Modular Protein Cages for Size-Selective RNA Packaging in Vivo

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Modular Protein Cages for Size-Selective RNA Packaging in Vivo Yusuke Azuma, Thomas G.W. Edwardson, Naohiro Terasaka, and Donald Hilvert J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b10798 • Publication Date (Web): 26 Dec 2017 Downloaded from http://pubs.acs.org on December 26, 2017

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Journal of the American Chemical Society

Modular Protein Cages for Size-Selective RNA Packaging in Vivo Yusuke Azuma‡, Thomas G.W. Edwardson‡, Naohiro Terasaka‡ and Donald Hilvert* Laboratory of Organic Chemistry, ETH Zurich, 8093 Zurich, Switzerland

Supporting Information Placeholder ABSTRACT: Protein cages have recently emerged as an important platform for nanotechnology development. Of the naturally existing protein cages, viruses are among the most efficient nanomachines, overcoming various barriers to achieve component replication and efficient self-assembly in complex biological milieu. We have designed an artificial system that can carry out the most basic steps of viral particle assembly in vivo. Our strategy is based on patchwork capsids formed from Aquifex aeolicus lumazine synthase and a circularly permuted variant with appended cationic peptides. These two-component protein containers self-assemble in vivo, capturing endogenous RNA molecules in a size-selective manner. By varying the number and design of the RNA-binding peptides displayed on the lumenal surface, the length of guest RNA can be further controlled. Using a fluorescent aptamer, we also show that short-lived RNA species are captured by the protein cage. This platform has potential as a model system for investigating virus assembly, as well as developing RNA regulation or sampling tools to augment biotechnology.

Viruses are striking examples of a most elegant nanotechnology. Based on widely accepted criteria for life, they are not living organisms, but rather highly specialized nanomachines that have been evolutionarily optimized to replicate using the resources available to them.1 The most vital step for successful propagation is the protection and transport of genetic information, and viruses achieve this by encapsidation of their genome by the protein shell for which it codes.2 Beyond the context of viruses, the ability to selectively package nucleic acids in a protein capsule has broad implications ranging from gene delivery to RNA biology.3-7 As such, the bottom-up reconstruction of virus-like structures from non-viral materials is not only useful to gain a deeper understanding of the physical principles governing virus assembly,8 but also to develop useful tools for biotechnology. Towards this goal we present the design and self-assembly of a non-viral protein cage that is capable of packaging RNA within its interior in a sequence-independent, size-selective manner based on electrostatic interactions in vivo. We opted to use cage-forming Aquifex aeolicus lumazine synthase (AaLS) as the starting scaffold for our nucleic acid loading system. This bacterial enzyme forms an icosahedral (T=1 state) capsid comprised of 60 identical subunits with a diameter of 16 nm.9,10 Due to extreme thermal stability, facile recombinant production, and high tolerance against both chemical and genetic modification, AaLS has been extensively employed for diverse applications including biomineralization,11 drug delivery,12 bioimaging,13 enzyme catalysis,14-16 and as a robust electrostatic encapsulation system.17-20 We have recently reported a circularly permuted variant of AaLS, cpAaLS(L8), which presents both N- and

C-termini between residues 119 and 120 on the lumenal surface and can co-assemble with wild-type AaLS (AaLS-wt) upon coexpression in E. coli to produce patchwork capsids.21 This modular system is well-suited to construct synthetic mimics of viruslike structures, as it allows control over both the number and type of nucleic acid binding domains (NBDs) presented on the interior surface (Figure 1a).

