Nanosized Particles Assembled by a Recombinant Virus Protein Are Able to Encapsulate Negatively Charged Molecules and Structured RNA

Figure 1. Structures of charged fluorescent molecules used in this study. Tetramethylrhodamine methyl ester (TMRM) has a net charge of +1, 5-carboxytetramethylrhodamine (TAMRA) has a net charge of 0, and fluorescein (FITC) has a net charge of −2.

Figure 2. Fluorescence emission spectra of particles assembled by the RNA-binding domain 1 of HCV core protein (p116) in the presence of charged molecules. (a) Fluorescence spectra of p116-particles assembled in the presence of FITC, TAMRA, and TMRM; (b) fluorescence spectra of p116 interacted with different amounts of FITC. In (a,b), p116 control is the particles formed by p116 alone; (c) the fluorescence intensity at 513 nm of p116-assembled particles interacted with different molar ratios of FITC.

Figure 3. Transmission electron micrographs of nanoparticles assembled by p116. (a) a TEM image of particles assembled by p116 alone. (b) a TEM image of particles assembled by p116 added with the fluorescent molecule FITC during the particle formation process. The TEM imaging was conducted with electrons with an energy of 100 KeV. The scale bar in each image represents 200 nm. According to TEM imaging, the protein is able to assemble into nanosized particles with or without interacting with the anionic molecules.

Figure 4. FITC fluorescence quenched by the quencher iodide. In the graphs, (▼) indicates the relative fluorescence intensity of free-form FITC in the presence of different quencher concentrations; (●) indicates the relative fluorescence intensity of FITC associated with p116-assembled particles in the presence of different quencher concentrations.

Figure 5. Atomic force microscopy (AFM) of the particles formed by p116 and p73. (a) An AFM image of particles assembled by p116 alone; (b) an AFM image of particles formed by p116 interacting with fluorescent tRNA during particle formation; (c) an AFM of particles assembled by p73 alone; (d) an AFM image of particles formed by p73 in the presence of fluorescent tRNA during the particle formation. In all these images, nucleocapsid-like particles were observed. The images shown are topographic images of AFM. Samples were imaged with a scan rate of 1 Hz and scan resolution of 512 × 512 pixels. The force applied by the AFM probe to the samples was 1 nN. The scale bar in each image represents 1 μm.

Figure 6. Fluorescence histograms of nucleocapsid-like particles in flow cytometry analysis. (a) Fluorescence distribution of the particles formed by p116 alone; (b) fluorescence distribution of particles formed by p116 with fluorescent-labeled tRNA; (c) fluorescence distribution of the particles formed by p73 alone; (d) fluorescence distribution of particles formed by p73 mixed with labeled tRNA.

Figure 7. tRNA fluorescence quenched by the quencher iodide. In the graphs, (—■—) indicates the relative fluorescence intensity of free-form fluorescent-labeled tRNA in the presence of different quencher concentrations; (—▼—) indicates the relative fluorescence intensity of labeled tRNA associated with p116-assembled particles in the presence of different quencher concentrations. Each point is an average of three repeats; errors are standard deviations.