Programming the Cellular Uptake of Protein-Based Viromimetic Nanoparticles for Enhanced Delivery.

Viral mimetics is a noteworthy strategy to design efficient delivery systems without the safety drawbacks and engineering difficulties of modifying viral vectors. The triblock polypeptide CSB was previously designed de novo to self-assemble with DNA into nanocomplexes called artificial virus-like particles (AVLPs) due to their similarities to viral particles. Here, we show how we can incorporate new blocks into the CSB polypeptide to enhance its transfection without altering its self-assembly capabilities and the stability and morphology of the AVLPs. The addition of a short peptide (aurein) and/or a large protein (transferrin) to the AVLPs improved their internalization and specific targeting to cells by up to 11 times. Overall, these results show how we can further program the cellular uptake of the AVLPs with a wide range of bioactive blocks. This can pave the way to develop programmable and efficient gene delivery systems.

Multiplex gRNAs Synergically Enhance Detection of SARS-CoV-2 by CRISPR-Cas12a.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) diagnostic methods have a large potential to effectively detect SARS-CoV-2 with sensitivity and specificity nearing 100%, comparable to quantitative polymerase chain reaction. Yet, there is room for improvement. Commonly, one guide CRISPR RNA (gRNA) is used to detect the virus DNA and activate Cas collateral activity, which cleaves a reporter probe. In this study, we demonstrated that using 2-3 gRNAs in parallel can create a synergistic effect, resulting in a 4.5 × faster cleaving rate of the probe and increased sensitivity compared to using individual gRNAs. The synergy is due to the simultaneous activation of CRISPR-Cas12a and the improved performance of each gRNA. This approach was able to detect as few as 10 viral copies of the N-gene of SARS-CoV-2 RNA after a preamplification step using reverse transcription loop-mediated isothermal amplification. The method was able to accurately detect 100% of positive and negative clinical samples in ∼25 min using a fluorescence plate reader and ∼45 min with lateral flow strips.

Extending resolution within a single imaging frame.

The resolution of fluorescence microscopy images is limited by the physical properties of light. In the last decade, numerous super-resolution microscopy (SRM) approaches have been proposed to deal with such hindrance. Here we present Mean-Shift Super Resolution (MSSR), a new SRM algorithm based on the Mean Shift theory, which extends spatial resolution of single fluorescence images beyond the diffraction limit of light. MSSR works on low and high fluorophore densities, is not limited by the architecture of the optical setup and is applicable to single images as well as temporal series. The theoretical limit of spatial resolution, based on optimized real-world imaging conditions and analysis of temporal image stacks, has been measured to be 40 nm. Furthermore, MSSR has denoising capabilities that outperform other SRM approaches. Along with its wide accessibility, MSSR is a powerful, flexible, and generic tool for multidimensional and live cell imaging applications.

Diastereomeric Separation of Chiral fac-Tricarbonyl(iminopyridine) Rhenium(I) Complexes and Their Cytotoxicity Studies: Approach toward an Action Mechanism against Glioblastoma.

A series of new (tricarbonyl)rhenium(I) complexes were synthesized using chiral bidentate ligands (+)/(-)-iminopyridines (LR/LS). The reaction yielded a mixture of mononuclear Re(I) diastereoisomers, formulated as fac-[Br(CO)3Re(S/R)L(S/R)]. Each single diastereoisomer was isolated and fully characterized. X-ray crystallography and circular dichroism spectra verified their enantiomeric nature. The cytotoxicity of each complex was evaluated against six cancer cell lines. The effect of the two complexes on viability, proliferation, and migration was analyzed on glioblastoma cell lines (U251 and LN229). Changes in the expression of histones, apoptotic, and key signaling proteins, as well as alterations in DNA structure, were also observed. These experiments showed that the chirality associated with both metal and ligand has a strong influence on cytotoxicity.

Diagnostics of COVID-19 Based on CRISPR-Cas Coupled to Isothermal Amplification: A Comparative Analysis and Update.

The emergence of the COVID-19 pandemic prompted fast development of novel diagnostic methods of the etiologic virus SARS-CoV-2. Methods based on CRISPR-Cas systems have been particularly promising because they can achieve a similar sensitivity and specificity to the benchmark RT-qPCR, especially when coupled to an isothermal pre-amplification step. Furthermore, they have also solved inherent limitations of RT-qPCR that impede its decentralized use and deployment in the field, such as the need for expensive equipment, high cost per reaction, and delivery of results in hours, among others. In this review, we evaluate publicly available methods to detect SARS-CoV-2 that are based on CRISPR-Cas and isothermal amplification. We critically analyze the steps required to obtain a successful result from clinical samples and pinpoint key experimental conditions and parameters that could be optimized or modified to improve clinical and analytical outputs. The COVID outbreak has propelled intensive research in a short time, which is paving the way to develop effective and very promising CRISPR-Cas systems for the precise detection of SARS-CoV-2. This review could also serve as an introductory guide to new labs delving into this technology.

