Phase separation by the SARS-CoV-2 nucleocapsid protein: Consensus and open questions.

In response to the recent SARS-CoV-2 pandemic, a number of labs across the world have reallocated their time and resources to better our understanding of the virus. For some viruses, including SARS-CoV-2, viral proteins can undergo phase separation: a biophysical process often related to the partitioning of protein and RNA into membraneless organelles in vivo. In this review, we discuss emerging observations of phase separation by the SARS-CoV-2 nucleocapsid (N) protein-an essential viral protein required for viral replication-and the possible in vivo functions that have been proposed for N-protein phase separation, including viral replication, viral genomic RNA packaging, and modulation of host-cell response to infection. Additionally, since a relatively large number of studies examining SARS-CoV-2 N-protein phase separation have been published in a short span of time, we take advantage of this situation to compare results from similar experiments across studies. Our evaluation highlights potential strengths and pitfalls of drawing conclusions from a single set of experiments, as well as the value of publishing overlapping scientific observations performed simultaneously by multiple labs.

Aptamer/antibody sandwich method for digital detection of SARS-CoV2 nucleocapsid protein.

Nucleocapsid protein (N protein) is the most abundant protein in SARS-CoV2 and is highly conserved, and there are no homologous proteins in the human body, making it an ideal biomarker for the early diagnosis of SARS-CoV2. However, early detection of clinical specimens for SARS-CoV2 remains a challenge due to false-negative results with viral RNA and host antibodies based testing. In this manuscript, a microfluidic chip with femtoliter-sized wells was fabricated for the sensitive digital detection of N protein. Briefly, ß-galactosidase (ß-Gal)-linked antibody/N protein/aptamer immunocomplexes were formed on magnetic beads (MBs). Afterwards, the MBs and ß-Gal substrate fluorescein-di-ß-d-galactopyranoside (FDG) were injected into the chip together. Each well of the chip would only hold one MB as confined by the diameter of the wells. The MBs in the wells were sealed by fluorocarbon oil, which confines the fluorescent (FL) product generated from the reaction between ß-Gal and FDG in the individual femtoliter-sized well and creates a locally high concentration of the FL product. The FL images of the wells were acquired using a conventional inverted FL microscope. The number of FL wells with MBs (FL wells number) and the number of wells with MBs (MBs wells number) were counted, respectively. The percentage of FL wells was calculated by dividing (FL wells number) by (MBs wells number). The higher the percentage of FL wells, the higher the N protein concentration. The detection limit of this digital method for N protein was 33.28 pg/mL, which was 300 times lower than traditional double-antibody sandwich based enzyme-linked immunosorbent assay (ELISA).