxycytidine base to be extrahelical so it can enter into the enzyme's active site where it is excised off the DNA backbone to afford an abasic site. The thermodynamic and nuclear magnetic resonance results presented here describe the effect of a 5-hydroxy-2′-deoxycytidine·2′-deoxyguanosine base pair on the stability of two different DNA duplexes. The results demonstrate that the lesion is highly destabilizing and that the energy barrier for the unstacking of 5-hydroxy-2′-deoxycytidine from the DNA duplex may be low. This could provide a thermodynamic mode of adduct identification by DNA glycosylases that requires the lesion to be extrahelical.

The Journal of Physical Chemistry B, 2011

As part of an ongoing effort to explore the effect of major groove electrostatics on the thermody... more As part of an ongoing effort to explore the effect of major groove electrostatics on the thermodynamic stability and structure of DNA, a 7-deaza-2'-deoxyadenosine:dT (7-deaza-dA:dT) base pair in the Dickerson-Drew dodecamer (DDD) was studied. The removal of the electronegative N7 atom on dA and the replacement with an electropositive C-H in the major groove was expected to have a significant effect on major groove electrostatics. The structure of the 7-deaza-dA:dT base pair was determined at 1.1 Å resolution in the presence of Mg(2+). The 7-deaza-dA, which is isosteric for dA, had minimal effect on the base pairing geometry and the conformation of the DDD in the crystalline state. There was no major groove cation association with the 7-deaza-dA heterocycle. In solution, circular dichroism showed a positive Cotton effect centered at 280 nm and a negative Cotton effect centered at 250 nm that were characteristic of a right-handed helix in the B-conformation. However, temperature-dependent NMR studies showed increased exchange between the thymine N3 imino proton of the 7-deaza-dA:dT base pair and water, suggesting reduced stacking interactions and an increased rate of base pair opening. This correlated with the observed thermodynamic destabilization of the 7-deaza-dA modified duplex relative to the DDD. A combination of UV melting and differential scanning calorimetry experiments were conducted to evaluate the relative contributions of enthalpy and entropy in the thermodynamic destabilization of the DDD. The most significant contribution arose from an unfavorable enthalpy term, which probably results from less favorable stacking interactions in the modified duplex, which was accompanied by a significant reduction in the release of water and cations from the 7-deaza-dA modified DNA.

Nucleic Acids Research, 2011

The oxidation of DNA resulting from reactive oxygen species generated during aerobic respiration ... more The oxidation of DNA resulting from reactive oxygen species generated during aerobic respiration is a major cause of genetic damage that, if not repaired, can lead to mutations and potentially an increase in the incidence of cancer and aging. A major oxidation product generated in cells is 8-oxoguanine (oxoG), which is removed from the nucleotide pool by the enzymatic hydrolysis of 8-oxo-2 0 -deoxyguanosine triphosphate and from genomic DNA by 8-oxoguanine-DNA glycosylase. Finding and repairing oxoG in the midst of a large excess of unmodified DNA requires a combination of rapid scanning of the DNA for the lesion followed by specific excision of the damaged base. The repair of oxoG involves flipping the lesion out of the DNA stack and into the active site of the 8-oxoguanine-DNA glycosylase. This would suggest that thermodynamic stability, in terms of the rate for local denaturation, could play a role in lesion recognition. While prior X-ray crystal and NMR structures show that DNA with oxoG lesions appears virtually identical to the corresponding unmodified duplex, thermodynamic studies indicate that oxoG has a destabilizing influence. Our studies show that oxoG destabilizes DNA (""G of 2-8 kcal mol À1 over a 16-116 mM NaCl range) due to a significant reduction in the enthalpy term. The presence of oxoG has a profound effect on the level and nature of DNA hydration indicating that the environment around an oxoGC is fundamentally different than that found at GC. The temperature-dependent imino proton NMR spectrum of oxoG modified DNA confirms the destabilization of the oxoGC pairing and those base pairs that are 5 0 of the lesion. The instability of the oxoG modification is attributed to changes in the hydrophilicity of the base and its impact on major groove cation binding.

