Purification of DNA Nanoparticles Using Photocleavable Biotin Tethers.

The number of applications of self-assembled deoxyribonucleic acid (DNA) origami nanoparticles (DNA NPs) has increased drastically, following the development of a variety of single-stranded template DNA (ssDNA) that can serve as the scaffold strand. In addition to viral genomes, such as M13 bacteriophage and lambda DNAs, enzymatically produced ssDNA from various template sources is rapidly gaining traction and being applied as the scaffold for DNA NP preparation. However, separating fully formed DNA NPs that have custom scaffolds from crude assembly mixes is often a multistep process of first separating the ssDNA scaffold from its enzymatic amplification process and then isolating the assembled DNA NPs from excess precursor strands. Only then is the DNA NP sample ready for downstream characterization and application. In this work, we highlight a single-step purification of custom sequence- or M13-derived scaffold-based DNA NPs using photocleavable biotin tethers. The process only requires an inexpensive ultraviolet (UV) lamp, and DNA NPs with up to 90% yield and high purity are obtained. We show the versatility of the process in separating two multihelix bundle structures and a wireframe polyhedral architecture.

Visualization of Engineered M13 Phages Bound to Bacterial Targets by Transmission Electron Microscopy.

The filamentous phage M13 is one of the most well-studied and characterized phages, particularly since it was introduced as a scaffold for phage display, a technique to express and evolve fusion proteins on the M13 phage's coat to study protein or peptide binding interactions. Since phages can be engineered or evolved to specifically bind to a variety of targets, engineered M13 phages have been explored for applications such as drug delivery, biosensing, and cancer therapy, among others. Specifically, with the rising challenge of antimicrobial resistance among bacteria, chimeric M13 phages have been explored both as detection and therapeutic agents due to the flexibility in tuning target specificity. Transmission electron microscopy (TEM) is a powerful tool enabling researchers to directly visualize and characterize binding of phages to bacterial surfaces. However, the filamentous phage structure poses a challenge for this technique, as the phages have similar morphology to bacterial structures such as pili. In order to differentiate between bacterial structures and the filamentous phages, here we describe a protocol to prepare TEM samples of engineered M13 phages bound to bacterial cells, in which the phage virions have been specifically labeled by decoration of the major capsid proteins with gold nanoparticles. This protocol enables clear visualization and unambiguous identification of attached filamentous phages within the context of bacterial cells expressing numerous pili.

An Engineered M13 Filamentous Nanoparticle as an Antigen Carrier for a Malignant Melanoma Immunotherapeutic Strategy.

Bacteriophages, prokaryotic viruses, hold great potential in genetic engineering to open up new avenues for vaccine development. Our study aimed to establish engineered M13 bacteriophages expressing MAGE-A1 tumor peptides as a vaccine for melanoma treatment. Through in vivo experiments, we sought to assess their ability to induce robust immune responses. Using phage display technology, we engineered two M13 bacteriophages expressing MAGE-A1 peptides as fusion proteins with either pVIII or pIIII coat proteins. Mice were intraperitoneally vaccinated three times, two weeks apart, using two different engineered bacteriophages; control groups received a wild-type bacteriophage. Serum samples taken seven days after each vaccination were analyzed by ELISA assay, while splenocytes harvested seven days following the second boost were evaluated by ex vivo cytotoxicity assay. Fusion proteins were confirmed by Western blot and nano-LC-MS/MS. The application of bacteriophages was safe, with no adverse effects on mice. Engineered bacteriophages effectively triggered immune responses, leading to increased levels of anti-MAGE-A1 antibodies in proportion to the administered bacteriophage dosage. Anti-MAGE-A1 antibodies also exhibited a binding capability to B16F10 tumor cells in vitro, as opposed to control samples. Splenocytes demonstrated enhanced CTL cytotoxicity against B16F10 cells. We have demonstrated the immunogenic capabilities of engineered M13 bacteriophages, emphasizing their potential for melanoma immunotherapy.

Bacteriophage Engineering for Improved Copper Ion Binding.

