In Vitro Reconstitution and Purification of Viral Nucleocapsid-Like Particles

Begin with a recombinantly expressed, RNA-free nucleoprotein–phosphoprotein complex that is specific to a virus.

Incubate the complex with virus-specific RNA oligonucleotides to initiate the assembly of nucleocapsid-like particles.

During assembly, the viral RNA displaces the phosphoprotein fragment, allowing the nucleoprotein to enclose the RNA, forming nucleocapsid-like particles.

Centrifuge the sample to remove any precipitate.

Take a pre-equilibrated size-exclusion chromatography column packed with a porous resin, and load the supernatant onto it.

As the sample flows through the column, large nucleocapsid complexes are excluded from the pores and elute first.

Medium-sized protein complexes and small RNA molecules enter the pores and elute later.

Monitor the absorbance of the eluate to generate an elution profile with distinct peaks.

The first peak shows assembled nucleocapsids, followed by smaller protein complexes and free RNA.

Collect the fraction that corresponds to the nucleocapsid peak for validation and further analysis.

For virus specific nucleocapsid assembly, mix and incubate the purified N0P complex with RNA oligo at a 1:1.5 molecular ratio at room temperature for one hour. Pre