MS2 Viruslike Particles: A Robust, Semisynthetic Targeted Drug Delivery Platform

We show that viruslike particles (VLPs) reassembled <i>in vitro</i> with the RNA bacteriophage MS2 coat protein and an RNA conjugate encompassing a siRNA and a known capsid assembly signal can be targeted to HeLa cells by covalent attachment of human transferrin. The siRNA VLPs protect their cargoes from nuclease, have a double-stranded conformation in the capsid and carry multiple drug and targeting ligands. The relative efficiency of VLP reassembly has been assessed, and conditions have been determined for larger scale production. Targeted VLPs have been purified away from unmodified VLPs for the first time allowing improved analysis of the effects of this synthetic virion system. The particles enter cells via receptor-mediated endocytosis and produce siRNA effects at low nanomolar concentrations. Although less effective than a commercial cationic lipid vector at siRNA delivery, the smaller amounts of internalized RNA with VLP delivery had an effect as good as if not better than the lipid transfection route. This implies that the siRNAs delivered by this route are more accessible to the siRNA pathway than identical RNAs delivered in complex lipid aggregates. The data suggest that the MS2 system continues to show many of the features that will be required to create an effective targeted drug delivery system. The fluorescence assays of siRNA effects described here will facilitate the combinatorial analysis of both future formulations and dosing regimes

Constraints on the RNA organization consistent with the tomogram.

<p>Each possible RNA organization is characterized by which long edges (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004146#pcbi.1004146.g001" target="_blank">Fig. 1B</a>, purple edges) are occupied in the polyhedral shell of the icosahedrally-averaged density. Long connections are labelled by the numbers of the five-fold vertices (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004146#pcbi.1004146.g001" target="_blank">Fig. 1D</a>) they connect (x•y connecting five-fold vertices x and y). Constraints imposed in the analysis are indicated in the first row, with green indicating an occupied edge, and red an unoccupied edge. The five paths meeting these constraints are characterized according to occupied and non-occupied edges. The last row shows edges shared by all five paths.</p

Hamiltonian path solution identified by the method.

<p>(A) The best match with the C5 averaged data (Path 4) starts and finishes at vertex 5 adjacent to the MP (cyan). Following the colouring convention in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004146#pcbi.1004146.g003" target="_blank">Fig. 3</a>, red dashed lines show unoccupied and green lines occupied constraints; other occupied connections implied by our analysis are shown in black. The position of TR, the strongest PS, is denoted in yellow [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004146#pcbi.1004146.ref015" target="_blank">15</a>]; heterodimers are coloured in green-blue and homodimers in pink. (B) An alternative embedding of the same (geometric) path with a different orientation relative to MP. The path (Path 3) starts and finishes at vertex 9; hence the occupation of the connections differs from Path 4 in (A), even though the overall geometry of the path is the same.</p

Classification of polyhedral edges as occupied and non-occupied.

<p>A comparison of the density profiles of the sampled long edge connections. The mean of a fitted normal distribution (y-axis) is scattered with a skewness parameter (x-axis). Connections with negative skew are disregarded as no statement about occupancy can be deduced in this case. From the remainder, two groups of four and five connections are identified as occupied (in the green circle) and non-occupied (red circle), respectively. These are used as constraints in the analysis.</p

The model system—bacteriophage MS2.

<p>(A) The viral capsid is formed from 60 asymmetric and 29 symmetric copies of the CP dimers, with one MP that takes the place of a symmetric dimer (PDBID 2MS2). The genomic RNA is organized inside the particles in two shells, with the outer shell adopting the shape of a polyhedral cage in icosahedrally-averaged reconstructions. (B) Depiction of the polyhedral cage, showing long (purple) and short (orange) PS-PS RNA connections. (C) Asymmetrically averaged tomogram of bacteriophage MS2 bound to its receptor, the bacterial F-pilus. The portion of the electron density corresponding to the CP shell (and bacterial pilus) is shown in blue; green depicts the density for genomic RNA (and presumably some elements of the MP), which forms the basis for the analysis described in this study. The RNA density forms a shell that is intimately associated with the inside surface of the capsid. (D) A planar representation of protein container and polyhedral RNA organization, showing the relative positions of the 60 polyhedral vertices (PS positions, indicated as yellow circles) in contact with the 60 asymmetric CP dimers.</p

Symmetry averaging identifies Path 4 as the correct solution.

<p>C5-averaged densities in 1-D projection for tomographic data and the path solutions listed in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004146#pcbi.1004146.g003" target="_blank">Fig. 3</a> are compared. The vertical axis shows the radial distance from the centre of the capsid in angstrom, and the horizontal axis corresponds to the C5-averaged density at that radial distance in arbitrary units; density profiles for tomogram and path solutions are normalized by equalizing the maximum densities. Density profiles are shown for: (A) the average of all possible 40,678 Hamiltonian paths; (B) the average of all paths consistent with RNA interaction with the MP; (C-G) the density profiles for the five paths in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004146#pcbi.1004146.g003" target="_blank">Fig. 3</a> individually; (H) the C5 cryo-EM reconstruction from the tomogram, adapted from [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004146#pcbi.1004146.ref020" target="_blank">20</a>]. Path 4 (cf. <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004146#pcbi.1004146.g005" target="_blank">Fig. 5A</a>), identical to Path 3 (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004146#pcbi.1004146.g005" target="_blank">Fig. 5B</a>) from a geometric point of view but positioned differently within the density with respect to MP, provides the closest fit with the cryo-EM data.</p

Toggled RNA Aptamers Against Aminoglycosides Allowing Facile Detection of Antibiotics Using Gold Nanoparticle Assays

We have used systematic evolution of ligands by exponential enrichment (SELEX) to isolate RNA aptamers against aminoglycoside antibiotics. The SELEX rounds were toggled against four pairs of aminoglycosides with the goal of isolating reagents that recognize conserved structural features. The resulting aptamers bind both of their selection targets with nanomolar affinities. They also bind the less structurally related targets, although they show clear specificity for this class of antibiotics. We show that this lack of aminoglycoside specificity is a common property of aptamers previously selected against single compounds and described as “specific”. Broad target specificity aptamers would be ideal for sensors detecting the entire class of aminoglycosides. We have used ligand-induced aggregation of gold-nanoparticles coated with our aptamers as a rapid and sensitive assay for these compounds. In contrast to DNA aptamers, unmodified RNA aptamers cannot be used as the recognition ligand in this assay, whereas 2′-fluoro-pyrimidine derivatives work reliably. We discuss the possible application of these reagents as sensors for drug residues and the challenges for understanding the structural basis of aminoglycoside-aptamer recognition highlighted by the SELEX results