In this study, fd viruses are genetically modified to display seven cropped versions (H, HG, HGF, HGFA, HGFAN, HGFANV and HGFANVA) of the previously identified Cu(II) specific peptide (HGFANVA). Atomic force microscopy (AFM) imaging reveals the typical filamentous structures of recombinant phages with thicknesses of ≈2-5 nm in dry state. Scanning electron microscopy (SEM) imaging shows that HGFANVA viruses form larger elongated assemblies than H viruses that are deposited with a mineral layer after Cu(II) treatment. C and N peaks are detected for virus samples through Energy dispersive X-ray spectroscopy (EDX) analyses confirming the presence of phage organic material. Cu peak is only detected for engineered viruses after Cu(II) exposure. Enzyme-linked immunosorbent assay (ELISA) analyses show the selective Cu(II) binding of engineered phages. Agarose gel electrophoresis (AGE) and zeta potential analyses reveal negative surface charges of engineered viral constructs. Positively charged Cytopore beads are coated with bacteriophages and used for Cu(II) ion sorption studies. ICP-MS analyses clearly show the improved Cu(II) binding of engineered viruses with respect to wild-type fd phages. Such bottom-up constructed, genetically engineered virus-based biomaterials may be applied in bioremediation studies targeting metal species from environmental samples.

Designing M13 Bacteriophage and Fe-Nanonest Self-Assembly System for Universal and Facile Preparation of Metal Single Atoms as Stable Mimicking Enzymes.

Metal single-atom catalysts (MSACs) possess multiple advantages in chemical synthesis; their efficient fabrication routes, however, remain a challenge to date. Here, an interdisciplinary design using M13 bacteriophage virus as a biotemplate to carry Fe nanoclusters, which we figuratively call "Fe-nanonests", is proposed to enable facile and versatile synthesis of MSACs. The feasibility and generality of this self-assembly method was demonstrated by the observation of six different metal single atoms (MSAs) including Ag, Pt, Pd, Zn, Cu, and Ni. With Pd as a representative, key factors dominating the fabrication were determined. The Pd single atoms exhibited excellent horseradish peroxidase (HRP)-like activity, which was further improved by 50% via genetic editing of the M13 pVIII protein terminals. Excellent stability was also observed in the quantification of acid phosphatase, a cancer predictor. X-ray absorption near-edge structure spectroscopy has been applied to the analysis of Pd single atoms as well, and the Pd-N4 coordination explained the mechanism of high HRP-like catalytic activity. The MSAs synthesized by the M13 phage and Fe-nanonest self-assembly method show promising prospects in non-cold-chain medical detection applications.

Serine-mediated hydrazone ligation displaying insulin-like peptides on M13 phage pIII.

Phage display has emerged as a tool for the discovery of therapeutic antibodies and proteins. However, the effective display and engineering of structurally complex proteins, such as insulin, pose significant challenges due to the sequence of insulin, which is composed of two peptide chains linked by three disulfide bonds. In this study, we developed a new approach for the display of insulin-like peptides on M13 phage pIII, employing N-terminal serine-mediated hydrazone ligation. The insulin-displaying phage retains the biological binding affinity of human insulin. To address the viability loss after ligation, we introduced a trypsin-cleavable spacer on pIII, enabling insulin-displayed phage library selection. This method offers a general pathway for the display of structurally complex proteins on pIII, enhancing the practicality of selecting chemically modified phage libraries and opening avenues for the engineering of new insulin analogs for the treatment of diabetes by using phage display.

Biomarker Discovery by ORFeome Phage Display.

Phage display is an efficient and robust method for protein-protein interaction studies. Although it is mostly used for antibody generation, it can be also utilized for the discovery of immunogenic proteins that could be used as biomarkers. Through this technique, a genome or metagenome is fragmented and cloned into a phagemid vector. The resulting protein fragments from this genetic material are displayed on M13 phage surface, while the corresponding gene fragments are packaged. This packaging process uses the pIII deficient helperphage, called Hyperphage (M13KO7 ΔpIII), so open reading frames (ORFs) are enriched in these libraries, giving the name to this method: ORFeome phage display. After conducting a selection procedure, called "bio-panning," relevant immunogenic peptides or protein fragments are selected using purified antibodies or serum samples, and can be used as potential biomarkers. As ORFeome phage display is an in vitro method, only the DNA or cDNA of the species of interest is needed. Therefore, this approach is also suitable for organisms that are hard to cultivate, or metagenomic samples, for example. An additional advantage is that the biomarker discovery is not limited to surface proteins due to the presentation of virtually every kind of peptide or protein fragment encoded by the ORFeome on the phage surface. At last, the selected biomarkers can be the start for the development of diagnostic assays, vaccines, or protein interaction studies.

