CAREER: Structure-Specific Fluorescence Spectroscopy to Dissect Conformational Heterogeneity in Macromolecules

With support from the Chemical Measurement and Imaging Program in the Division of Chemistry, and partial co-funding from the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences, Dr. Julia Widom and her research group at the University of Oregon (UO) are developing measurement and analysis methods to resolve with high spatial and temporal resolution the diverse structures adopted by biological macromolecules. Biological macromolecules frequently transition across multiple conformational structures, and the nature and prevalence of these structures is of great significance to both their intrinsic biological functions, and their potential applications as drug targets, biomarkers, and building blocks in nanostructures. With a focus on RNA and DNA, the Widom Lab is establishing methods based on high-resolution laser spectroscopy that extract distinct signatures from macromolecules with different structures, and is expanding the toolbox of well-characterized molecular probes that can be used for such measurements. In addition, Dr. Widom is partnering with the UO undergraduate Chemistry Club to engage in outreach to middle- and high-school students in rural Oregon communities through remote, interactive chemistry activities, site visits to UO by rural students, and collaboration with the chemistry program at Eastern Oregon University. Most existing spectroscopic techniques are sensitive to either the local (Angstrom length-scale) or global (nanometer length-scale) structure of the system being measured. To overcome this limitation, Dr. Widom’s research group is developing a method based on measurement of fluorescence spectra on an ultrafast timescale, utilizing excited-state energy transfer between two probes to separate signals from tightly and loosely folded conformational subpopulations. Global fitting of the resulting time-resolved emission spectra connects the local environments of the two probes to the global conformations of the macromolecule within which they reside. Structured DNA and RNA oligomers with at least two well-defined global conformations will be used as model systems, followed by expansion of the technique to study the binding of small molecules to RNA. The Widom Lab will also investigate the suitability of different fluorescent probes for applications involving nucleic acids, analyzing their photophysical properties and their impacts on RNA structure. If successful, this project has the potential to provide new tools for the analysis of structural heterogeneity in complex chemical, biological and material systems and thus have far-reaching scientific broader impact. 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.