Biosensing Properties of Fluorescent DNA Base Analogs

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Chemically modified DNA bases can facilitate mutations that cause serious health effects such as cancer, or produce useful fluorescent derivatives that are ideal candidates for characterizing biomolecular interactions. My NSERC-funded research program is concerned with the design and synthesis of structurally modified DNA oligonucleotides that contain fluorescent base analogs. Such oligonucleotides are utilized to expand our understanding of mutagenicity mediated by bulky DNA adducts (addition products) and serve as new biomolecular tools for sensing applications. ******Part 1 of this grant focuses on the structural and fluorescent properties of bulky DNA adducts that are produced by human carcinogens. Specifically, bulky aryl groups attached to the C8-site of 2'-deoxyguanosine (dG) produce C8-aryl-dG lesions, which is a common DNA adduct type. For these lesions, adduct conformation can dictate mutagenic potency. Novel C8-aryl-dG adducts are designed to possess fluorescent properties that can serve as a sensitive indicator of adduct conformation within the DNA duplex and when the adduct is processed by DNA polymerases. The short-term goal of this project is relate their fluorescence to adduct conformation and in vitro mutagenicity. The long-term goal is to be able to utilize fluorescent C8-aryl-dG adducts within cellular DNA to gain an understanding of how adduct conformation impacts cellular processes, such as DNA replication and DNA repair.******Part 2 of this grant examines the biosensing properties of visible fluorescent dyes within DNA aptamers. Aptamers are single-stranded oligonucleotides that bind a variety of targets with high affinity and specificity. The dyes incorporated into the DNA aptamers are designed to possess emission sensitivity to target binding, which serves as a diagnostic signal. The short-term goal of this project is to establish strategies for incorporation of fluorescent dyes site-specifically into DNA aptamers for detection of food toxins and proteins as a proof-of-concept for establishing food safety and detecting biomarkers related to disease. This involves optimization of: dye stability, site-specific emission response and detection limits. The long-term goal of this project is the development of various fluorescent aptasensors for incorporation into affordable, easy-to-use hand-held detection kits for point-of-care diagnostics. ******The work outlined is carried out by a talented group of undergraduate and graduate students who learn the basics of nucleic acid synthesis and how to characterize the molecular recognition properties of the resulting modified DNAs using a wide variety of instrumental techniques. The students receive guidance not only from the principal investigator of this application, but also from two postdoctoral fellows with expertise in nucleic acid chemistry and dye synthesis.