Final Report Summary - DNA-AMP (DNA Adduct Molecular Probes: Elucidating the Diet-Cancer Connection at Chemical Resolution) DNA adducts are potentially powerful personalized biomarkers for carcinogen exposure and cancer susceptibility, however, their extremely low physiological levels make it difficult to relate their presence and biological influence with the mutation and disease risk. Our research focuses on establishing unconventional approaches to detect and evaluate the contribution of nitrosamine-DNA adducts to mutation induction. The aims of the project were threefold, centering on the creation, characterization and applications of a novel class of DNA modification devised to bind selectively with health-relevant DNA adduts. The first aim of the project was to elucidate the basis of stability and determine optimal chemical structures for DNA adduct:synthetic nucleoside probe base pairs. Several novel nucleobase surrogates were designed and synthesized in the course of the research. It was established that while limited stabilization of adduct-containing DNA was achieved on the basis of complementary hydrogen bonding, hydrophobic aromatic ring systems were most effective in the stabilization of targets. Furthermore, an optimal orientation for such structures was identified and insight into the physical basis of the stability was obtained, involving inter-strand pi-stacking interactions as a major contributor. The second aim was to characterize the mechanism of polymerase-mediated adduct-probe paired DNA synthesis and establish polymerase-mediated strategies for detecting low-level DNA alkylation in biological samples. First, a mechanistic characterization of this process was needed to define scope, in terms of nucleoside structure, and provide a sound basis for the design of new bioanalytical methodologies that rely on synthetic nucleoside probes. In this regard, tremendous progress was made regarding both the insertion and extension process of DNA synthesis on modified templates, and in a breakthrough result, we reported the first demonstration of amplifying alkylated DNA, accomplished by combining a novel polymerase and synthetic triphosphate that is templated by the DNA adduct and serves as a marker for DNA damage in its copies. The results were published in the Journal of the American Chemical Society and received notable media attention in Europe and the USA (for example: Damaged DNA amplified, https://www.ethz.ch/en/news-and-events/eth-news/news/2015/01/damaged-dna-amplified.html). The final aim was to create and validate DNA hybridization probes for sequence-specific detection of a bulky DNA adducts in p53. We first established a fundamental basis for sequence-specific reversible aggregation of gold nanoparticles. A colorimetric probe for the detection of a mutagenic DNA adduct within a sequence was created. The probe involves incorporation of a synthetic nucleoside that selectively pairs opposite a target DNA adduct into oligonucleotides conjugated to gold nanoparticles. A major implication of this result is the first reported means for detecting DNA adducts within a DNA sequence and in a mixture with unmodified DNA. The broad impacts of this research is a fundamental understanding of physical and chemical interactions that dictate how chemicals alter the structure and properties of DNA as well as a basis for advancing biomonitoring strategies for detecting DNA adducts in biospecimins as a prognostic marker for cancer risk.