Sensor development for multiple simultaneous classifications using genetically engineered M13 bacteriophages.

Sensors for detecting infinitesimal amounts of chemicals in air have been widely developed because they can identify the origin of chemicals. These sensing technologies are also used to determine the variety and freshness of fresh food and detect explosives, hazardous chemicals, environmental hormones, and diseases using exhaled gases. However, there is still a need to rapidly develop portable and highly sensitive sensors that respond to complex environments. Here, we show an efficient method for optimising an M13 bacteriophage-based multi-array colourimetric sensor for multiple simultaneous classifications. Apples, which are difficult to classify due to many varieties in distribution, were selected for classifying targets. M13 was adopted to fabricate a multi-array colourimetric sensor using the self-templating process since a chemical property of major coat protein p8 consisting of the M13 body can be manipulated by genetic engineering to respond to various target substances. The twenty sensor units, which consisted of different types of manipulated M13, exhibited colour changes because of the change of photonic crystal-like nanostructure when they were exposed to target substances associated with apples. The classification success rate of the optimal sensor combinations was achieved with high accuracy for the apple variety (100%), four standard fragrances (100%), and aging (84.5%) simultaneously. We expect that this optimisation technique can be used for rapid sensor development capable of multiple simultaneous classifications in various fields, such as medical diagnosis, hazardous environment monitoring, and the food industry, where sensors need to be developed in response to complex environments consisting of various targets.

Unraveling the potential of M13 phages in biomedicine: Advancing drug nanodelivery and gene therapy.

M13 phages possessing filamentous phage genomes offer the benefits of selective display of molecular moieties and delivery of therapeutic agent payloads with a tolerable safety profile. M13 phage-displayed technology for resembling antigen portions led to the discovery of mimetic epitopes that applied to antibody-based therapy and could be useful in the design of anticancer vaccines. To date, the excremental experiences have engaged the M13 phage in the development of innovative biosensors for detecting biospecies, biomolecules, and human cells with an acceptable limit of detection. Addressing the emergence of antibiotic-resistant bacteria, M13 phages are potent for packaging the programmed gene editing tools, such as CRISPR/Cas, to target multiple antimicrobial genes. Moreover, their display potential in combination with nanoparticles inspires new approaches for engineering targeted theragnostic platforms targeting multiple cellular biomarkers in vivo. In this review, we present the available data on optimizing the use of bacteriophages with a focus on the to date experiences with M13 phages, either as monoagent or as part of combination regimens in the practices of biosensors, vaccines, bactericidal, modeling of specific antigen epitopes, and phage-guided nanoparticles for drug delivery systems. Despite increasing research interest, a deep understanding of the underlying biological and genetic behaviors of M13 phages is needed to enable the full potential of these bioagents in biomedicine, as discussed here. We also discuss some of the challenges that have thus far limited the development and practical marketing of M13 phages.

Effects of the Magnetic Orientation of M13 Bacteriophage on Phage Display Selection.

Although phage display selection using a library of M13 bacteriophage has become a powerful tool for finding peptides that bind to target materials on demand, a remaining concern of this method is the interference by the M13 main body, which is a huge filament >103  times larger than the displayed peptide, and therefore would nonspecifically adhere to the target or sterically inhibit the binding of the displayed peptide. Meanwhile, filamentous phages are known to be orientable by an external magnetic field. If M13 filaments are magnetically oriented during the library selection, their angular arrangement relative to the target surface would be changed, being expected to control the interference by the M13 main body. This study reports that the magnetic orientation of M13 filaments vertical to the target surface significantly affects the selection. When the target surface was affinitive to the M13 main body, this orientation notably suppressed the nonspecific adhesion. Furthermore, when the target surface was less affinitive to the M13 main body and intrinsically free from the nonspecific adhesion, this orientation drastically changed the population of M13 clones obtained through library selection. The method of using no chemicals but only a physical stimulus is simple, clean, and expected to expand the scope of phage display selection.

Identification of the First Sulfobetaine Hydrogel-Binding Peptides via Phage Display Assay.

Using the M13 phage display, a series of 7- and 12-mer peptides which interact with new sulfobetaine hydrogels are identified. Two peptides each from the 7- and 12-mer peptide libraries bind to the new sulfobetaine hydrogels with high affinity compared to the wild-type phage lacking a dedicated hydrogel binding peptide. This is the first report of peptides binding to zwitterionic sulfobetaine hydrogels and the study therefore opens up the pathway toward new phage or peptide/hydrogel hybrids with high application potential.

