Tricyclic nucleobase analogues have emerged as a promising category of fluorescent bases which minimize perturbation of B-form DNA and retain fluorescence when base paired and stacked. They are useful for studying changes in structure, conformation, and dynamics of nucleic acids relevant to function, such as the dynamics of lesions and the conformational effects of protein binding to the duplex. One such analog, 8-Cl-tC^O, exhibits vibrational fine structure in its absorption and fluorescence emission spectrum upon incorporation into duplex DNA, regardless of neighboring base identity. 8-Cl-tC^O shows enhanced vibrational fine structure when base paired with guanine or adenine but exhibits a smooth fluorescence spectrum in hydrogen bonding solvents or when placed opposite an abasic site. The origin and mechanism behind this phenomenon are poorly understood. Current data suggests that the vibrational fine structure manifests only under certain conditions of hydrogen bonding interactions located at the Watson-Crick face. To evaluate this hypothesis, we are placing 8-Cl-tC^Oin a variety of base-pairing and differentially solvated environments including hairpins and single-stranded DNA oligos. Base pairing partners for 8-Cl-tC^O will consist of the four natural nucleobases in addition to inosine and alkylated nucleobases typical of damaged DNA, such as O⁶-methylguanine. Emission and time-resolve fluorescence spectra will be studied in addition to temperature-dependent CD and fluorescence anisotropy spectra. A deeper understanding of the emergence of vibrational fine structure in 8-Cl-tC^Oas a response to base pairing and stacking will enable the use of this unique spectral feature as a reporter on local environments, such as the structural and dynamic consequences of DNA mismatches and damage.

Raymond Lee

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