RNA: packaged and protected by VLPs

Abstract

Virus Like Particles (VLPs) are devices for RNA packaging, protection and delivery, with utility in fundamental research, drug discovery, and disease treatment. Using E. coli for combined expression and packaging of non-viral RNAs into Qβ VLPs, we investigated the extent of chemical protection conferred by packaging of RNA in VLPs. We also probed relationships between packaging efficiency and RNA size, sequence and intrinsic compaction. We observe that VLP packaging protects RNA against assault by small diffusible damaging agents such as hydroxyl radicals and divalent cations. By contrast, the extent of unmediated cleavage, in the absence of reactive species, is the same for RNA that is free or packaged within VLPs, and is very slow. In vivo packaging of RNA within VLPs appears to be more efficient for intrinsically compact RNAs, such as rRNA, and less efficient for unstructured, elongated RNA such as mRNA. Packaging efficiency is reduced by addition of the ribosome binding site to a target RNA. The Qβ hairpin is necessary but not sufficient for efficient packaging.

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

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-mRNA_GFP-hp, (g) mRNA_GFP-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-mRNA_GFP-hp. Lane 3: RNAs from purified Qβ CP co-expressed with mRNA_GFP-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-mRNA_GFP-hp (851 nt). Band B: mRNA_GFP-hp (833 nt, without the RBS). The open arrow indicates the a-rRNA-hp (747 nt). The closed arrow indicates the Qβ CP mRNA.