Construction, Characterization, and Application of a Nonpathogenic Virus-like Model for SARS-CoV-2 Nucleocapsid Protein by Phage Display.

With the outbreak and spread of COVID-19, a deep investigation of SARS-CoV-2 is urgent. Direct usage of this virus for scientific research could provide reliable results and authenticity. However, it is strictly constrained and unrealistic due to its high pathogenicity and infectiousness. Considering its biosafety, different systems and technologies have been employed in immunology and biomedical studies. In this study, phage display technology was used to construct a nonpathogenic model for COVID-19 research. The nucleocapsid protein of SARS-CoV-2 was fused with the M13 phage capsid p3 protein and expressed on the M13 phages. After validation of its successful expression, its potential as the standard for qPCR quantification and affinity with antibodies were confirmed, which may show the possibility of using this nonpathogenic bacteriophage to replace the pathogenic virus in scientific research concerning SARS-CoV-2. In addition, the model was used to develop a system for the classification and identification of different samples using ATR-FTIR, which may provide an idea for the development and evaluation of virus monitoring equipment in the future.

Phage-free production of artificial ssDNA with Escherichia coli.

Artificial single-stranded DNA (ssDNA) with user-defined sequences and lengths up to the kilobase range is increasingly needed in mass quantities to realize the potential of emerging technologies such as genome editing and DNA origami. However, currently available biotechnological approaches for mass-producing ssDNA require dedicated, and thus costly, fermentation infrastructure, because of the risk of cross-contaminating manufacturer plants with self-replicating phages. Here we overcome this problem with an efficient, scalable, and cross-contamination-free method for the phage-free biotechnological production of artificial ssDNA with Escherichia coli. Our system utilizes a designed phagemid and an optimized helper plasmid. The phagemid encodes one gene of the M13 phage genome and a freely chosen custom target sequence, while the helper plasmid encodes the other genes of the M13 phage. The phagemid particles produced with this method are not capable of self-replication in the absence of the helper plasmid. This enables cross-contamination-free biotechnological production of ssDNA at any contract manufacturer. Furthermore, we optimized the process parameters to reduce by-products and increased the maximal product concentration up to 83 mg L-1 of ssDNA in a stirred-tank bioreactor, thus realizing up to a 40-fold increase in maximal product concentration over previous scalable phage-free ssDNA production methods.

Solid state NMR chemical shift assignment of the non-structural single-stranded DNA binding protein gVp from fd bacteriophage.

The non-structural gene V protein (pV, gVp) from fd virus is a non-specific single-stranded DNA binding protein. The role of gVp is to sequester the single-stranded DNA thus reducing the generation of the replicative DNA form and leading to the formation of progeny phage. In this study, we assigned the 13C and 15N resonances of the crystalline unbound protein by magic-angle spinning solid-state NMR. The secondary structure predicted by the NMR shifts is in excellent agreement with the X-ray structure of the same 87-residue protein.

Orthogonal nanoarchitectonics of M13 phage for receptor targeted anticancer photodynamic therapy.

Photodynamic therapy (PDT) represents a promising therapeutic modality for cancer. Here we used an orthogonal nanoarchitectonics approach (genetic/chemical) to engineer M13 bacteriophages as targeted vectors for efficient photodynamic killing of cancer cells. M13 was genetically refactored to display on the phage tip a peptide (SYPIPDT) able to bind the epidermal growth factor receptor (EGFR). The refactored M13EGFR phages demonstrated EGFR-targeted tropism and were internalized by A431 cancer cells, that overexpress EGFR. Using an orthogonal approach to the genetic display, M13EGFR phages were then chemically modified, conjugating hundreds of Rose Bengal (RB) photosensitizing molecules on the capsid surface, without affecting the selective recognition of the SYPIPDT peptides. Upon internalization, the M13EGFR-RB derivatives generated intracellularly reactive oxygen species, activated by an ultralow intensity white light irradiation. The killing activity of cancer cells is observed at picomolar concentrations of the M13EGFR phage.

Systematic molecular evolution enables robust biomolecule discovery.

