Our research in the area of biochemical toxicology focuses on the structural and biological impact of C8-aryl-2′-deoxyguanosine (C8-aryl-dG) adducts (addition products) that are a common lesion type and may contain N-, O- or C-aryl linkages (denoted N-, O- and C-linked adducts). For N-linked adducts, three common structural motifs in duplex DNA (i.e., the major-groove B-type, the intercalated base-displaced stacked S-type, or the minor-groove wedge W-type) have been characterized. In general, N-linked C8-aryl-dG adducts that exhibit potent mutagenicity have planar polycyclic structures and favor the S-type or W-type duplexes with the bulky lesion in the syn-conformation. N-linked C8-aryl-dG lesions also have the propensity to cause two-base deletions within XCG (X = adduct) sequences. The long-term goal of this project is to advance our fundamental understanding of how bulky C8-aryl-dG adducts containing various linkage types are processed in cells. To achieve this goal, synthetic organic chemistry is utilized to convert dG into various C8-aryl-dG adducts. The modified nucleoside is converted into a phosphoramidite and incorporated into oligonucleotide substrates using solid-phase DNA synthesis to afford adducted DNAs containing a single C8-aryl-dG adduct. Optical spectroscopies (UV-vis, fluorescence and circular dichroism) are then used to determine the impact of the lesion on duplex DNA. The adducted DNAs are also used as substrates in primer-extension assays with various DNA polymerase enzymes to determine the biological impact of the modified base. Students interested in molecular toxicology, organic and biochemistry, are encouraged to read some of our recent publications and inquire about possible projects in this area of biochemical toxicology.
|Manderville, R.A.; Wetmore, S.D. Understanding the Mutagenicity of O-Linked and C-Linked Guanine DNA Adducts: A Combined Experimental and Computational Approach. Chem. Res. Toxicol. 2017, 30, 177-188.|
|Manderville, R.A.; Wetmore, S.D. Mutagenicity of Ochratoxin A: Role for a Carbon-Linked C8-Deoxyganosine Adduct? J. Agric. Food Chem. 2017, 65, 7097-7105.|
|Sproviero, M.; Verwey, A.M.R.; Witham, A.A.; Manderville, R.A.; Sharma, P.; Wetmore, S.D. Enhancing Bulge Stabilization through Linear Extension of C8-Aryl-Guanine Adducts to Promote Polymerase Blockage or Strand Realignment to Produce a C:C Mismatch. Chem. Res. Toxicol. 2015, 28, 1647-1658.|
|Witham, A.A.; Verwey, A.M.R.; Sproviero, M.; Manderville, R.A.; Sharma, P.; Wetmore, S.D. Chlorine functionalization of a model phenolic C8-guanine adduct increases conformational rigidity and blocks extension by a Y-family DNA polymerase. Chem. Res. Toxicol. 2015, 28, 1346-1356.|
Sproviero, M.; Verwey, A.M.R.; Rankin, K.M.; Witham, A.A.; Soldatov, D.V.; Manderville, R.A.; Fekry, M.I.; Sturla, S.J.; Sharma, P.; Wetmore, S.D. Structural and biochemical impact of C8-aryl-guanine adducts within the NarI recognition DNA sequence: influence of aryl ring size on targeted and semi-targeted mutagenicity. Nucleic Acids Res. 2014, 42, 13405-13421.
Fluorescence is one of the most powerful bioanalytical methods and fluorescent biosensors are designed to change their fluorescent intensities or wavelengths in response to physiological changes including pH, solvent polarity, viscosity, redox reactions, metal ions and apoptosis. This aspect of our research focuses on the utility of internal fluorescent DNA bases to report molecular target binding by DNA aptamers. DNA aptamers are selected in vitro from random libraries for their ability to bind molecular targets with high affinity and specificity. Modification of DNA aptamers with internal fluorescent replacements can be used for diagnostics. The challenge is to select a fluorescent dye with sufficient brightness, chemical and photochemical stability, and emission sensitivity to target binding without perturbing aptamer affinity for the target. Our published research in this area has focused on defining the fluorescent sensing properties of aryl-modified nucleobases within DNA aptamers. While these bases undergo excitation in the UV (320-350 nm) and lack sufficient brightness for real-life applications, we demonstrated their utility for monitoring aptamer-target binding through a change in DNA topology (duplex→G-quadruplex (GQ) exchange) and through direct interaction of the modified nucleobase with the target. We are now in the process of synthesizing and testing visibly emissive dyes that undergo changes in excitation wavelength and emission intensity upon aptamer binding to protein targets. Students interested in organic chemistry and biochemistry are encouraged to read some of our recent publications and inquire about possible projects in this area of research.
|Fadock, K.L.; Manderville, R.A. DNA Aptamer-Target Binding Motif Revealed Using a Fluorescent Guanine Probe: Implications for Food Toxin Detection. ACS Omega 2017, 2, 4955-4963.|
|Manderville, R.A.; Wetmore, S.D. C-Linked 8-Aryl Guanine Nucleobase Adducts: Biological Outcomes and Utility as Fluorescent Probes. Chem. Sci. 2016, 7, 3482-3493.|
|Cservenyi, T.Z.; Van Riesen, A.J.; Berger, F.D.; Desoky, A.; Manderville, R.A. A Simple Molecular Rotor for Defining Nucleoside Environment within a DNA Aptamer-Protein Complex. ACS Chem. Biol. 2016, 11, 2576-2582.|
|Fadock, K. L., Manderville, R. A., Sharma, P., and Wetmore, S. D. Optimization of fluorescent 8-heteroaryl-guanine probes for monitoring protein-mediated duplex→G-quadruplex exchange. Org. Biomol. Chem. 2016, 14, 4409-4419.|
Blanchard, D.J.M.; Cservenyi, T.Z.; Manderville, R.A. Dual fluorescent deoxyguanosine mimics for FRET detection of G-quadruplex folding. Chem. Commun. 2015, 51, 16829-16831.