RESUMEN

Designing proteins that fold and assemble over different length scales provides a way to tailor the mechanical properties and biological performance of hydrogels. In this study, we designed modular proteins that self-assemble into fibrillar networks and, as a result, form hydrogel materials with novel properties. We incorporated distinct functionalities by connecting separate self-assembling (A block) and cell-binding (B block) domains into single macromolecules. The number of self-assembling domains affects the rigidity of the fibers and the final storage modulus G' of the materials. The mechanical properties of the hydrogels could be tuned over a broad range (G' = 0.1 - 10 kPa), making them suitable for the cultivation and differentiation of multiple cell types, including cortical neurons and human mesenchymal stem cells. Moreover, we confirmed the bioavailability of cell attachment domains in the hydrogels that can be further tailored for specific cell types or other biological applications. Finally, we demonstrate the versatility of the designed proteins for application in biofabrication as 3D scaffolds that support cell growth and guide their function. STATEMENT OF SIGNIFICANCE: Designed proteins that enable the decoupling of biophysical and biochemical properties within the final material could enable modular biomaterial engineering. In this context, we present a designed modular protein platform that integrates self-assembling domains (A blocks) and cell-binding domains (B blocks) within a single biopolymer. The linking of assembly domains and cell-binding domains this way provided independent tuning of mechanical properties and inclusion of biofunctional domains. We demonstrate the use of this platform for biofabrication, including neural cell culture and 3D printing of scaffolds for mesenchymal stem cell culture and differentiation. Overall, this work highlights how informed design of biopolymer sequences can enable the modular design of protein-based hydrogels with independently tunable biophysical and biochemical properties.

Asunto(s)

RESUMEN

Diagnostic medical imaging utilizes magnetic resonance (MR) to provide anatomical, functional, and molecular information in a single scan. Nanoparticles are often labeled with Gd(III) complexes to amplify the MR signal of contrast agents (CAs) with large payloads and high proton relaxation efficiencies (relaxivity, r1). This study examined the MR performance of two structurally unique cages, AaLS-13 and OP, labeled with Gd(III). The cages have characteristics relevant for the development of theranostic platforms, including (i) well-defined structure, symmetry, and size; (ii) the amenability to extensive engineering; (iii) the adjustable loading of therapeutically relevant cargo molecules; (iv) high physical stability; and (v) facile manufacturing by microbial fermentation. The resulting conjugates showed significantly enhanced proton relaxivity (r1 = 11-18 mM-1 s-1 at 1.4 T) compared to the Gd(III) complex alone (r1 = 4 mM-1 s-1). Serum phantom images revealed 107% and 57% contrast enhancements for Gd(III)-labeled AaLS-13 and OP cages, respectively. Moreover, proton nuclear magnetic relaxation dispersion (1H NMRD) profiles showed maximum relaxivity values of 50 mM-1 s-1. Best-fit analyses of the 1H NMRD profiles attributed the high relaxivity of the Gd(III)-labeled cages to the slow molecular tumbling of the conjugates and restricted local motion of the conjugated Gd(III) complex.

Asunto(s)

RESUMEN

Although viruses have been successfully repurposed as vaccines, antibiotics, and anticancer therapeutics, they also raise concerns regarding genome integration and immunogenicity. Virus-like particles and non-viral protein cages represent a potentially safer alternative but often lack desired functionality. Here, we investigated the utility of a new enzymatic bioconjugation method, called lysine acylation using conjugating enzymes (LACE), to chemoenzymatically modify protein cages. We equipped two structurally distinct protein capsules with a LACE-reactive peptide tag and demonstrated their modification with diverse ligands. This modular approach combines the advantages of chemical conjugation and genetic fusion and allows for site-specific modification with recombinant proteins as well as synthetic peptides with facile control of the extent of labeling. This strategy has the potential to fine-tune protein containers of different shape and size by providing them with new properties that go beyond their biologically native functions.

