Compartmentalization is one of the defining features of life. Through intracellular spatial control, cells are able to organize and regulate their metabolism. One of the most broadly used ...organizational principles in nature is encapsulation. Cellular processes can be encapsulated within either membrane-bound organelles or proteinaceous compartments that create distinct microenvironments optimized for a given task. Further challenges addressed through intracellular compartmentalization are toxic or volatile pathway intermediates, slow turnover rates and competing side reactions. This review highlights a selection of naturally occurring membrane- and protein-based encapsulation systems in microbes and their recent applications and emerging opportunities in synthetic biology. We focus on examples that use engineered cellular organization to control metabolic pathway flux for the production of useful compounds and materials.
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•Cells use intracellular compartmentalization to organize and regulate metabolism.•Encapsulation in membrane- and protein-based compartments is widespread in prokaryotes.•Bacterial microcompartments, viral capsids and ferritins have been used to engineer synthetic compartmentalization.•Membranous prokaryotic compartments (acidocalcisomes, pirellulosomes, thylakoids and magnetosomes) represent interesting systems for future engineering efforts.
Ribonuclease S (RNase S) is an enzyme that exhibits anticancer activity by degrading RNAs within cancer cells; however, the cellular uptake efficiency is low due to its small molecular size. Here we ...generated RNase S‐decorated artificial viral capsids with a size of 70–170 nm by self‐assembly of the β‐annulus‐S‐peptide followed by reconstitution with S‐protein at neutral pH. The RNase S‐decorated artificial viral capsids are efficiently taken up by HepG2 cells and exhibit higher RNA degradation activity in cells compared with RNase S alone. Cell viability assays revealed that RNase S‐decorated capsids have high anticancer activity comparable to that of standard anticancer drugs.
RNase S‐decorated artificial viral capsid that exhibited enzyme activity were constructed by reconstituting β‐annulus‐S‐peptide with S‐protein at neutral pH. The RNase S‐decorated capsids taken up by HepG2 cells exhibited higher RNA degradation and anticancer activity in cells compared with RNase S alone.
Noroviruses are the main cause of viral gastroenteritis with new variants emerging frequently. There are three norovirus genogroups infecting humans. These genogroups are divided based on the ...sequence of their major capsid protein, which is able to form virus-like particles (VLPs) when expressed recombinantly. VLPs of the prototypical GI.1 Norwalk virus are known to disassemble into specific capsid protein oligomers upon alkaline treatment. Here, native mass spectrometry and electron microscopy on variants of GI.1 and of GII.17 were performed, revealing differences in terms of stability between these groups. Beyond that, these experiments indicate differences even between variants within a genotype. The capsid stability was monitored in different ammonium acetate solutions varying both in ionic strength and pH. The investigated GI.1 West Chester isolate showed comparable disassembly profiles to the previously studied GI.1 Norwalk virus isolate. However, differences were observed with the West Chester being more sensitive to alkaline pH. In stark contrast to that, capsids of the variant belonging to the currently prevalent genogroup GII were stable in all tested conditions. Both variants formed smaller capsid particles already at neutral pH. Certain amino acid substitutions in the S domain of West Chester relative to the Norwalk virus potentially result in the formation of these T = 1 capsids.
The self-assembling process of a simple 3D molecular model that reproduces the hexagonal lattice that HIV-1 has in its immature stage is presented. The system is made of two overlapped repulsive ...spheres as a building block, both spheres are decorated with attractive patches simulating the main HIV-1 Gag interactions. If the overlapped spheres have the same diameter size, a flat lattice is assembled, while a curvature is induced in the resulting lattice when different diameters are used. Thus, the importance of this model lies on the fact that only by changing the overlapped spheres diameters it is possible to form icosahedral capsids according to the Kaspar and Klug model, known as T = 1, T = 3 and T = 4 without the use of any scaffold, such as the surface of a sphere. Only one molecular species is used to assemble hexagons and pentagons. WCA potential is used for repulsive particles and LJ for the attractive ones. Only two kinds of patches are needed to form a capsid. Two patches to form pairs, which form hexagons and pentagons, and the one that forms triplets, which connects the polygons together. These interactions are based on previous reports of the HIV-1 Gag protein.