A kinetic model for the impact of packaging signal mimics on genome encapsulation.

Inspired by recent experiments on the spontaneous assembly of virus-like particles from a solution containing a synthetic coat protein and double-stranded DNA, we put forward a kinetic model that has as main ingredients a stochastic nucleation and a deterministic growth process. The efficiency and rate of DNA packaging strongly increase after tiling the DNA with CRISPR-Cas proteins at predesignated locations, mimicking assembly signals in viruses. Our model shows that treating these proteins as nucleation-inducing diffusion barriers is sufficient to explain the experimentally observed increase in encapsulation efficiency, but only if the nucleation rate is sufficiently high. We find an optimum in the encapsulation kinetics for conditions where the number of packaging signal mimics is equal to the number of nucleation events that can occur during the time required to fully encapsulate the DNA template, presuming that the nucleation events can only take place adjacent to a packaging signal. Our theory is in satisfactory agreement with the available experimental data.

An artificial virus-like triblock protein shows low in vivo humoral immune response and high stability.

The use of viral vectors for in vivo gene therapy can be severely limited by their immunogenicity. Non-viral vectors may represent an alternative, however, reports analyzing their immunogenicity are still lacking. Here, we studied the humoral immune response in a murine model triggered by artificial virus-like particles (AVLPs) carrying plasmid or antisense DNA. The AVLPs were assembled using a family of modular proteins based on bioinspired collagen-like and silk-like sequences that produce virus-like particles. We compared our AVLPs against an Adeno Associated Virus 1 (AAV), a widely used viral vector for in vivo gene delivery that has been approved by the FDA and EMA for gene therapy. We found that a 1000-fold higher mass of AVLPs than AAV are necessary to obtain similar specific antibody titters. Furthermore, we studied the stability of AVLPs against relevant biological reagents such as heparin and fetal bovine serum to ensure nucleic acid protection in biological media. Our study demonstrates that the AVLPs are stable in physiological conditions and can overcome safety limitations such as immunogenicity. The scarce humoral immunogenicity and high stability found with AVLPs suggest that they have potential to be used as stealth non-viral gene delivery systems for in vivo studies or gene therapy.

Strategies to Build Hybrid Protein-DNA Nanostructures.

Proteins and DNA exhibit key physical chemical properties that make them advantageous for building nanostructures with outstanding features. Both DNA and protein nanotechnology have growth notably and proved to be fertile disciplines. The combination of both types of nanotechnologies is helpful to overcome the individual weaknesses and limitations of each one, paving the way for the continuing diversification of structural nanotechnologies. Recent studies have implemented a synergistic combination of both biomolecules to assemble unique and sophisticate protein-DNA nanostructures. These hybrid nanostructures are highly programmable and display remarkable features that create new opportunities to build on the nanoscale. This review focuses on the strategies deployed to create hybrid protein-DNA nanostructures. Here, we discuss strategies such as polymerization, spatial directing and organizing, coating, and rigidizing or folding DNA into particular shapes or moving parts. The enrichment of structural DNA nanotechnology by incorporating protein nanotechnology has been clearly demonstrated and still shows a large potential to create useful and advanced materials with cell-like properties or dynamic systems. It can be expected that structural protein-DNA nanotechnology will open new avenues in the fabrication of nanoassemblies with unique functional applications and enrich the toolbox of bionanotechnology.

CRISPR-Guided Programmable Self-Assembly of Artificial Virus-Like Nucleocapsids.

Designer virus-inspired proteins drive the manufacturing of more effective, safer gene-delivery systems and simpler models to study viral assembly. However, self-assembly of engineered viromimetic proteins on specific nucleic acid templates, a distinctive viral property, has proved difficult. Inspired by viral packaging signals, we harness the programmability of CRISPR-Cas12a to direct the nucleation and growth of a self-assembling synthetic polypeptide into virus-like particles (VLP) on specific DNA molecules. Positioning up to ten nuclease-dead Cas12a (dCas12a) proteins along a 48.5 kbp DNA template triggers particle growth and full DNA encapsidation at limiting polypeptide concentrations. Particle growth rate is further increased when dCas12a is dimerized with a polymerization silk-like domain. Such improved self-assembly efficiency allows for discrimination between cognate versus noncognate DNA templates by the synthetic polypeptide. CRISPR-guided VLPs will help to develop programmable bioinspired nanomaterials with applications in biotechnology as well as viromimetic scaffolds to improve our understanding of viral self-assembly.

