Editing VLP Expression and Purification (section)

== Expression&nbsp; of Qβ VLP and GFP-containing VLP ==

E. coli containing pCDF-CP vector was inoculated in NZY medium with streptomycin (50μg mL-1) and incubated overnight at 37 °C. Expression was induced by 1:100 dilution of overnight culture in 500 mL of ZYM-5052 auto-induction medium.123 Production culture was incubated 24 h at 37 °C. Cells were collected by centrifugation at 4 °C and 6500xg for 30 min. Cells were resuspended in an equal volume of Qβ buffer (10 mM MgCl2 and 20 mM Tris-HCl, pH7.5) and lysed by sonication at 40 watts for three minutes with 10s on/off intervals. The lysate was centrifuged to remove cell debris for 30 minutes at 23,430xg. 2M Ammonium sulfate precipitation followed by a 30-minute centrifugation at 23,430xg was used to obtain crude VLPs. Crude VLPs were resuspended in 1 mL Qβ buffer and extracted with 1:1 n-butanol:chloroform. Organic extraction was repeated three times. The aqueous layer was subsequently purified by step sucrose gradient ultracentrifugation (10-40% w:v) in a SW41Ti rotor (Beckman) at 40K RPM for two hours. VLPs were precipitated from the sucrose fraction with 20% w:v PEG8000. VLPs were resuspended in 25 mL of Qβ buffer, combined, and dialyzed against 2L dialysis buffer (20 mM Tris, pH 7.5 and 50 mM NaCl) overnight. This procedure was repeated an extra three times for 2 h prior to the use of VLPs in analyses. VLP concentration was assessed by Pierce BCA Protein Assay kit. GFP-containing VLP was expressed as described above using E.&nbsp; &nbsp;coli BL21(DE3) pCDF-CP/pET28-b(+)-GFP strain selected with streptomycin/kanamycin (50μg mL-1).

=== Conjugation of Dylight 633 NHS Ester to Qβ-VLPs pression of Qβ VLP and GFPcontaining<br/>VLP ===

<br/>Conjugation of DyLight 633 N-hydroxysuccinimide (NHS) ester to VLPs was performed<br/>according to manufacturer protocols with slight modifications. VLPs were dialyzed against 0.05<br/>M sodium borate buffer at pH 8.5 (labeling buffer) to remove the Tris present in Qβ buffer.<br/>VLPs (100 μL of 1 mg mL-1) were incubated with 200 μL DyLight 633 NHS ester reagent (1 mg<br/>mL-1) at room temperature for one hour. Buffer exchange was performed to remove non-reacted<br/>reagent using centrifugal filter unit with a 100 kDa molecular-weight cutoff. DyLight 633-<br/>labeled Qβ-VLPs were stored at -80 °C following GFP in vitro encapsidation.

=== In vitro encapsulation of GFP within Qβ-VLPs ===

<br/>VLPs were expressed and purified as described above. DyLight 633-labeled VLPs (10mg/mL)<br/>were incubated in disassembly buffer (20 mM Tris-HCl, 50 mM NaCl, 6M urea and 10 mM<br/>dithiothreitol) at 4 °C for one hour to disassemble VLP into CP dimers and posteriorly dialyzed<br/>against 10 mM acetic acid with 50 mM NaCl. Dialyzed CP dimers were then applied to a<br/>Sephadex G75 column and concentrated by a centrifugal filter unit (3.5 kDa MWCO).<br/>Subsequently, DyLight 633 labeled Qβ-VLPs were combined with a five-fold molar excess of<br/>GFP in dialysis tubing (3.5 kDa MWCO). VLP dimers and GFP were dialyzed against 2L of<br/>reassembling buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl) three times at 4 °C for at least 24<br/>h. Excess GFP and unassembled dimers were removed by centrifugation (100 kDa MWCO).



=== Fluorescence microscopy ===

<br/>To confirm that internalization is not affected by either attachment of fluorescent probes to the<br/>exterior or in vitro reassembly of VLP, in vivo prepared GFP-containing VLP was added to PC3<br/>cells to a final concentration of 360 nM and cells incubated without transfection reagent at 37 °C<br/>and 5% CO2 for 48 h. After incubation, the cells were washed three times with PBS to remove<br/>residual VLPs. Fluorescence images of PC3 cells were captured using a fluorescence microscope<br/>(Olympus U-LH100HG, Japan)



=== In vitro transcription of RNAilet7 ===

<br/>DNA containing the T7 promoter, the RNAilet7, and T7 terminator generated by PCR was used as<br/>template for in vitro transcription (Primer: RNAi-Fwd and RNAi-Rev, Table S1). The DNA was<br/>purified with the QIAquick PCR Purification Kit (Qiagen). Approximately 1000 ng of DNA per&nbsp;20 μL reaction volume was transcribed for 4 h at 37 °C with the MEGAscript High Yield&nbsp;Transcription Kit (Applied Biosystems), followed by incubation with TURBO DNase for 15 min<br/>to 1 h at 37 °C. RNA was pelleted by ammonium acetate precipitation, and the pellet was washed<br/>with 80% ethanol before drying via Speedvac or lyophilization. Pellets were resuspended in<br/>nuclease-free water and further purified by illustra™ NAP-5™ columns to remove<br/>unincorporated nucleotides.





co-expressed Qβ CP and the RNAi scaffold in E. coli resulted in&nbsp;overexpression of RNAi scaffold is overexpressed and accumulation in<br/>E. coli&nbsp; package inside VLPs .



=== Immunization ===

Balb/c mice (n = 3) can be immuned subcutaneously on day 0 and 14 with 25 µg proteins of AP205 fusion VLP. Dilute VLP (proteins)&nbsp; to a final volume of 200 µl in PBS, and inject 100 µl&nbsp; &nbsp;in the left and right inguinal region of each animal. Antibody titer in blood can be measure&nbsp; &nbsp;on day 14 and 21