Fig. 4: Plugged antibody nanoparticles electrostatically package protein and nucleic acid cargoes.
From: Computational design of non-porous pH-responsive antibody nanoparticles
a, Designed positively charged trimer variants were assembled by incorporating the designed tetramer, pegRNA and antibody to EGFR (α-EGFR) to form O432-17(+) nucleocapsids. b,c, O432-17(+) nucleocapsids and O42.1 assembled in vitro with RNA were treated with Benzonase or RNAse A for 1 h, electrophoresed on non-denaturing 0.8% agarose gels and stained with SYBR Gold (b; nucleic acid) and Coomassie (c; protein). d, Designed negatively charged trimer variants were assembled with designed tetramer, pos36GFP and human Fc to form O432-17(–) nanoparticles with in vitro-packaged pos36GFP. e,f, SEC chromatograms of in vitro packaging reactions involving O432-17(–) and pos36GFP were performed in either 200 mM NaCl (e) or 1 M NaCl (f). g, As a comparison, SEC chromatograms of in vitro-assembled O42.1 with pos36GFP in 200 mM NaCl showed no co-migration of pos36GFP. Absorbance was monitored at 280 nm (black) and 488 nm (green). h, O432-17(–) nanoparticles for in vitro release of pos36GFP were assembled with negatively charged trimer, designed tetramer, pos36GFP and a 1:1 mixture of a Myc-targeted monoclonal antibody (α-Myc) and mRuby2-Fc. i, As a comparison, O42.1 was assembled in vitro with O42.1 C4, pos36GFP and a 1:1 mixture of antibody to Myc and mRuby2-Fc. j, Experimental design for in vitro release of encapsulated pos36GFP in acidic conditions. Assembled nanoparticles were immobilized on Myc-peptide-coated S. cerevisiae yeast cells. Cells and supernatant were collected by centrifugation and resuspended and incubated in acidic conditions. The supernatant containing released components and cargo was buffer exchanged to pH 8, and fluorescence intensity was analyzed. k, Fluorescence intensity of the supernatant of O432-17(–) and O42.1 assembled with pos36GFP and mRuby2-Fc before and after acidic incubation. mRuby2-Fc fluorescence was used as an indicator of nanoparticle assembly; a positive fluorescence signal indicates nanoparticle disassembly. Positive pos36GFP fluorescence indicates release of pos36GFP cargo. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P < 0.0001, two-way ANOVA with Tukey’s correction for multiple comparisons. n.s., not significant. Data are presented as mean values ± s.e.m. and measured over three independent samples in duplicate (Supplementary Table 6). UCSF ChimeraX 1.6 (ref. 54) and the PyMOL Molecular Graphics System version 2.5 (Schrödinger) were used to create a, d, h and i. j was created using BioRender. GraphPad Prism version 9.3.1 (GraphPad Software) was used to create k. RFUs, relative fluorescence units.