Asunto(s)

RESUMEN

Proteins that self-assemble into polyhedral shell-like structures are useful molecular containers both in nature and in the laboratory. Here we review efforts to repurpose diverse protein cages, including viral capsids, ferritins, bacterial microcompartments, and designed capsules, as vaccines, drug delivery vehicles, targeted imaging agents, nanoreactors, templates for controlled materials synthesis, building blocks for higher-order architectures, and more. A deep understanding of the principles underlying the construction, function, and evolution of natural systems has been key to tailoring selective cargo encapsulation and interactions with both biological systems and synthetic materials through protein engineering and directed evolution. The ability to adapt and design increasingly sophisticated capsid structures and functions stands to benefit the fields of catalysis, materials science, and medicine.

Asunto(s)

RESUMEN

Well-defined containers constructed from multiple protein subunits are a unique class of nanomaterial useful in supramolecular chemistry and biology. These protein cages are widespread in nature, where they are responsible for a diversity of important tasks. As such, producing our own designer protein cages, complete with bespoke functionalities, is a promising avenue to new nanodevices, biotechnology and therapies. Herein, we describe how an artificial, computationally designed protein cage can be rationally engineered using supramolecular intuition to produce new functional capsules. Positive supercharging the interior cavity of this porous protein cage enables the efficient encapsulation of oligonucleotides by electrostatically-driven self-assembly. Moreover, the resulting cargo-loaded cages enter mammalian cells and release their cargo, for example siRNA which modulates gene expression. To expand the cargo scope of this proteinaceous container, a higher level of supramolecular complexity can also be introduced. Encapsulation of anionic surfactants affords protein-scaffolded micelles, which are capable of sequestering poorly water-soluble small molecules within their hydrophobic cores. These hybrid particles stably carry bioactive cargo and deliver it intracellularly, thereby increasing potency. Further development of these genetically-encoded materials is ongoing towards specific applications ranging from cell biology to medicine.

Asunto(s)

RESUMEN

Expanding protein design to include other molecular building blocks has the potential to increase structural complexity and practical utility. Nature often employs hybrid systems, such as clathrin-coated vesicles, lipid droplets, and lipoproteins, which combine biopolymers and lipids to transport a broader range of cargo molecules. To recapitulate the structure and function of such composite compartments, we devised a supramolecular strategy that enables porous protein cages to encapsulate poorly water-soluble small molecule cargo through templated formation of a hydrophobic surfactant-based core. These lipoprotein-like complexes protect their cargo from sequestration by serum proteins and enhance the cellular uptake of fluorescent probes and cytotoxic drugs. This design concept could be applied to other protein cages, surfactant mixtures, and cargo molecules to generate unique hybrid architectures and functional capabilities.

Asunto(s)

RESUMEN

The structural and functional diversity of proteins combined with their genetic programmability has made them indispensable modern materials. Well-defined, hollow protein capsules have proven to be particularly useful due to their ability to compartmentalize macromolecules and chemical processes. To this end, viral capsids are common scaffolds and have been successfully repurposed to produce a suite of practical protein-based nanotechnologies. Recently, the recapitulation of viromimetic function in protein cages of nonviral origin has emerged as a strategy to both complement physical studies of natural viruses and produce useful scaffolds for diverse applications. In this perspective, we review recent progress toward generation of virus-like behavior in nonviral protein cages through rational engineering and directed evolution. These artificial systems can aid our understanding of the emergence of viruses from existing cellular components, as well as provide alternative approaches to tackle current problems, and open up new opportunities, in medicine and biotechnology.

Asunto(s)

RESUMEN

Oligonucleotide therapeutics have transformative potential in modern medicine but are poor drug candidates in themselves unless fitted with compensatory carrier systems. We describe a simple approach to transform a designed porous protein cage into a nucleic acid delivery vehicle. By introducing arginine mutations to the lumenal surface, a positively supercharged capsule is created, which can encapsidate oligonucleotides in vitro with high binding affinity. We demonstrate that the siRNA-loaded cage is taken up by mammalian cells and releases its cargo to induce RNAi and knockdown gene expression. These general concepts could also be applied to alternative scaffold designs, expediting the development of artificial protein cages toward delivery applications.