Journal of the American Chemical Society, 2009

The replacement of the 7-N atom on guanine (G) with a C-H to give 7-deazaguanine (c 7 G) alters t... more The replacement of the 7-N atom on guanine (G) with a C-H to give 7-deazaguanine (c 7 G) alters the electronic properties of the heterocyclic base and eliminates a potential major groove cation binding site, which affects the organization of salts and water in the major groove. This has a destabilizing effect on DNA. We report herein the characterization of DNA oligomers containing 7-(aminomethyl)-7-deazaguanine (1) residues using a variety of spectroscopic and thermodynamic approaches. 1 is an intramolecular model for the major groove binding of cations and basic amino acid residues to G. In contrast to c 7 G, the tethering of a cation in the major groove using 1 affords goldbi@pitt.edu. ¥ contributed equally Supporting Information.

The incorporation of 7-deazapurine modifications into DNA is frequently used in biochemical studi... more The incorporation of 7-deazapurine modifications into DNA is frequently used in biochemical studies, especially to investigate the effect of the modifications on the DNA duplex stability. It is assumed that 7-deaza-2'-deoxyadenosine (7-deaza-dA) forms a normal Watson-Crick base pair with thymine. In this work, the high resolution crystallographic structure of the 7-deaza-dA modified Dickerson-Drew Dodecamer (DDD) is reported. The incorporation of the 7-deaza-2'-deoxyadenosine modification has minimal effect on the structure; a cation-binding site adjacent to the lesion is maintained. NMR melting studies as well as UV thermal melting experiments show a destabilization of a 7-deaza-2'-deoxyadenosine modified DNA duplex relative to an unmodified duplex.

The Journal of Physical Chemistry B, 2011

Nucleic Acids Research, 2011

The oxidation of DNA resulting from reactive oxygen species generated during aerobic respiration ... more The oxidation of DNA resulting from reactive oxygen species generated during aerobic respiration is a major cause of genetic damage that, if not repaired, can lead to mutations and potentially an increase in the incidence of cancer and aging. A major oxidation product generated in cells is 8-oxoguanine (oxoG), which is removed from the nucleotide pool by the enzymatic hydrolysis of 8-oxo-2 0-deoxyguanosine triphosphate and from genomic DNA by 8-oxoguanine-DNA glycosylase. Finding and repairing oxoG in the midst of a large excess of unmodified DNA requires a combination of rapid scanning of the DNA for the lesion followed by specific excision of the damaged base. The repair of oxoG involves flipping the lesion out of the DNA stack and into the active site of the 8-oxoguanine-DNA glycosylase. This would suggest that thermodynamic stability, in terms of the rate for local denaturation, could play a role in lesion recognition. While prior X-ray crystal and NMR structures show that DNA with oxoG lesions appears virtually identical to the corresponding unmodified duplex, thermodynamic studies indicate that oxoG has a destabilizing influence. Our studies show that oxoG destabilizes DNA (""G of 2-8 kcal mol À1 over a 16-116 mM NaCl range) due to a significant reduction in the enthalpy term. The presence of oxoG has a profound effect on the level and nature of DNA hydration indicating that the environment around an oxoGC is fundamentally different than that found at GC. The temperature-dependent imino proton NMR spectrum of oxoG modified DNA confirms the destabilization of the oxoGC pairing and those base pairs that are 5 0 of the lesion. The instability of the oxoG modification is attributed to changes in the hydrophilicity of the base and its impact on major groove cation binding.

