key: cord-305378-jmcuq9c5
authors: Chen, Hui; Cui, Yang; Han, Xuling; Hu, Wei; Sun, Min; Zhang, Yong; Wang, Pei-Hui; Song, Guangtao; Chen, Wei; Lou, Jizhong
title: Liquid–liquid phase separation by SARS-CoV-2 nucleocapsid protein and RNA
date: 2020-09-08
journal: Cell Res
DOI: 10.1038/s41422-020-00408-2
sha: 
doc_id: 305378
cord_uid: jmcuq9c5

nan

well refolded proteins were concentrated and further purified with anion exchange chromatography column and size exclusion chromatography. All proteins were quantified by A260/A280 (below 0.55) measurement to guarantee the exclusion of nucleic acid.

The N protein RNA N1541 (1541 nt; Supplementary information, Table S1 ) and A85

(85 nt; Supplementary information, Table S1 ) were in vitro transcribed and purified as previously described 2 . H20 (20 nt), A20 (20 nt) and poly-U RNA oligos of different lengths (U10, U20, U40, U60; Supplementary information, Table S1) with HEX labeling were purchased from Sangon Biotech (Shanghai China). All RNA sequences were listed in Supplementary information, Table S1 .

All the proteins were constructed with C-terminal cystine and conjugated with maleimide-Alexa fluorescence according to the manufacturer's instruction. Briefly, 80 μM proteins were buffer change into labeling buffer (1× PBS, pH 7.4, 1 mM TCEP), 1 mM C5-maleimide Alexa488 or C2-maleimide Alexa647 were added and incubated 2 h at room temperature. Excess dye was removed by using a zeba spin desalting column and finally stored at -80 °C.

Phase separation assays were performed in 1× PBS (pH 7.4) supplemented with 2.5% PEG8000 unless otherwise noted. A microwell 96-well optical-bottom plates with polymer base was coated with 1% BSA at 4 °C overnight and then washed with PBS three times. Phase separation was induced with RNA addition and then transferred in 96-well plate at room temperature for 1 h. Imaging was performed with Olympus FV1200 equipped with a 60× oil immersion objective, and 3-6 regions were randomly selected for image acquisition. All images were processed with FV10-ASW, the percentage area of the image occupied by droplets was calculated based on the method from the published research 3 .

For negative stain EM, approximately 5 μL of sample were applied to glow-discharged copper EM grids covered with a thin layer of continuous carbon film, and then the grids were stained with 5 μL of 2% uranyl acetate. The images were recorded at a magnification of 28,000, yielding a pixel size of 3.68 Å on Talos F200C 200-kV electron microscope equipped with a 4 K × 4 K Ceta camera (FEI).

Samples were prepared by mixing indicated concentration of protein and RNA.

Approximately 3 μL of sample were applied to glow-discharged holey carbon grids 

The particle size of N protein was detected by a DynaPro NanoStar (Wyatt Technology) which was equipped with a 660 nm laser, detector angle was fixed at 90°.

Every single experiment was consisted of ten independent repeats of 10-s acquirement with a 1-s interval. Particle size was characterized from signal intensity with isotropic spheres model based on cumulant fit.

Assembly of severe acute respiratory syndrome coronavirus RNA packaging signal into virus-like particles is nucleocapsid dependent

The initiation, propagation and dynamics of CRISPR-SpyCas9 R-loop complex