Figure 1. Design and structure of a nucleic-acid packaging protein capsule. (a) Patchwork capsid formation of AaLS-wt with cpAaLS-RX possessing an oligoarginine tag (RX) in E. coli cells, where endogenous nucleic acids are spontaneously encapsulated and protected from nuclease digestion. (b) Sequences of the three nucleotide binding tags fused to the C terminus of cpAaLS, showing linker (purple) and oligoarginine (blue). (c) Negatively stained transmission electron micrographs of AaLS-wt:cpAaLS and AaLS-wt:cpAaLS-R9 (scale bars = 50 nm). Viruses employ various mechanisms for selective packaging of cognate nucleic acid in complex biological milieu.2 While some large viruses utilize sophisticated machinery, such as the packaging motors of bacteriophages,22 others, especially small RNA viruses, rely on sequence-specific protein-nucleic acid interactions23-27 or simply electrostatics.28,29 Inspired by NBDs found in such small viruses, we employed a series of simple oligoarginines (R3, R6, R9) to drive the encapsulation of nucleic acids through electrostatic interactions. These peptide tags were genetically fused to the C-terminus of cpAaLS via a flexible seven amino acid linker, yielding cpAaLS-RX, where X specifies the number of arginine residues (Figure 1b). The cpAaLS-RX variants were co-expressed with AaLS-wt in E. coli cells, and His-tagged AaLS-wt containing assemblies were purified by Ni-NTA affinity and size-exclusion chromatography (SEC), as previously reported.21 SDS-PAGE analysis of the isolated assemblies indicated the presence of both AaLS-wt and cpAaLS-RX, in a ca. 1:1 molar ratio of each protein (Figure S1). Irrespective of arginine tag length, all variants self-assemble into cage structures with sizes corresponding to wild-type assembly, as

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judged by negative stain transmission electron microscopy (TEM), SEC, native agarose gel electrophoresis (AGE) and dynamic light scattering (DLS) (Figure 1c, S2-S8). These results are surprising based on previous engineering of AaLS, where the introduction of four glutamic acids per subunit led to drastic changes in morphology.30 Even though AaLS-wt:cpAaLS-R9 has a net charge of approximately +270, localized within the 8 nm diameter inner cavity, no such change was observed. It is likely that the greater conformational freedom of the peptide appendage, as opposed to mutations along the protein main chain, as well as the presence of charge compensating guest molecules, contributes to structural integrity. These results underscore the robustness of the AaLS-wt:cpAaLS system, as well as its suitability for producing protein cages presenting different peptide tags while retaining a consistent morphology.

Figure 2. Analysis of nucleic acid content and stability. Native agarose gel electrophoresis (AGE) of AaLS-wt (WT) and AaLSwt:cpAaLS-R9 patchwork (WT:R9) assemblies, stained with Coomassie Blue (a) or GelRed (b). Total nucleic acid (NA) from E.coli is also provided (the rightmost lane). R, D, and B correspond to prior treatment of the sample with RNase A, DNase I or benzonase, respectively. (The dark shadow around the lanes of (a) is a staining artifact.) With the assembly of stable patchwork capsids validated, we moved to investigate the loading of nucleic acid from the host cells. The A260/A280 ratio was used to determine relative nucleic acid content in each sample, and the values obtained suggested that the amount of nucleic acid depends on the length of the arginine tag (Table S1). The association of nucleic acid with the patchwork capsids was further confirmed by native AGE visualized with both Coomassie Blue for protein and GelRed for nucleic acid (Figure 2a and S3). Additionally, treatment with RNase A, DNase I and benzonase, an engineered nuclease that digests both DNA and RNA,31 resulted in no degradation or reduction in nucleic acid content, showing that the protein cages provide cargo protection. To determine the identity of the guest molecules, the nucleic acid cargo was extracted from the purified AaLSwt:cpAaLS-R9 sample and treated with each of the nucleases, verifying that the cargo consisted exclusively of RNA (Figure 2b). Furthermore, from the RNA band distribution, it was evident that a fairly narrow size range, falling between 50 and 300 nt, was captured within the patchwork capsid. Based on this initial result, we were interested to probe the potential of our system for sizeselective RNA encapsidation. Based on previous studies on the importance of the protein:RNA ratio in viral capsid assembly,32-34 it would be expected that the maximum length of encapsulated RNA molecules, in nucleotides, approaches the number of positively charged residues present in the protein cage. Thus, one would expect the maximum RNA lengths to be ca. 180 nt for appropriate charge compensation in AaLS-wt:cpAaLS-R6, and 270 nt for and AaLS-wt:cpAaLSR9 co-assemblies, respectively. Denaturing polyacrylamide gel electrophoresis (PAGE) on RNA extracts were in line with this