Delivery of Antisense DNA into Pathogenic Parasite Trypanosoma cruzi Using Virus-Like Protein-Based Nanoparticles.

Trypanosoma cruzi, which causes Chagas disease, is one of the most lacerating parasites in terms of health and social impacts. New approaches for its study and treatment are urgently needed since in more than 50 years only two drugs have been approved. Genetic approaches based on antisense oligonucleotides (AONs) are promising; however, to harness their full potential the development of effective carriers is paramount. Here, we report the use of an engineered virus-like protein C-BK12 to transfect AONs into T. cruzi. Using gel electrophoresis, Dynamic Light Scattering, and atomic force microscopy, we found that C-BK12 binds AONs and forms 10-25 nm nanoparticles (NPs), which are very stable when incubated in biological media, only releasing up to 25% of AON. Fluorescence microscopy and qPCR revealed that the NPs successfully delivered AONs into epimastigotes and reduced the expression of a target gene down to 68%. Importantly, the protein did not show cytotoxicity. The combination of high stability and capability to transfect and knock down gene expression without causing cell damage and death makes the protein C-BK12 a promising starting point for the further development of safe and effective carriers to deliver AONs into T. cruzi for biological studies.

Protein-coated dsDNA nanostars with high structural rigidity and high enzymatic and thermal stability.

DNA nanotechnology creates precise shape-specific nanostructures through the self-assembly of short ssDNA oligonucleotides. One such shape, which has relevant biomedical applications due to its multivalency, is the star. However, building star-like nanostructures with a large size (>100 nm) using ssDNA is complex and challenging. This study presents a novel strategy to prepare stiff and large dsDNA nanostars by assembling duplex DNA fragments into star-shapes that are subsequently coated with a virus-inspired protein. The protein binds dsDNA and overcomes the high structural flexibility of naked dsDNA. The nanostar-like dsDNA templates with up to six arms were prepared by self-assembly of PCR-produced dsDNA fragments (211 to 722 bp) with a central DNA junction. Through gel electrophoresis and Atomic Force Microscopy it is demonstrated that single dsDNA nanostars are self-assembled and coated with the protein, and this has a large stiffening effect on the nanostar. Furthermore, the coating significantly enhances stability at high temperatures and protects nanostars against nuclease degradation for at least 10 hours. This study shows that DNA-binding proteins can be harnessed as structural "rigidifiers" of flexible branched dsDNA templates. This strategy opens a way to prepare structurally defined hybrid protein-dsDNA nanostructures that could be exploited as building blocks for novel DNA nanomaterials.

Nature of Amorphous Hydrophilic Block Affects Self-Assembly of an Artificial Viral Coat Polypeptide.

Consensus motifs for sequences of both crystallizable and amorphous blocks in silks and natural structural analogues of silks vary widely. To design novel silklike polypeptides, an important question is therefore how the nature of either the crystallizable or the amorphous block affects the self-assembly and resulting physical properties of silklike polypeptides. We address herein the influence of the amorphous block on the self-assembly of a silklike polypeptide that was previously designed to encapsulate single DNA molecules into rod-shaped viruslike particles. The polypeptide has a triblock architecture, with a long N-terminal amorphous block, a crystallizable midblock, and a C-terminal DNA-binding block. We compare the self-assembly behavior of a triblock with a very hydrophilic collagen-like amorphous block (GXaaYaa)132 to that of a triblock with a less hydrophilic elastin-like amorphous block (GSGVP)80. The amorphous blocks have similar lengths and both adopt a random coil structure in solution. Nevertheless, atomic force microscopy revealed significant differences in the self-assembly behavior of the triblocks. If collagen-like amorphous blocks are used, there is a clear distinction between very short polypeptide-only fibrils and much longer fibrils with encapsulated DNA. If elastin-like amorphous blocks are used, DNA is still encapsulated, but the polypeptide-only fibrils are now much longer and their size distribution partially overlaps with that of the encapsulated DNA fibrils. We attribute the difference to the more hydrophilic nature of the collagen-like amorphous block, which more strongly opposes the growth of polypeptide-only fibrils than the elastin-like amorphous blocks. Our work illustrates that differences in the chemical nature of amorphous blocks can strongly influence the self-assembly and hence the functionality of engineered silklike polypeptides.