Nucleic Acids Research, 2007

The incorporation of 7-deazaguanine modifications into DNA is frequently used to probe protein re... more The incorporation of 7-deazaguanine modifications into DNA is frequently used to probe protein recognition of H-bonding information in the major groove of DNA. While it is generally assumed that 7-deazaguanine forms a normal Watson-Crick base pair with cytosine, detailed thermodynamic and structural analyses of this modification have not been reported. The replacement of the 7-N atom on guanine with a C-H, alters the electronic properties of the heterocycle and eliminates a major groove cation-binding site that could affect the organization of salts and water in the major groove. We report herein the characterization of synthetic DNA oligomers containing 7-deazaguanine using a variety of complementary approaches: UV thermal melting, differential scanning calorimetry (DSC), circular dichroism (CD), chemical probing and NMR. The results indicate that the incorporation of a 7-deazaguanine modification has a significant effect on the dynamic structure of the DNA at the flanking residue. This appears to be mediated by changes in hydration and cation organization.

Journal of the American Chemical Society, 2009

The replacement of the 7-N atom on guanine (G) with a C-H to give 7-deazaguanine (c 7 G) alters t... more The replacement of the 7-N atom on guanine (G) with a C-H to give 7-deazaguanine (c 7 G) alters the electronic properties of the heterocyclic base and eliminates a potential major groove cation binding site, which affects the organization of salts and water in the major groove. This has a destabilizing effect on DNA. We report herein the characterization of DNA oligomers containing 7-(aminomethyl)-7-deazaguanine (1) residues using a variety of spectroscopic and thermodynamic approaches. 1 is an intramolecular model for the major groove binding of cations and basic amino acid residues to G. In contrast to c 7 G, the tethering of a cation in the major groove using 1 affords

Biophysical Journal, 2010

G6-53 unfolds at~250 pN. Using their characteristic unfolding forces as a reporter, we were able ... more G6-53 unfolds at~250 pN. Using their characteristic unfolding forces as a reporter, we were able to directly quantify the partitioning of G6-53 between the apo and Ni2þ bound states at different Ni2þ concentration and measure the binding affinity of Ni2þ to G6-53. The distinct unfolding forces of apo and holo forms of G6-53 also allow us to discriminate different species in the process of folding and Ni2þ binding and measure their kinetic evolution. We unfolded G6-53 by force and waited to allow it to fold and bind with Ni2þ. We found that the unfolded G6-53 folds to apo form before incorporating Ni2þ. The folding rate of G6-53 is independent of Ni2þ concentration, while the binding rate of Ni2þ to apo form of G6-53 is directly proportional to the Ni2þ concentration. Our kinetic data can be fully described using a ''folding before binding'' model. We anticipate that this novel assay will find unique applications in the study of various protein-ligand interactions.

Biochemistry, 2013

A cationic 7-aminomethyl-7-deaza-2′-deoxyguanosine (7amG) was incorporated site-specifically into... more A cationic 7-aminomethyl-7-deaza-2′-deoxyguanosine (7amG) was incorporated site-specifically into the selfcomplementary duplex d(G 1 A 2 G 3 A 4 X 5 C 6 G 7 C 8 T 9 C 10 T 11 C 12) 2 (X = 7amG). This construct placed two positively charged amines adjacent to the major groove edges of two symmetryrelated guanines, providing a model for probing how cation binding in the major groove modulates the structure and stability of DNA. Molecular dynamics calculations restrained by nuclear magnetic resonance (NMR) data revealed that the tethered cationic amines were in plane with the modified base pairs. The tethered amines did not form salt bridges to the phosphodiester backbone. There was also no indication of the amines being capable of hydrogen bonding to flanking DNA bases. NMR spectroscopy as a function of temperature revealed that the X 5 imino resonance remained sharp at 55°C. Additionally, two 5′-neighboring base pairs, A 4 :T 9 and G 3 :C 10 , were stabilized with respect to the exchange of their imino protons with solvent. The equilibrium constant for base pair opening at the A 4 :T 9 base pair determined by magnetization transfer from water in the absence and presence of added ammonia base catalyst decreased for the modified duplex compared to that of the A 4 :T 9 base pair in the unmodified duplex, which confirmed that the overall fraction of the A 4 :T 9 base pair in the open state of the modified duplex decreased. This was also observed for the G 3 :C 10 base pair, where αK op for the G 3 :C 10 base pair in the modified duplex was 3.0 × 10 6 versus 4.1 × 10 6 for the same base pair in the unmodified duplex. In contrast, equilibrium constants for base pair opening at the X 5 :C 8 and C 6 :G 7 base pairs did not change at 15°C. These results argue against the notion that electrostatic interactions with DNA are entirely entropic and suggest that major groove cations can stabilize DNA via enthalpic contributions to the free energy of duplex formation.