(Figure 3b), although the presence of a range of sizes reveals that while some capsids may contain one long RNA, others presumably contain multiple short oligonucleotides. To further investigate the possibility of controlling cargo sizeselectivity, we designed two new RNA-binding peptides for fusion to cpAaLS, GGSR9 and HlxR9, which have the same charge density as R9 but include a flexible (Gly-Gly-Ser) or rigid (Hlx, with high helical propensity)35 sixteen-residue linker between capsomer and cationic peptide (Figure 3a). These variants were expected to alter the available cavity volume, which, alongside the number of positive residues presented, is likely a crucial factor for cargo size-selection. Patchwork capsids with extended linkers, AaLS-wt:cpAaLSGGSR9 or AaLS:cpAaLS-HlxR9, were expressed at 1:1 ratios with AaLS-wt (Figure S9). Analysis by SEC, AGE, TEM and DLS confirmed capsid sizes in line with the 60-mer wild-type-like structure, further highlighting the tolerance of the AaLS scaffold (Figures S8, S10-S13). Interestingly, both AaLS-wt:cpAaLSGGSR9 and AaLS-wt:cpAaLS-HlxR9 show lower A260/A280 values of 0.98 and 0.87 (Table S1), respectively (compared to 1.18 for AaLS-wt:cpAaLS-R9), suggesting that available cavity volume is also a determinant of RNA content. Denaturing PAGE analysis of the extracted guest RNAs revealed that oligonucleotides with a range of lengths were encapsulated, with the smallest fragments around 25 nt in each case (Figure 3b). When looking at the upper size-limit of encapsulated cargo, however, clear variation was evident between different capsids, with an inverse relationship between peptide size and maximum RNA length. The upper cut-off drops from ca. 300 to 130 nt between the samples R9, GGSR9 and HlxR9. The rationale for these three variants is that by altering the length and flexibility of the peptides, we can limit the available volume of the cavity and therefore the length of cargo molecules. The RNA analysis supports this hypothesis and is a notable result, as it is feasible that at some stage in the evolutionary trajectory of viruses, sizecompatibility was a selection criterion for protein cages that could package and propagate their own genetic material.

Figure 3. Cargo size-selectivity by altering number and identity of RNA-binding tags. (a) RNA binding tags with the same net charge but different length and flexibility. (b) Denaturing PAGE analysis of RNA extracts from capsids with four different tags. (c) Denaturing PAGE on RNA extracts from AaLS-wt:cpAaLS-R9 patchwork assemblies with varying ratio of cpAaLS-R9. An advantage of the patchwork system is the capability to tune the molar ratio of AaLS-wt to cpAaLS, thus controlling the number of peptide tags presented on the lumenal surface. This is easily


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Journal of the American Chemical Society achieved by varying the concentration of the chemical inducers, in this case isopropyl β-D-1-thiogalactopyranoside (IPTG) for the gene expression of AaLS-wt, and tetracycline for cpAaLS. To investigate the effect of the number of oligoarginine tags per cage on the encapsulated cargo, we prepared a series of AaLSwt:cpAaLS-R9 co-assemblies at a fixed concentration of IPTG (0.1 mM) but different tetracycline concentrations (10-500 ng/mL) to induce expression in E. coli. After isolation of these capsids, the ratio of cpAaLS-R9 to AaLS-wt was assessed by SDS-PAGE, confirming the increase in relative cpAaLS-R9 content at higher inducer concentrations (Figure S14). All SEC, AGE and TEM data revealed that while the majority of capsids retained wild-type size, a population of larger species also formed at the highest cpAaLS-R9 to AaLS-wt ratios (Figures S15-S17). Nevertheless, DLS of purified samples revealed homogenous capsids with wild-type-like diameters (Figure S18). As expected, an increase in A260/A280, from 0.86 to 1.27 (Table S2), was observed as the content of cpAaLS-R9 in the capsid increased, again highlighting the importance of charge compensation between capsid and guest. Interestingly, the cpAaLS-R9 content of the co-assemblies does not change the maximum length cutoff of guest RNA, but changes the overall size distribution of the cargo: an enrichment of smaller fragments with increasing ratio of cpAaLS-R9 was observed (Figure 3c). In contrast to the non-RNA-binding peptide linkers, GGS and Hlx, which add steric bulk and thus limit the RNA content, these results suggest that additional polyarginines can more efficiently condense RNA, but with a preference for smaller fragments as the available space becomes more limited.