Asunto(s)

RESUMEN

Nanoscale compartments formed by hierarchical protein self-assembly are valuable platforms for nanotechnology development. The well-defined structure and broad chemical functionality of protein cages, as well as their amenability to genetic and chemical modification, have enabled their repurposing for diverse applications. In this review, we summarize progress in the engineering of the cage-forming enzyme lumazine synthase. This bacterial nanocompartment has proven to be a malleable scaffold. The natural protein has been diversified to afford a family of unique proteinaceous capsules that have been modified, evolved and assembled with other components to produce nanoreactors, artificial organelles, delivery vehicles and virus mimics.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

The development of nucleic acid therapeutics has been hampered by issues associated with their stability and in vivo delivery. To address these challenges, we describe a new strategy to engineer DNA structures with strong binding affinity to human serum albumin (HSA). HSA is the most abundant protein in the blood and has a long circulation half-life (19 days). It has been shown to hinder phagocytosis, is retained in tumors, and aids in cellular penetration. Indeed, HSA has already been successfully used for the delivery of small-molecule drugs and nanoparticles. We show that conjugating dendritic alkyl chains to DNA creates amphiphiles that exhibit high-affinity (Kd in low nanomolar range) binding to HSA. Notably, complexation with HSA did not hinder the activity of silencing oligonucleotides inside cells, and the degradation of DNA strands in serum was significantly slowed. We also show that, in a site-specific manner, altering the number and orientation of the amphiphilic ligand on a self-assembled DNA nanocube can modulate the affinity of the DNA cage to HSA. Moreover, the serum half-life of the amphiphile bound to the cage and the protein was shown to reach up to 22 hours, whereas unconjugated single-stranded DNA was degraded within minutes. Therefore, adding protein-specific binding domains to DNA nanostructures can be used to rationally control the interface between synthetic nanostructures and biological systems. A major challenge with nanoparticles delivery is the quick formation of a protein corona (i.e., protein adsorbed on the nanoparticle surface) upon injection to biological media. We foresee such DNA cage-protein complexes as new tools to study the role of this protein adsorption layer with important implications in the efficient delivery of RNAi therapeutics in vitro and in vivo.

Asunto(s)

RESUMEN

Correction for 'Antisense precision polymer micelles require less poly(ethylenimine) for efficient gene knockdown' by Johans J. Fakhoury, et al., Nanoscale, 2015, 7, 20625-20634.

RESUMEN

DNA base-pairing is the central interaction in DNA assembly. However, this simple four-letter (A-T and G-C) language makes it difficult to create complex structures without using a large number of DNA strands of different sequences. Inspired by protein folding, we introduce hydrophobic interactions to expand the assembly language of DNA nanotechnology. To achieve this, DNA cages of different geometries are combined with sequence-defined polymers containing long alkyl and oligoethylene glycol repeat units. Anisotropic decoration of hydrophobic polymers on one face of the cage leads to hydrophobically driven formation of quantized aggregates of DNA cages, where polymer length determines the cage aggregation number. Hydrophobic chains decorated on both faces of the cage can undergo an intrascaffold "handshake" to generate DNA-micelle cages, which have increased structural stability and assembly cooperativity, and can encapsulate small molecules. The polymer sequence order can control the interaction between hydrophobic blocks, leading to unprecedented "doughnut-shaped" DNA cage-ring structures. We thus demonstrate that new structural and functional modes in DNA nanostructures can emerge from the synergy of two interactions, providing an attractive approach to develop protein-inspired assembly modules in DNA nanotechnology.

Asunto(s)

RESUMEN

DNA nanotechnology offers unparalleled precision and programmability for the bottom-up organization of materials. This approach relies on pre-assembling a DNA scaffold, typically containing hundreds of different strands, and using it to position functional components. A particularly attractive strategy is to employ DNA nanostructures not as permanent scaffolds, but as transient, reusable templates to transfer essential information to other materials. To our knowledge, this approach, akin to top-down lithography, has not been examined. Here we report a molecular printing strategy that chemically transfers a discrete pattern of DNA strands from a three-dimensional DNA structure to a gold nanoparticle. We show that the particles inherit the DNA sequence configuration encoded in the parent template with high fidelity. This provides control over the number of DNA strands and their relative placement, directionality and sequence asymmetry. Importantly, the nanoparticles produced exhibit the site-specific addressability of DNA nanostructures, and are promising components for energy, information and biomedical applications.