Biochemistry, 2008

Site-specific insertion of 5-(3-aminopropyl)-2′-deoxyuridine (Z3dU) and 7-deaza-dG into the Dicke... more Site-specific insertion of 5-(3-aminopropyl)-2′-deoxyuridine (Z3dU) and 7-deaza-dG into the Dickerson-Drew dodecamers 5′-d(C

Theory and Practice of Contemporary Pharmaceutics, 2004

The Journal of Physical Chemistry B, 2010

In many high-resolution structures of DNA there are ordered waters associated with the floor of t... more In many high-resolution structures of DNA there are ordered waters associated with the floor of the minor groove and extending outward in several layers. It is thought that this hydration structure, along with cations, reduces the Coulombic repulsion of the interstrand phosphates. In previous studies, the replacement of the 3-N atom of adenine with a C-H to afford 3-deazaadenine was shown to decrease the thermodynamic stability of DNA via a reduction in the enthalpic term. Using spectroscopic and calorimetric methods, we report herein a rigorous examination of the thermodynamics of DNA with 3-deazaadenine modifications, and report for the first time how the presence of a minor groove methyl group, i.e., 3-methyl-3-deazaadeine, affects DNA stability, hydration, and cation binding. The methylation of adenine at the N3-position to yield N3-methyladenine represents an important reaction in the toxicity of many anticancer compounds. This minor groove lesion is unstable and cannot be readily studied in terms of its effect on DNA stability or structure. Our studies show that 3-methyl-3-deazaadenine, an isostere of N3-methyladenine, significantly destabilizes DNA (DeltaDeltaG > 4 kcal x mol(-1)) due to a significant drop in the enthalpy (DeltaH) term, which is associated with a lower hydration of the duplex relative to the unfolded state.

Biochemistry, 2012

Oxidation of DNA due to exposure to reactive oxygen species is a major source of DNA damage. One ... more Oxidation of DNA due to exposure to reactive oxygen species is a major source of DNA damage. One of the oxidation lesions formed, 5-hydroxy-2′-deoxycytidine, has been shown to miscode by some replicative DNA polymerases but not by error prone polymerases capable of translesion synthesis. The 5-hydroxy-2′-deoxycytidine lesion is repaired by DNA glycosylases that require the 5-hydroxycytidine base to be extrahelical so it can enter into the enzyme's active site where it is excised off the DNA backbone to afford an abasic site. The thermodynamic and nuclear magnetic resonance results presented here describe the effect of a 5-hydroxy-2′-deoxycytidine·2′-deoxyguanosine base pair on the stability of two different DNA duplexes. The results demonstrate that the lesion is highly destabilizing and that the energy barrier for the unstacking of 5-hydroxy-2′-deoxycytidine from the DNA duplex may be low. This could provide a thermodynamic mode of adduct identification by DNA glycosylases that requires the lesion to be extrahelical.

