Fundamental Molecular Mechanisms of DNA Damage

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DNA and RNA are linear oligomers of adenosine (A), cytidine (C), and guanidine (G) nucleotides, with either uridine (U) or thymidine (T) as the fourth nucleotide in RNA and DNA, respectively. In DNA, hydrogen bonding between nucleobases on two oligomeric strands forms the iconic double-stranded B-form helix, with C pairing with G and A pairing with T. The consequent overlap and interactions between the nucleobase electronic states results in hypochromism, exciton splitting and ultrafast (sub-ps) electronic excited-state relaxation, and leads to difficulties in predicting DNA's chemical and photophysical behavior.***     DNA exhibits many damage products from such insults as UV and other radiation, reactive oxygen species and other chemicals. Our research program goal is to measure and understand molecular-level damage in nucleic acids. Our approach combines measurement of damage from fluorescent probes and multiplex platforms, with elucidation of the femtosecond (fs) initial excited-state structural dynamics and damage reaction mechanisms from resonance Raman spectroscopy.***     Most techniques for DNA damage detection suffer from either too specific or too general detection of damage, are complex or time-consuming, can only detect damage in a few sequences simultaneously, and/or introduce additional lesions. We have developed simple fluorescent mix-and-read assays of single- and double-stranded DNA damage and applied them in a microplate platform to characterize damage in over 20 oligomers simultaneously. Our assays are selective for a single damage lesion within 10 kb and have sensitivity in the fmol range. Expanding this approach to hundreds and thousands of sequences simultaneously, as proposed here, can yield a critical understanding of how damage depends on DNA structure and composition.***      Current techniques for characterizing initial excited-state structural dynamics, such as time-resolved spectroscopy, suffer from non-linear artifacts and insufficient time or spectral resolution. We use resonance Raman intensities to probe the fs initial excited-state structural dynamics of nucleic acid components. Our previous work has shown that UV-induced damage in nucleobases is highly dependent on structure, particularly the mass of nucleobase substituents. In this proposal, we continue to explore the causal relationship between nucleobase structure, initial excited-state structural dynamics and photochemistry. These experiments provide useful tests of computational models, many of which remain controversial and unproven.***     Specific objectives for this proposal include (1) characterizing the fs initial excited-state structural dynamics of nucleic acid components, (2) expanding our assays of damage to cellular DNA, and (3) developing parallel platforms to simultaneously probe sequence- and structure-dependent damage in multiple DNA sequences to a wide range of insults.**