Real-Time Assembly of Viruslike Nucleocapsids Elucidated at the Single-Particle Level.

While the structure of a multitude of viral particles has been resolved to atomistic detail, their assembly pathways remain largely elusive. Key unresolved issues are particle nucleation, particle growth, and the mode of genome compaction. These issues are difficult to address in bulk approaches and are effectively only accessible by the real-time tracking of assembly dynamics of individual particles. This we do here by studying the assembly into rod-shaped viruslike particles (VLPs) of artificial capsid polypeptides. Using fluorescence optical tweezers, we establish that small oligomers perform one-dimensional diffusion along the DNA. Larger oligomers are immobile and nucleate VLP growth. A multiplexed acoustic force spectroscopy approach reveals that DNA is compacted in regular steps, suggesting packaging via helical wrapping into a nucleocapsid. By reporting how real-time assembly tracking elucidates viral nucleation and growth principles, our work opens the door to a fundamental understanding of the complex assembly pathways of both VLPs and naturally evolved viruses.

Template-Free Self-Assembly of Artificial De Novo Viral Coat Proteins into Nanorods: Effects of Sequence, Concentration, and Temperature.

The self-assembly of protein polymers is a promising route to prepare sophisticated functional nanostructures. However, the interplay between protein self-assembly by itself and its co-assembly with a template is not well understood. Silk-based protein polymers that co-assemble with DNA to form rod-like artificial viruses are herein developed and the effects of silk block length, concentration, and temperature in the self-assembly of the proteins alone are characterized by using a combination of bulk dynamic light scattering (DLS) and single-molecule atomic force microscopy (AFM). Protein nanorods were slowly formed (up to hours) through the interaction of the silk-like blocks. The proteins present a silk-length dependent critical elongation concentration, and above it the amount and size of nanorods rapidly increase. Temperature-dependent light scattering data was adequately fitted into a cooperative model of nucleation-elongation. These results are also important to understand the self-assembly of designed viral coat proteins with DNA templates to form artificial virus-like particles and help us to define general guidelines to design proteins with the ability to precisely organize matter at the nanoscale.

Peptide-siRNA Supramolecular Particles for Neural Cell Transfection.

Small interfering ribonucleic acid (siRNA)-based gene knockdown is an effective tool for gene screening and therapeutics. However, the use of nonviral methods has remained an enormous challenge in neural cells. A strategy is reported to design artificial noncationic modular peptides with amplified affinity for siRNA via supramolecular assembly that shows efficient protein knockdown in neural cells. By solid phase synthesis, a sequence that binds specifically double-stranded ribonucleic acid (dsRNA) with a self-assembling peptide for particle formation is integrated. These supramolecular particles can be further functionalized with bioactive sequences without affecting their biophysical properties. The peptide carrier is found to silence efficiently up to 83% of protein expression in primary astroglial and neuronal cell cultures without cytotoxicity. In the case of neurons, a reduction in electrical activity is observed once the presynaptic protein synaptophysin is downregulated by the siRNA-peptide particles. The results demonstrate that the supramolecular particles offer an siRNA delivery platform for efficient nonviral gene screening and discovery of novel therapies for neural cells.

Force and Scale Dependence of the Elasticity of Self-Assembled DNA Bottle Brushes.

As a model system to study the elasticity of bottle-brush polymers, we here introduce self-assembled DNA bottle brushes, consisting of a DNA main chain that can be very long and still of precisely defined length, and precisely monodisperse polypeptide side chains that are physically bound to the DNA main chains. Polypeptide side chains have a diblock architecture, where one block is a small archaeal nucleoid protein Sso7d that strongly binds to DNA. The other block is a net neutral, hydrophilic random coil polypeptide with a length of exactly 798 amino acids. Light scattering shows that for saturated brushes the grafting density is one side chain per 5.6 nm of DNA main chain. According to small-angle X-ray scattering, the brush diameter is D = 17 nm. By analyzing configurations of adsorbed DNA bottle brushes using AFM, we find that the effective persistence of the saturated DNA bottle brushes is Peff = 95 nm, but from force-extension curves of single DNA bottle brushes measured using optical tweezers we find Peff = 15 nm. The latter is equal to the value expected for DNA coated by the Sso7d binding block alone. The apparent discrepancy between the two measurements is rationalized in terms of the scale dependence of the bottle-brush elasticity using theory previously developed to analyze the scale-dependent electrostatic stiffening of DNA at low ionic strengths.

