Fig. 5. Single-droplet normal Raman spectra of FUS in the presence of RNA.

a Average single-droplet normal Raman spectra in the presence of 25 ng/μL, 50 ng/μL, and 100 ng/μL poly-U RNA (spectra recorded at 500 mW laser power with a ×100 objective; number of droplets, n = 3). Solid lines represent the mean, whereas shaded region represents the standard deviation (n = 3). All spectra are normalized with respect to the phenylalanine ring breathing band at 1003 cm⁻¹ marked by an asterisk. b Stacked average single-droplet normal Raman spectra for different concentrations of RNA are shown in blue (0 ng/μL RNA), red (25 ng/μL RNA), purple (50 ng/μL RNA), and olive (100 ng/μL RNA) for comparison (the dotted line shows the shift in amide I in the presence of RNA). c Shoulder band at ~782 cm⁻¹ indicated by a red arrow corresponding to the uracil ring breathing mode for FUS droplets in the presence of 100 ng/μL RNA. d Another RNA marker band at ~1230 cm⁻¹ corresponding to the uracil ring stretching mode. e Raman difference plot (between 0 ng/ μL RNA and 100 ng/ μL RNA) of single-droplet normal Raman spectra of droplets in the absence and presence of 100 ng/μL RNA (arrows indicate the differences of interest). Arrows at 780 cm⁻¹ and 1230 cm⁻¹ represent the RNA marker bands. Arrows at 1325 cm⁻¹ and 1660 cm⁻¹ denotes greater α-helical content within FUS droplets in the absence of RNA. f Linear plot of RNA marker band at 1230 cm⁻¹ versus concentration of RNA (ng/μL) used. g Plot of ratio of intensity at 1230 cm⁻¹ and 1450 cm⁻¹ versus RNA: protein. h Zoomed in amide I region for FUS droplets at different concentrations of RNA indicating blue-shift of band maxima. i Zoomed in amide III region band for FUS droplets in the absence and presence of RNA (100 ng/μL). Arrow indicates a shoulder at ~1248 cm⁻¹ denoting small contribution of β-sheets in addition to random-coil structures within droplets in the absence of RNA. See “Methods” for details of data acquisition, processing, and analysis.