Measurements of BPDE-DNA adducts by solid state nonopore & deep sequencing (PQ3

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Measurements of BPDE-DNA adducts by solid state nanopore and deep sequencing (PQ3) Potent carcinogen Benzo[a]pyrene (BaP) is found in heavily polluted air, smoked food, and tobacco smoke. Once inside cells, BaP can form stable benzo[a]pyrene dihydrodiol epoxide (BPDE) DNA adducts leading to the insertion of incorrect bases during replication. BPDE-DNA adducts are considered biomarkers of exposure to BaP and increased concentrations of BPDE-DNA adducts in white blood cells and in tissues such as lung and esophageal are associated with increased risk of cancer. Current methods for BPDE adduct measurements either involve highly radioactive reagents and are non-specific, or require highly specialized equipment and prohibitively large amounts of DNA for most epidemiological studies. Novel sensors for specific biomonitoring of genomic BPDE adduct which do not require hazardous reagents, or cumbersome procedures will speed the links between BaP exposure and development of various cancers. Furthermore, it is difficult to assess the associations of adduct at specific genomic loci and various cancers. The binding of BPDE on the genomic DNA is not random. Recent reports have demonstrated a preferential distribution of the BPDE-DNA complexes on the methylated CpG dinucleotides and on the mutational hotspots in tumor suppressor p53 and ras oncogene. However, with the existing methodologies BPDE adducts can only be examined at a very limited number of genomic loci. Such investigations would require laborious techniques involving highly radioactive reagents and sequencing gels. Novel non-intensive methodologies for estimating BPDE concentrations on the genome could provide new tools for identifying potential links between BaP exposure and specific mutations and epigenetic alterations that cause cancer. The objective of this proposal is to develop new solid state sensors and novel "omic" -style methodologies for the measurement of DNA adducts. Both approaches will be specifically tuned to BPDE-DNA measurements and will have the sensitivity needed for subsequent studies in human cancer using surgical tissues without requiring prohibitively large amounts of DNA. Aim 1 will develop a solid state nanopore measurement system for single molecule detection of BPDE-DNA adducts. Aim 2 will develop a protocol for genome mapping BPDE adducts in DNA from cell lines exposed to BaP or BPDE. This research will provide the "proof of concept" and scientific evidence upon which subsequent experiments can be designed for assessing BaP exposures in specific populations. Achieving these goals will open new areas of research and provide valuable new tools for fast and detailed measurements of long and short term exposure to the BaP. The approaches introduced here can be extended to most other toxins and carcinogens that form DNA adducts. These findings will benefit diverse areas of cancer and scientific research, such as toxicology, where adducts are studied.