Correction to Force and Scale Dependence of the Elasticity of Self-Assembled DNA Bottle Brushes.

[This corrects the article DOI: 10.1021/acs.macromol.7b01795.].

Virus-Like Particles of mRNA with Artificial Minimal Coat Proteins: Particle Formation, Stability, and Transfection Efficiency.

RNA has enormous potential as a therapeutic, yet, the successful application depends on efficient delivery strategies. In this study, we demonstrate that a designed artificial viral coat protein, which self-assembles with DNA to form rod-shaped virus-like particles (VLPs), also encapsulates and protects mRNA encoding enhanced green fluorescent protein (EGFP) and luciferase, and yields cellular expression of these proteins. The artificial viral coat protein consists of an oligolysine (K12) for binding to the oligonucleotide, a silk protein-like midblock S10 = (GAGAGAGQ)10 that self-assembles into stiff rods, and a long hydrophilic random coil block C that shields the nucleic acid cargo from its environment. With mRNA, the C-S10-K12 protein coassembles to form rod-shaped VLPs each encapsulating about one to five mRNA molecules. Inside the rod-shaped VLPs, the mRNAs are protected against degradation by RNAses, and VLPs also maintain their shape following incubation with serum. Despite the lack of cationic surface charge, the mRNA VLPs transfect cells with both EGFP and luciferase, although with a much lower efficiency than obtained by a lipoplex transfection reagent. The VLPs have a negligible toxicity and minimal hemolytic activity. Our results demonstrate that VLPs yield efficient packaging and shielding of mRNA and create the basis for implementation of additional virus-like functionalities to improve transfection and cell specificity, such as targeting functionalities.

Engineered Diblock Polypeptides Improve DNA and Gold Solubility during Molecular Assembly.

Programmed molecular recognition is being developed for the bionanofabrication of mixed organic/inorganic supramolecular assemblies for applications in electronics, photonics, and medicine. For example, DNA-based nanotechnology seeks to exploit the easily programmed complementary base-pairing of DNA to direct assembly of complex, designed nanostructures. Optimal solution conditions for bionanofabrication, mimicking those of biological systems, may involve high concentrations of biomacromolecules (proteins, nucleic acids, etc.) and significant concentrations of various ions (Mg2+, Na+, Cl-, etc.). Given a desire to assemble diverse inorganic components (metallic nanoparticles, quantum dots, carbon nanostructures, etc.), it will be increasingly difficult to find solution conditions simultaneously compatible with all components. Frequently, the use of chemical surfactants is undesirable, leaving a need for the development of alternative strategies. Herein, we discuss the use of artificial, diblock polypeptides in the role of solution compatibilizing agents for molecular assembly. We describe the use of two distinct diblock polypeptides with affinity for DNA in the stabilization of DNA origami and DNA-functionalized gold nanoparticles (spheres and rods) in solution, protection of DNA from enzymatic degradation, as well as two 3D tetrahedral DNA origamis. We present initial data showing that the diblock polypeptides promote the formation in the solution of desired organic/inorganic assemblies.

Precise Coating of a Wide Range of DNA Templates by a Protein Polymer with a DNA Binding Domain.

Emerging DNA-based nanotechnologies would benefit from the ability to modulate the properties (e.g., solubility, melting temperature, chemical stability) of diverse DNA templates (single molecules or origami nanostructures) through controlled, self-assembling coatings. We here introduce a DNA coating agent, called C8-BSso7d, which binds to and coats with high specificity and affinity, individual DNA molecules as well as folded origami nanostructures. C8-BSso7d coats and protects without condensing, collapsing or destroying the spatial structure of the underlying DNA template. C8-BSso7d combines the specific nonelectrostatic DNA binding affinity of an archeal-derived DNA binding domain (Sso7d, 7 kDa) with a long hydrophilic random coil polypeptide (C8, 73 kDa), which provides colloidal stability (solubility) through formation of polymer brushes around the DNA templates. C8-BSso7d is produced recombinantly in yeast and has a precise (but engineerable) amino acid sequence of precise length. Using electrophoresis, AFM, and fluorescence microscopy we demonstrate protein coat formation with stiffening of one-dimensional templates (linear dsDNA, supercoiled dsDNA and circular ssDNA), as well as coat formation without any structural distortion or disruption of two-dimensional DNA origami template. Combining the programmability of DNA with the nonperturbing precise coating capability of the engineered protein C8-BSso7d holds promise for future applications such as the creation of DNA-protein hybrid networks, or the efficient transfection of individual DNA nanostructures into cells.