Synthetic biology centers on the design and modular assembly of biological parts so as to construct artificial biological systems. Over the past decade, synthetic biology has blossomed into a highly ...productive field, yielding advances in diverse areas such as neuroscience, cell-based therapies, and chemical manufacturing. Similarly, the field of gene therapy has made enormous strides both in proof-of-concept studies and in the clinical setting. One viral vector of increasing interest for gene therapy is the adenovirus (Ad). A major part of the Ad’s increasing momentum comes from synthetic biology approaches to Ad engineering. Convergence of gene therapy and synthetic biology has enhanced Ad vectors by mitigating Ad toxicity in vivo, providing precise Ad tropisms, and incorporating genetic circuits to make smart therapies which adapt to environmental stimuli. Synthetic biology engineering of Ad vectors may lead to superior gene delivery and editing platforms which could find applications in a wide range of therapeutic contexts.
The ground state dominance approximation (GSDA) has been extensively used to study the assembly of viral shells. In this work we employ the self-consistent field theory (SCFT) to investigate the ...adsorption of RNA onto positively charged spherical viral shells and examine the conditions when GSDA does not apply and SCFT has to be used to obtain a reliable solution. We find that there are two regimes in which GSDA does work. First, when the genomic RNA length is long enough compared to the capsid radius, and second, when the interaction between the genome and capsid is so strong that the genome is basically localized next to the wall. We find that for the case in which RNA is more or less distributed uniformly in the shell, regardless of the length of RNA, GSDA is not a good approximation. We observe that as the polymer-shell interaction becomes stronger, the energy gap between the ground state and first excited state increases and thus GSDA becomes a better approximation. We also present our results corresponding to the genome persistence length obtained through the tangent-tangent correlation length and show that it is zero in case of GSDA but is equal to the inverse of the energy gap when using SCFT.
Bacteriophage MS2 was used to construct a targeted, multivalent photodynamic therapy vehicle for the treatment of Jurkat leukemia T cells. The self-assembling spherical virus capsid was modified on ...the interior surface with up to 180 porphyrins capable of generating cytotoxic singlet oxygen upon illumination. The exterior of the capsid was modified with ∼20 copies of a Jurkat-specific aptamer using an oxidative coupling reaction targeting an unnatural amino acid. The capsids were able to target and selectively kill more than 76% of the Jurkat cells after only 20 min of illumination. Capsids modified with a control DNA strand did not target Jurkat cells, and capsids modified with the aptamer were found to be specific for Jurkat cells over U266 cells (a control B cell line). The doubly modified capsids were also able to kill Jurkat cells selectively even when mixed with erythrocytes, suggesting the possibility of using our system to target blood-borne cancers or other pathogens in the blood supply.
Under many in vitro conditions, some small viruses spontaneously encapsidate a single stranded (ss) RNA into a protein shell called the capsid. While viral RNAs are found to be compact and highly ...branched because of long distance base-pairing between nucleotides, recent experiments reveal that in a head-to-head competition between an ssRNA with no secondary or higher order structure and a viral RNA, the capsid proteins preferentially encapsulate the linear polymer! In this paper, we study the impact of genome stiffness on the encapsidation free energy of the complex of RNA and capsid proteins. We show that an increase in effective chain stiffness because of base-pairing could be the reason why under certain conditions linear chains have an advantage over branched chains when it comes to encapsidation efficiency. While branching makes the genome more compact, RNA base-pairing increases the effective Kuhn length of the RNA molecule, which could result in an increase of the free energy of RNA confinement, that is, the work required to encapsidate RNA, and thus less efficient packaging.
Targeting ligands are used in drug delivery to improve drug distribution to desired cells or tissues and to facilitate cellular entry. In vivo biopanning, whereby billions of potential ligand ...sequences are screened in biologically-relevant and complex conditions, is a powerful method for identification of novel target ligands. This tool has impacted drug delivery technologies and expanded our arsenal of therapeutics and diagnostics. Within this review we will discuss current in vivo panning technologies and ways that these technologies can be improved to advance next-generation drug delivery strategies.
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