Evolution occurs when selective pressures from the environment shape inherited variation over time. Within the laboratory, evolution is commonly used to engineer proteins and RNA, but experimental constraints have limited the ability to reproducibly and reliably explore factors such as population diversity, the timing of environmental changes and chance on outcomes. We developed a robotic system termed phage- and robotics-assisted near-continuous evolution (PRANCE) to comprehensively explore biomolecular evolution by performing phage-assisted continuous evolution in high-throughput. PRANCE implements an automated feedback control system that adjusts the stringency of selection in response to real-time measurements of each molecular activity. In evolving three distinct types of biomolecule, we find that evolution is reproducibly altered by both random chance and the historical pattern of environmental changes. This work improves the reliability of protein engineering and enables the systematic analysis of the historical, environmental and random factors governing biomolecular evolution.

DNA-Encoded Multivalent Display of Chemically Modified Protein Tetramers on Phage: Synthesis and in Vivo Applications.

Phage display links the phenotype of displayed polypeptides with the DNA sequence in the phage genome and offers a universal method for the discovery of proteins with novel properties. However, the display of large multisubunit proteins on phages remains a challenge. A majority of protein display systems are based on monovalent phagemid constructs, but methods for the robust display of multiple copies of large proteins are scarce. Here, we describe a DNA-encoded display of a ∼ 200 kDa tetrameric l-asparaginase protein on M13 and fd phages produced by ligation of SpyCatcher-Asparaginase fusion (ScA) and PEGylated-ScA (PEG-ScA) to barcoded phage clones displaying SpyTag peptide. Starting from the SpyTag display on p3 or p8 coat proteins yielded constructs with five copies of ScA displayed on p3 (ScA-p3), ∼100 copies of ScA on p8 protein (ScA-p8) and ∼300 copies of PEG-ScA on p8 protein (PEG-ScA-p8). Display constructs of different valencies and chemical modifications on protein (e.g., PEGylation) can be injected into mice and analyzed by deep sequencing of the DNA barcodes associated with phage clones. In these multiplexed studies, we observed a density and protein-dependent clearance rate in vivo. Our observations link the absence of PEGylation and increase in density of the displayed protein with the increased rate of the endocytosis by cells in vivo. In conclusion, we demonstrate that a multivalent display of l-asparaginase on phages could be used to study the circulation life of this protein in vivo, and such an approach opens the possibility to use DNA sequencing to investigate multiplexed libraries of other multisubunit proteins in vivo.

Phage-delivered CRISPR-Cas9 for strain-specific depletion and genomic deletions in the gut microbiome.

Mechanistic insights into the role of the human microbiome in the predisposition to and treatment of disease are limited by the lack of methods to precisely add or remove microbial strains or genes from complex communities. Here, we demonstrate that engineered bacteriophage M13 can be used to deliver DNA to Escherichia coli within the mouse gastrointestinal (GI) tract. Delivery of a programmable exogenous CRISPR-Cas9 system enables the strain-specific depletion of fluorescently marked isogenic strains during competitive colonization and genomic deletions that encompass the target gene in mice colonized with a single strain. Multiple mechanisms allow E. coli to escape targeting, including loss of the CRISPR array or even the entire CRISPR-Cas9 system. These results provide a robust and experimentally tractable platform for microbiome editing, a foundation for the refinement of this approach to increase targeting efficiency, and a proof of concept for the extension to other phage-bacterial pairs of interest.

Membrane Insertion of the M13 Minor Coat Protein G3p Is Dependent on YidC and the SecAYEG Translocase.

The minor coat protein G3p of bacteriophage M13 is the key component for the host interaction of this virus and binds to Escherichia coli at the tip of the F pili. As we show here, during the biosynthesis of G3p as a preprotein, the signal sequence interacts primarily with SecY, whereas the hydrophobic anchor sequence at the C-terminus interacts with YidC. Using arrested nascent chains and thiol crosslinking, we show here that the ribosome-exposed signal sequence is first contacted by SecY but not by YidC, suggesting that only SecYEG is involved at this early stage. The protein has a large periplasmic domain, a hydrophobic anchor sequence of 21 residues and a short C-terminal tail that remains in the cytoplasm. During the later synthesis of the entire G3p, the residues 387, 389 and 392 in anchor domain contact YidC in its hydrophobic slide to hold translocation of the C-terminal tail. Finally, the protein is processed by leader peptidase and assembled into new progeny phage particles that are extruded out of the cell.