DNA Photoproducts as Intrinsic Probes of Non-B DNA Conformations

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With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. John-Stephen Taylor at Washington University-Saint Louis to develop methods for establishing the existence and locations of G-quadruplexes in cells. The double-helical B-DNA structure discovered by Watson and Crick revolutionized the understanding of how genetic information is replicated and expressed as RNA and proteins in cells. G-quadruplexes are labile four-stranded DNA structures that have more recently been found, along with other non-B-DNA structures, to add new dimensions to the fundamental understanding of how the expression of genetic information is controlled. The study uses a unique reaction of G-quadruplexes with light that leads to indelible and unique molecular imprints in DNA. These distinctive imprints provide direct evidence for the existence of G-quadruplexes in cells, allowing their positions and dynamic behavior to be investigated within the human genome. The existence of G-quadruplexes and other non-B DNA structures lead to better understanding of their roles in disease development. Graduate students working on this project receive multidisciplinary training in synthetic organic chemistry, photochemistry, automated DNA synthesis, molecular and cellular biology, and mass spectrometry. In addition, teaching modules and tutorials for high school students are developed that use physical and computer models to understand G-quadruplex structures and how their photochemistry differs from that of standard B-DNA. This project builds on a long-standing program in the principal investigator's laboratory to use chemical and biophysical tools to study the details of complex nucleic acis structures, with a particular focus on G-quadruplex structures. The scientific approach takes advantage of the observation that UV irradiation of G-quadruplex-forming sequences containing thymine (T) and cytosine (C) bases in adjacent lateral loops results in the formation of stable anti-cyclobutane pyrimidine dimers (anti-CPDs), photoproducts that cannot form in canonical B-DNA. Highly sensitive methods are developed to detect these anti-CPDs photoproducts in cells using post-labeling and isotope dilution mass spectrometric methods. Their positions within the genome are located through ligation-mediated polymerase chain reaction (PCR) methods. Structure-photoreactivity relationships that serve as unique molecular signatures of G-quadruplexes and other related non-B DNA conformations are determined using enzyme-coupled mass spectrometry assays of site-specific isotopically labeled DNA. The broader impacts include presenting research opportunities to students from traditionally underrepresented communities and the development of new materials to teach concepts of DNA structure that align with educational standards.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.