Analyzing Structural Heterogeneity in RNA Using Fluorescence-Detected Circular Dichroism Spectroscopy
Julia R Widom1,2, Taylor L Coulson3
1Department of Chemistry and Biochemistry, University of Oregon, Eugene, OR, USA. jwidom@uoregon.edu.
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Circular dichroism (CD) spectroscopy is widely used to assess the conformations of biological macromolecules, while its fluorescence-detected variant, fluorescence-detected CD (FDCD), provides structural information that is specific to the fluorescent species in a heterogeneous system. We report a detailed protocol for measuring FDCD spectra using the widespread Jasco J-1500 CD spectrophotometer and discuss experimental considerations specific to RNA. We also report procedures for processing and analyzing the resulting data, which are applicable to data recorded on any instrument. Data may be interpreted qualitatively under the simple guideline that a larger FDCD than CD signal means that species with large CD signals are brighter than those with weak CD signals. In addition, established theory can be used to determine the prevalence of different structures from measurements of FDCD and other spectroscopic quantities such as CD and relative fluorescence quantum yield, or to solve for the CD spectra of individual conformational subpopulations.
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