Asunto(s)

RESUMEN

Therapeutic nucleic acids are powerful molecules for shutting down protein expression. However, their cellular uptake is poor and requires transport vectors, such as cationic polymers. Of these, poly(ethylenimine) (PEI) has been shown to be an efficient vehicle for nucleic acid transport into cells. However, cytotoxicity has been a major hurdle in the development of PEI-DNA complexes as clinically viable therapeutics. We have synthesized antisense-polymer conjugates, where the polymeric block is completely monodisperse and sequence-controlled. Depending on the polymer sequence, these can self-assemble to produce micelles of very low polydispersity. The introduction of linear poly(ethylenimine) to these micelles leads to aggregation into size-defined PEI-mediated superstructures. Subsequently, both cellular uptake and gene silencing are greatly enhanced over extended periods compared to antisense alone, while at the same time cellular cytotoxicity remains very low. In contrast, gene silencing is not enhanced with antisense polymer conjugates that are not able to self-assemble into micelles. Thus, using antisense precision micelles, we are able to achieve significant transfection and knockdown with minimal cytotoxicity at much lower concentrations of linear PEI then previously reported. Consequently, a conceptual solution to the problem of antisense or siRNA delivery is to self-assemble these molecules into 'gene-like' micelles with high local charge and increased stability, thus reducing the amount of transfection agent needed for effective gene silencing.

Asunto(s)

RESUMEN

Polymer self-assembly and DNA nanotechnology have both proved to be powerful nanoscale techniques. To date, most attempts to merge the fields have been limited to placing linear DNA segments within a polydisperse block copolymer. Here we show that, by using hydrophobic polymers of a precisely predetermined length conjugated to DNA strands, and addressable 3D DNA prisms, we are able to effect the formation of unprecedented monodisperse quantized superstructures. The structure and properties of larger micelles-of-prisms were probed in depth, revealing their ability to participate in controlled release of their constituent nanostructures, and template light-harvesting energy transfer cascades, mediated through both the addressability of DNA and the controlled aggregation of the polymers.

Asunto(s)

RESUMEN

Inspired by biological polymers, sequence-controlled synthetic polymers are highly promising materials that integrate the robustness of synthetic systems with the information-derived activity of biological counterparts. Polymer-biopolymer conjugates are often targeted to achieve this union; however, their synthesis remains challenging. We report a stepwise solid-phase approach for the generation of completely monodisperse and sequence-defined DNA-polymer conjugates using readily available reagents. These polymeric modifications to DNA display self-assembly and encapsulation behavior-as evidenced by HPLC, dynamic light scattering, and fluorescence studies-which is highly dependent on sequence order. The method is general and has the potential to make DNA-polymer conjugates and sequence-defined polymers widely available.

Asunto(s)

RESUMEN

Nature uses a combination of non-covalent interactions to create a hierarchy of complex systems from simple building blocks. One example is the selective association of the hydrophobic side chains that are a strong determinant of protein organization. Here, we report a parallel mode of assembly in DNA nanotechnology. Dendritic alkyl-DNA conjugates are hybridized to the edges of a DNA cube. When four amphiphiles are on one face, the hydrophobic residues of two neighbouring cubes engage in an intermolecular 'handshake', resulting in a dimer. When there are eight amphiphiles (four on the top and bottom cube faces, respectively), they engage in an intramolecular 'handshake' inside the cube. This forms the first example of a monodisperse micelle within a DNA nanostructure that encapsulates small molecules and releases them by DNA recognition. Creating a three-dimensional pattern of hydrophobic patches, like side chains in proteins, can result in specific, directed association of hydrophobic domains with orthogonal interactions to DNA base-pairing.

Asunto(s)