Figure 3.

Locating encapsulated mRNA in LNPs by cryo-EM. The effect of thionine on mRNA-LNPs of different morphologies reveals that lipid-dissociated mRNA may reside in bleb compartments (a) or may be more lipid-associated in spherical (b) or less prominently blebbed particles (c). The black arrow in (a) indicates the distinctive mottled mass density of mRNA inside the bleb cavity, which itself is distinguished by a thick, dark periphery. The blue arrow indicates the significant contrast enhancement that occurs when thionine dye is present, thereby identifying mRNA within the bleb. (d) Charge-driven migration of mRNA from the bleb into the body of the LNP. When the mRNA-LNP sample of (a) (no dye) was dialyzed into pH 5 buffer, the images of (d) resulted. The mottled density is now associated with the body of the LNP (green arrow), leaving the bleb cavity devoid of mRNA (white arrow). Addition of thionine to this sample produced no notable contrast change (right), indicating that thionine did not displace lipid.