RNA: packaged and protected by VLPs

Figures

• 

  Fig. 1. The Qβ hairpin (blue) in complex with the coat protein (two copies are shown) in the assembled Qβ VLP (PDB 4L8H).25

• 

  Fig. 2. The pCDF-CP and pET-RNA plasmids used to co-express VLP protein and RNA in E. coli. (a) The protein expression vector (pCDF-CP) encoding Qβ VLP. (b) The generic RNA production vector (pET-RNA) used to express the series of RNAs in this study. The red line on pET-RNA plasmid (b) indicates the RNA transcript. (c) The gene cloned to pCDF-1b to create pCDF-CP is Qβ coat protein. (d–h) Genes cloned to pET-28b (+) to create the series of pET-RNAs are (d) a-rRNA-hp, (e) 23S rRNA-hp, (f) RBS-mRNAGFP-hp, (g) mRNAGFP-hp, and (h) a-rRNA. ‘hp’ is Qβ hairpin; ‘RBS’ is ribosomal binding site.

• 

  Fig. 3. VLP packaging protects RNA. VLP–packaged a-rRNA-hp and free a-rRNA-hp were incubated under conditions (a) that promote Fenton chemistry (33 μM Fe-EDTA, H2O2 and atmospheric oxygen at 37 °C) at various timepoints, (b) that promote Fenton chemistry (varying [Fe-EDTA]) at constant time, (c) that promote inline cleavage (25 mM MgCl2 at 37 °C) at various timepoints, (d) that promote inline cleavage (varying [Mg2+] at 37 °C) at constant time, and (e) in the absence of divalent cations at neutral pH at various timepoints. For all experiments here, the uncleaved a-rRNA-hp was isolated on denaturing urea PAGE and quantitated with AlphaView® software. Error bars represent the 95% confidence interval of the quantitation.

• 

  Fig. 4. In vivo expressed RNA is packaged within VLPs to form VLP–RNA assemblies. (a) Purified Qβ VLPs analyzed by SDS-PAGE. The Qβ CP monomer is 14.5 kDa. Lane 1: protein sizing ladder. Lane 2: Qβ CP monomer from purified Qβ VLPs. (b) Transmission electron microscope images of Qβ VLP containing a-rRNA-hp. Scale bar = 50 nm. (c) In vivo expressed a-rRNA-hp, extracted from purified Qβ VLPs, subjected to electrophoresis on denaturing urea PAGE. Lane 1: RNA ladder, Lane 2: RNAs of Qβ CP expressed alone. Lane 3: RNAs of purified Qβ CP co-expressed with a-rRNA-hp. The open arrow indicates the a-rRNA-hp (747 nt). The closed arrow indicates the Qβ CP mRNA. (d) In vivo expressed 23S rRNA-hp, extracted from purified Qβ VLP, subjected to microfluidic-based chip electrophoresis. Lane 1: RNA sizing ladder, Lane 2: RNAs extracted from purified Qβ CP co-expressed with 23S rRNA-hp (3051 nt). Lane 3: 23S rRNA prepared by in vitro transcription (2966 nt). The open arrow in panel (d) indicates the 23S rRNA-hp. The closed arrow indicates the Qβ CP mRNA.

• 

  Fig. 5. Highly structured RNAs bearing the Qβ hp and lacking the RBS package efficiently in Qβ VLP in vivo. (a) Structured and unstructured RNAs, packaged in vivo by Qβ CP. Lane 1: RNA ladder. Lane 2: RNAs extracted from purified Qβ CP co-expressed with RBS-mRNAGFP-hp. Lane 3: RNAs from purified Qβ CP co-expressed with mRNAGFP-hp. Lane 4: RNAs from purified Qβ CP co-expressed with a-rRNA-hp. (b) RNAs with and without the Qβ hp, co-expressed in vivo with Qβ CP. Lane 1: RNA ladder. Lane 2: RNAs of purified Qβ CP co-expressed with a-rRNA-hp. Lane 3: RNAs of purified Qβ CP co-expressed with a-rRNA (without Qβ hp). Lane 4: RNAs of Qβ CP expressed alone. One hundred nanograms of total VLP–extracted RNA was loaded to each well for denaturing PAGE. Band A: RBS-mRNAGFP-hp (851 nt). Band B: mRNAGFP-hp (833 nt, without the RBS). The open arrow indicates the a-rRNA-hp (747 nt). The closed arrow indicates the Qβ CP mRNA.