## 3 Results and Discussion
### Complexation of mCherry mRNA and Artificial Viral Capsid Bearing dT\({}_{20}\)  
We initially attempted encapsulation of mRNA within the artificial viral capsid consisting of dT\({}_{20}\)-SS-\(\beta\)-annulus via hybridization between dT\({}_{20}\) and mRNA. However, when poly(A) and dT\({}_{20}\)-SS-\(\beta\)-annulus were mixed at equimolar base concentrations, capsid formation was insufficient (data are not shown). It is probably difficult to self-assemble among dT\({}_{20}\)-SS-\(\beta\)-annulus peptides hybridized on a large poly(A) molecule due to the excluded volume effect. Therefore, we developed an alternate strategy to encapsulate mRNA with dT\({}_{20}\)-SS-\(\beta\)-annulus and unmodified \(\beta\)-annulus peptide (INHVGGTGGAIMAPVAVTRQLVGS) (Figure 1). DLS and TEM images of the mixture of dT\({}_{20}\)-SS-\(\beta\)-annulus/unmodified \(\beta\)-annulus peptide at 1:9 molar ratio in PBS showed the formation of spherical co-assemblies of size \(45\pm 20\) nm (Figure 3). The peptides mixture at other molar ratios (dT\({}_{20}\)-SS-\(\beta\)-annulus:\(\beta\)-annulus = 1:4.5, 1:2, 1:1) also formed spherical co-assemblies of similar size (Figure S2). Next, we encapsulated mRNA coding in the mCherry fluorescent protein (mCherry mRNA) by co-assembly at different molar ratios of dT\({}_{20}\)-modified and unmodified \(\beta\)-annulus peptide. Figure 2: (**A**) Reverse-phase HPLC chart of (**a**) dT\({}_{20}\)-NH\({}_{2}\), (**b**) PySS-dT\({}_{20}\) detected at 260 nm, eluted with a linear gradient of CH\({}_{3}\)CN/0.1 M NH\({}_{4}\)HCO\({}_{2}\) aq (0/100 to 100/0 over 95 min), and (**c**) dT\({}_{20}\)-SS-\(\beta\)-annulus detected at 260 nm, eluted with a linear gradient of CH\({}_{3}\)CN/0.1 M NH\({}_{4}\)HCO\({}_{2}\) aq (10/90 to 100/0 over 95 min). (**B**) MALDI TOF-MS of the purified dT\({}_{20}\)-SS-\(\beta\)-annulus (matrix: 3-HPA). (**C,D**) TEM image and size distribution obtained from dynamic light scattering (DLS) for an aqueous solution of 50 \(\upmu\)M dT\({}_{20}\)-SS-\(\beta\)-annulus in PBS (pH 7.4) at 25 \({}^{\circ}\)C.  
An aqueous solution of mCherry mRNA in PBS was added to the lyophilized powder of dT\({}_{20}\)-SS-\(\beta\)-annulus:\(\beta\)-annulus peptide followed by dilution with PBS and incubation for 30 min at 25 \({}^{\circ}\)C to prepare capsids containing mCherry mRNA. The electrophoretic mobility shift assay (EMSA) showed a band of mCherry mRNA significantly shifted by complexation with a co-assembly mixture of dT\({}_{20}\)-SS-\(\beta\)-annulus:\(\beta\)-annulus peptide at a 1:9 molar ratio (Figure 4A, lane 4). In contrast, the other co-assembly mixtures showed only a slight retardation of mRNA (Figure 4A, lane 5-7), which reflect almost neutral net charge and relatively small size of the \(\beta\)-annulus peptide. The migration position of a mixed solution of unmodified \(\beta\)-annulus peptide and mCherry mRNA was not shifted from mCherry mRNA alone (Figure 4B). DLS and TEM images of the 1:9 co-assembly complexes with mCherry mRNA show the formation of spherical assemblies of size \(52\pm 10\) nm (Figure 5A,B). We previously reported that the N-terminus of the \(\beta\)-annulus peptide is directed toward the interior of the capsid and the surface \(\zeta\)-potential of the capsid is almost zero at neutral pH [25]. Thus, when negatively charged RNA is encapsulated at neutral pH, the artificial viral capsid should minimally migrate from the applied position due to the capsid charge shield. Therefore, EMSA results indicate that the dT\({}_{20}\)-SS-\(\beta\)-annulus:\(\beta\)-annulus peptide 1:9 co-assembly encapsulated mCherry mRNA via hybridization with dT\({}_{20}\) directed to the interior of capsids. EMSA shows that mCherry mRNA could be complexed with the 1:9 co-assembly at 4 \({}^{\circ}\)C, 25 \({}^{\circ}\)C, 37 \({}^{\circ}\)C, and 60 \({}^{\circ}\)C (Figure S3). However, complexation at 4 \({}^{\circ}\)C and 60 \({}^{\circ}\)C afforded large aggregates with bimodal size distribution (Figure S4).