quence. This allows the tracking of two distinct RNA species, BtBrT7 and BtBr, which both contain the fluorescent core but represent different stages of RNA processing (Figure 4a). The gene encoding BtBrT7 was cloned into the plasmid for AaLS-wt, also under the control of an IPTG inducible promoter, and coexpressed with both cage component proteins in E. coli cells. In the absence of capsids, time-dependent cleavage of the T7 terminator of BtBrT7 was observed in whole cell RNA extracts, where the Broccoli-specific fluorophore (DFHBI-1T) was used to visualize the denaturing PAGE gels (Figure 4b, lanes 1 and 2). However, co-expression with AaLS-wt:cpAaLS-R9 cages resulted in a marked increase in the population of unprocessed BtBrT7 (Figure 4b, lane 3), suggesting that AaLS-wt:cpAaLS-R9 traps the pre-mature construct before it can be processed. Furthermore, RNA extracted from AaLS-wt:cpAaLS-R9 contained additional intermediate products between the precursor and mature RNA, which were not detected in the total RNA extracts (Figure 4b, lane 4, labeled I). These intermediates presumably correspond to very short-lived species during the multiple processing steps and this result demonstrates that the AaLS-based cages can capture and protect unstable and transient RNA species, which are otherwise difficult to track. In summary, we have developed a simple and efficient system for in vivo encapsulation of RNA based on patchwork capsids formed from AaLS and peptide-tethered cpAaLS proteins. By varying the number or structure of RNA-binding peptides presented on the capsid lumen, it was possible to control the sizeselectivity of cargo molecules. Experiments with Broccoli RNA demonstrated that short-lived processing intermediates can be trapped, revealing the potential of the system to function as a novel RNA sampling technology. Nature provides a bounty of molecular machines, and we strive to understand the intricacies of their assembly, structure and function as a basis for the design of our own nanodevices. An important step towards this understanding is the generation of model systems enabling systematic investigation. The RNA loading cages presented here offer such a platform: to ask fundamental questions regarding viral evolution and assembly, as well as to develop novel tools for biotechnology. Furthermore, we anticipate that through development of this naïve system guest-specific interactions could be designed, providing further insights into viral evolution and affording cargo-specific protein cages for applications in RNA regulation and delivery.

ASSOCIATED CONTENT Figure 4. Packaging of RNA processing intermediates. (a) Cartoon of RNA precursor, BtBrT7, which can be captured by the protein cage and thus protected from processing to the mature BtBr. (b). Time dependent processing of BtBrT7 to BtBr analyzed by denaturing PAGE stained for Broccoli RNA. After 20hr, the RNA extracted from the capsid shows an additional intermediate product (labelled I). Alongside the size-controlled encapsulation of endogenous RNA within the patchwork capsids, an important aspect of the system was the protection of guest RNA from nucleases. Many RNA molecules exist as transient species, being quickly degraded or processed by enzymes in cells,36-38 and the AaLS-wt:cpAaLS cages could provide a useful method for capture and protective storage of short-lived RNA intermediates in vivo. To test this possibility, we produced a model RNA containing a tRNA scaffold which induces post-transcriptional cleavage of a 3′ T7 terminator sequence by endogenous nucleases,39 and the fluorescent aptamer Broccoli, an RNA mimic of green fluorescent protein,40 for detection of a specific RNA molecule (Figure S19). This tRNA-Broccoli construct, BtBrT7, is 169 nt in length, comprising a 122 nt tRNA-Broccoli core with a 47 nt 3′ T7 terminator se-

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental procedures, A260/A280 ratios, SDS-PAGE, Native AGE, SEC, DLS and TEM data (PDF)

AUTHOR INFORMATION ‡ These authors contributed equally and are listed alphabetically.

Corresponding Author *[email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT We thank the Scientific Center for Optical and Electron Microscopy (ScopeM), ETH Zurich for help with electron microscopy experiments. This work was supported by the ETH Zurich and the



European Research Council (Advanced ERC Grant ERC-AdG2012-321295 to D.H.). T.E. and N.T. are both very grateful for Human Frontier Science Program Long-term fellowships, and Y.A. for an Uehara Memorial Foundation Research Fellowship and an ETH Zurich Postdoctoral Fellowship (co-funded by the Marie Curie Actions program).

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Modular Protein Cages for Size-Selective RNA Packaging in Vivo

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