Yijin Liu, David M.J. Lilley Biophysical Journal

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Crystal Structures of Cyanine Fluorophores Stacked onto the End of Double-Stranded RNA Yijin Liu, David M.J. Lilley Biophysical Journal Volume 113, Issue 11, Pages 2336-2343 (December 2017) DOI: 10.1016/j.bpj.2017.10.002 Copyright © 2017 Biophysical Society Terms and Conditions

Figure 1 The chemical structures of Cy3 and Cy5 (left), and the sequence of the 33 nt stem-loop derived from 5S rRNA (right). Four forms of the RNA have been studied, with the four possible terminal basepairs. The fluorophores are connected to the 5′-terminus of the RNA (Cy) via one of the C3 linkers attached to the indole nitrogen atom. Biophysical Journal 2017 113, 2336-2343DOI: (10.1016/j.bpj.2017.10.002) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 2 The crystal structure of one RNA-protein complex with Cy3 attached to the 5′-terminus of the RNA. A parallel-eye stereoscopic view of the structure is shown, with the L5 protein depicted in cartoon mode with α-helices (yellow) and β-sheet (cyan). To see this figure in color, go online. Biophysical Journal 2017 113, 2336-2343DOI: (10.1016/j.bpj.2017.10.002) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 3 Cy3 at the RNA-protein interface. Cy3 is colored orange, and the terminal basepair of the RNA (U26-A58) colored green. The arch of the adjacent L5 protein molecule comprising amino acids 106–120 shown in blue encloses a cavity in which the fluorophore is located. The FO-FC electron density omit map is shown for the key elements of the structure. (A) A parallel-eye stereoscopic side view of Cy3 stacked on the end of the RNA in the complex, showing the arch of the protein. (B) Top view of the Cy3 stacked on the U26-A58 basepair. To see this figure in color, go online. Biophysical Journal 2017 113, 2336-2343DOI: (10.1016/j.bpj.2017.10.002) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 4 The structure of Cy5 attached to the RNA. Cy5 is colored orange, and the terminal basepair of the RNA (C26-G58) colored green. The FO-FC electron density omit map is shown for the fluorophore, and for the terminal basepair (in (C) only). Parallel-eye stereoscopic views are shown. (A and B) Cy5 stacked on the C26-G58 basepair at the RNA-protein end of the RNA in the complex. Side view (A) and top view (B). (C) Top view looking down onto the Cy5 stacked onto the terminal basepair at the RNA-RNA interface. Electron density is only visible for the distal indole ring stacked on G58. To see this figure in color, go online. Biophysical Journal 2017 113, 2336-2343DOI: (10.1016/j.bpj.2017.10.002) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 5 The locations of Cy3 (A) and Cy5 (B) observed stacked on the terminal basepair of the RNA helix in the crystal, compared with Cy5 stacked on the terminal basepair of a DNA helix observed previously by NMR (C) (10). To see this figure in color, go online. Biophysical Journal 2017 113, 2336-2343DOI: (10.1016/j.bpj.2017.10.002) Copyright © 2017 Biophysical Society Terms and Conditions