The Journal of Physical Chemistry B, 2011

As part of an ongoing effort to explore the effect of major groove electrostatics on the thermody... more As part of an ongoing effort to explore the effect of major groove electrostatics on the thermodynamic stability and structure of DNA, a 7-deaza-2'-deoxyadenosine:dT (7-deaza-dA:dT) base pair in the Dickerson-Drew dodecamer (DDD) was studied. The removal of the electronegative N7 atom on dA and the replacement with an electropositive C-H in the major groove was expected to have a significant effect on major groove electrostatics. The structure of the 7-deaza-dA:dT base pair was determined at 1.1 Å resolution in the presence of Mg(2+). The 7-deaza-dA, which is isosteric for dA, had minimal effect on the base pairing geometry and the conformation of the DDD in the crystalline state. There was no major groove cation association with the 7-deaza-dA heterocycle. In solution, circular dichroism showed a positive Cotton effect centered at 280 nm and a negative Cotton effect centered at 250 nm that were characteristic of a right-handed helix in the B-conformation. However, temperature-dependent NMR studies showed increased exchange between the thymine N3 imino proton of the 7-deaza-dA:dT base pair and water, suggesting reduced stacking interactions and an increased rate of base pair opening. This correlated with the observed thermodynamic destabilization of the 7-deaza-dA modified duplex relative to the DDD. A combination of UV melting and differential scanning calorimetry experiments were conducted to evaluate the relative contributions of enthalpy and entropy in the thermodynamic destabilization of the DDD. The most significant contribution arose from an unfavorable enthalpy term, which probably results from less favorable stacking interactions in the modified duplex, which was accompanied by a significant reduction in the release of water and cations from the 7-deaza-dA modified DNA.

Nucleic Acids Research, 2011

The oxidation of DNA resulting from reactive oxygen species generated during aerobic respiration ... more The oxidation of DNA resulting from reactive oxygen species generated during aerobic respiration is a major cause of genetic damage that, if not repaired, can lead to mutations and potentially an increase in the incidence of cancer and aging. A major oxidation product generated in cells is 8-oxoguanine (oxoG), which is removed from the nucleotide pool by the enzymatic hydrolysis of 8-oxo-2 0 -deoxyguanosine triphosphate and from genomic DNA by 8-oxoguanine-DNA glycosylase. Finding and repairing oxoG in the midst of a large excess of unmodified DNA requires a combination of rapid scanning of the DNA for the lesion followed by specific excision of the damaged base. The repair of oxoG involves flipping the lesion out of the DNA stack and into the active site of the 8-oxoguanine-DNA glycosylase. This would suggest that thermodynamic stability, in terms of the rate for local denaturation, could play a role in lesion recognition. While prior X-ray crystal and NMR structures show that DNA with oxoG lesions appears virtually identical to the corresponding unmodified duplex, thermodynamic studies indicate that oxoG has a destabilizing influence. Our studies show that oxoG destabilizes DNA (""G of 2-8 kcal mol À1 over a 16-116 mM NaCl range) due to a significant reduction in the enthalpy term. The presence of oxoG has a profound effect on the level and nature of DNA hydration indicating that the environment around an oxoGC is fundamentally different than that found at GC. The temperature-dependent imino proton NMR spectrum of oxoG modified DNA confirms the destabilization of the oxoGC pairing and those base pairs that are 5 0 of the lesion. The instability of the oxoG modification is attributed to changes in the hydrophilicity of the base and its impact on major groove cation binding.

Journal of the American Chemical Society, 2009

The replacement of the 7-N atom on guanine (G) with a C-H to give 7-deazaguanine (c 7 G) alters t... more The replacement of the 7-N atom on guanine (G) with a C-H to give 7-deazaguanine (c 7 G) alters the electronic properties of the heterocyclic base and eliminates a potential major groove cation binding site, which affects the organization of salts and water in the major groove. This has a destabilizing effect on DNA. We report herein the characterization of DNA oligomers containing 7-(aminomethyl)-7-deazaguanine (1) residues using a variety of spectroscopic and thermodynamic approaches. 1 is an intramolecular model for the major groove binding of cations and basic amino acid residues to G. In contrast to c 7 G, the tethering of a cation in the major groove using 1 affords goldbi@pitt.edu. ¥ contributed equally Supporting Information.