Sealing tape universal optical

RNA stock solutions were thawed, then annealed by heating at 95 °C for 3 min and transferring quickly on the ice. The RNAs were diluted to 1 mM concentration by their original buffer of 10 mM HEPES pH 7.0. LCD protein stock was freshly thawed and added together with the appropriate RNA solution into 1X PBS to reach a 1:2 protein:RNA molar ratio at 50 μM final concentration of LCD, in 50 μL final volume in a black 384-well clear-bottom plate (NUNC 384). The plate was covered with optical film (Corning Sealing Tape Universal Optical) and incubated in a plate reader (BMG LABTECH FLUOstar Omega) at 37 °C with shaking. Samples were taken for TEM screening after 12 h (day 1) and 4 days of incubation.

Purified LCD protein segment was diluted into 20 mM Tris pH 8.0, 300 mM NaCl buffer at 235 μM concentration. The proteins and ThT were then mixed to final concentrations of 300 μM ThT and 100 μM protein, in 1X PBS pH 7.4. A blank sample containing everything but the protein was prepared and measured as a buffer control. Fibril formation was carried out in parafilm-covered PCR tubes, incubated in a floor shaker (Torrey Pines Scientific Inc, Orbital mixing chilling/heating plate) at 37 °C, with fast mixing speed for 11 days. 30 μL of the samples were taken out of the tubes at days 1, 6, and 11of incubation and put in a black 384-well clear-bottom plate (NUNC 384) covered with optical film (Corning Sealing Tape Universal Optical) and incubated in a plate reader (BMG LABTECH FLUOstar Omega) at 37 °C, with 700 rpm double orbital shaking for 30 s before the measurement. ThT fluorescence was measured with excitation and emission wavelengths of 430 and 485 nm, respectively.

Purified NCAP protein and its segments were separately diluted into 20 mM Tris pH 8.0, 300 mM NaCl buffer at 235 μM concentration. S2hp RNA was diluted by 10 mM HEPES pH 7.0 buffer to 75 μM concentration. The proteins, RNA and ThT were then mixed to final concentrations of 300 μM ThT, 30 μM protein, and 0 or 7.5 μM RNA (as indicated in Fig. 1 and Supplementary Fig. 2), in 1X PBS pH 7.4. Blank samples containing everything but the protein were prepared. The reaction was carried out in a black 384-well clear-bottom plate (NUNC 384) covered with optical film (Corning Sealing Tape Universal Optical) and incubated in a plate reader (BMG LABTECH FLUOstar Omega) at 37 °C, with 700 rpm double orbital shaking for 30 s before each measurement. ThT fluorescence was measured with excitation and emission wavelengths of 430 and 485 nm, respectively. Measurements were made with technical triplicates for each sample. All triplicate values were averaged, and blank readings from samples without proteins were averaged and subtracted from the values of corresponding protein mixtures. The results were plotted against time. The experiment was repeated at least three times on different days.