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Antimicrobial resistance presents a substantial threat to global public health, demanding urgent attention and action. This study focuses on lanthipeptides, ribosomally encoded peptides that display significant structural diversity and hold promising potential as antibiotics. Genome mining was employed to locate biosynthetic gene clusters (BGCs) containing class II lanthipeptide synthetases encoded by lanM genes. A phylogenetic study analyzing homologous sequences of functional LanM sequences revealed a unique evolutionary clade of 17 LanM proteins associated with 12 Clostridium bacterial genomes. In silico exploration identified nine complete BGCs, including one super-cluster containing two co-localized operons from Clostridium cellulovorans 743B, that encode for two new peptides named clostrisin and cellulosin. Each operon was heterologously expressed in Escherichia coli. Molecular weights associated with the expected post-translational modifications of the purified lanthipeptide were confirmed by MS-MS/MS analysis for cellulosin, while clostrisin was not post-translationally modified. Both peptides demonstrated antimicrobial activity against multidrug-resistant bacteria, such as a clinical strain of Staphylococcus epidermidis MIQ43 and Pseudomonas aeruginosa PA14. This is the first report of lanthipeptides from the Clostridium genus produced with its native biosynthetic machinery, as well as chemically and biologically characterized. This study showcases the immense potential of genome mining in identifying new RiPP synthetases and associated bioactive peptides.

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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.

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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.

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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.

Assuntos

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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.

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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.

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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.

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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.

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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.

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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.

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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.