DNA-Dye Sensitized Photodamage Quantification Under Single Molecule Imaging Conditions (2011)

Undergraduates: Catherine Dial, Michael Tycon

Faculty Advisor: Christopher Fecko
Department: Chemistry

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Single-molecule imaging (SMI) studies allow for the visualization of individual DNA molecules. When combined with tracking of DNA binding proteins, it can provide information on their interactions. It is known that a fluorophores can transfer energy from its excited state to the DNA molecule, resulting in photodamage. Two forms are single strand breaks (ssb) and double strand breaks (dsb) along the DNA backbone, and it is the purpose of this study to quantify these forms of damage under condition relevant to the design and implementation of SMI. To mimic SMI conditions, supercoiled DNA was stained with YOYO-1, a common fluorescent dye, and subsequently irradiated. The difference between ssb and dsb in supercoiled DNA can be separated and quantified using gel electrophoresis. To study the extent of dye-mediated energy transfer, various DNA nucleotide to fluorescent dye molar ratios were mixed and irradiated for varying exposure times. It was found that increasing the quantity of dye bound to DNA increases the amount of damage. Increased irradiation times showed a similar damage dependence. To model damage reduction systems used in SMI, three oxygen scavengers- ?-mercaptoethanol, deoxygenated buffer and ascorbic acid were used. The use of ?-mercaptoethanol and ascorbic acid were shown to decrease the amount of damage. From this work it can be observed that DNA imaging conditions induce unintentional photodamage that may have to be accounted for in the design of SMI experiments.