Evolutionary Design of Self-Templated Supramolecular Fibrils Using M13 Bacteriophage for Tissue Engineering.

Biomaterials in nature form hierarchical structures and functions across various length scales through binding and assembly processes. Inspired by nature, we developed hierarchically organized tissue engineering materials through evolutionary screening and self-templating assembly. Leveraging the M13 bacteriophage (phage), we employed an evolutionary selection process against hydroxyapatite (HA) to isolate HA-binding phage (HAPh). The newly discovered phage exhibits a bimodal length, comprising 950 nm and 240 nm, where the synergistic effect of these dual lengths promotes the formation of supramolecular fibrils with periodic banded structures. The assembled HAPh fibrils show the capability of HA mineralization and the directional growth of osteoblast cells. When applied to a dentin surface, it induces the regeneration of dentin-like tissue structures, showcasing its potential applications as a scaffold in tissue engineering. The integration of evolutionary screening and self-templating assembly holds promise for the future development of hierarchically organized tissue engineering materials.

Virus-Based Separation of Rare Earth Elements.

The utilization of biomaterials for the separation of rare earth elements (REEs) has attracted considerable interest due to their inherent advantages, including diverse molecular structures for selective binding and the use of eco-friendly materials for sustainable systems. We present a pioneering methodology for developing a safe virus to selectively bind REEs and facilitate their release through pH modulation. We engineered the major coat protein of M13 bacteriophage (phage) to incorporate a lanthanide-binding peptide. The engineered lanthanide-binding phage (LBPh), presenting ∼3300 copies of the peptide, serves as an effective biological template for REE separation. Our findings demonstrate the LBPh's preferential binding for heavy REEs over light REEs. Moreover, the LBPh exhibits remarkable robustness with excellent recyclability and stability across multiple cycles of separations. This study underscores the potential of genetically integrating virus templates with selective binding motifs for REE separation, offering a promising avenue for environmentally friendly and energy-efficient separation processes.

A new standardization for the use of chicken embryo: selection of target from the phage display library and infection.

The filamentous bacteriophage M13KO7 (M13) is the most used in phage display (PD) technology and, like other phages, has been applied in several areas of medicine, agriculture, and in the food industry. One of the advantages is that they can modulate the immune response in the presence of pathogenic microorganisms, such as bacteria and viruses. This study evaluated the use of phage M13 in the chicken embryos model. We inoculated 13-day-old chicken embryos with Salmonella Pullorum (SP) and then evaluated survival for the presence of phage M13 or E. coli ER2738 (ECR) infected with M13. We found that the ECR bacterium inhibits SP multiplication in 0.32 (M13-infected ECR) or 0.44 log UFC/mL (M13-uninfected ECR) and that the ECR-free phage M13 from the PD library can be used in chicken embryo models. This work provides the use of the chicken embryo as a model to study systemic infection and can be employed as an analysis tool for various peptides that M13 can express from PD selection. KEY POINTS: • SP-infected chicken embryo can be a helpful model of systemic infection for different tests. • Phage M13 does not lead to embryonic mortality or cause serious injury to embryos. • Phage M13 from the PD library can be used in chicken embryo model tests.

Specific interaction between the DSPHTELP peptide and various functional groups.

M13 bacteriophages serve as a versatile foundation for nanobiotechnology due to their unique biological and chemical properties. The polypeptides that comprise their coat proteins, specifically pVIII, can be precisely tailored through genetic engineering. This enables the customized integration of various functional elements through specific interactions, leading to the development of innovative hybrid materials for applications such as energy storage, biosensing, and catalysis. Notably, a certain genetically engineered M13 bacteriophage variant, referred to as DSPH, features a pVIII with a repeating DSPHTELP peptide sequence. This sequence facilitates specific adhesion to single-walled carbon nanotubes (SWCNTs), primarily through π-π and hydrophobic interactions, though the exact mechanism remains unconfirmed. In this study, we synthesized the DSPHTELP peptide (an 8-mer peptide) and analyzed its interaction forces with different functional groups across various pH levels using surface forces apparatus (SFA). Our findings indicate that the 8-mer peptide binds most strongly to CH3 groups (Wad = 13.74 ± 1.04 mJ m-2 at pH 3.0), suggesting that hydrophobic interactions are indeed the predominant mechanism. These insights offer both quantitative and qualitative understanding of the molecular interaction mechanisms of the 8-mer peptide and clarify the basis of its specific interaction with SWCNTs through the DSPHTELP M13 bacteriophage.

Weak-cooperative binding of a long single-stranded DNA chain on a surface.

Binding gene-wide single-stranded nucleic acids to surface-immobilized complementary probes is an important but challenging process for biophysical studies and diagnostic applications. The challenge comes from the conformational dynamics of the long chain that affects its accessibility and weakens its hybridization to the probes. We investigated the binding of bacteriophage genome M13mp18 on several different 20-mer probes immobilized on the surface of a multi-spot, label-free biosensor, and observed that only a few of them display strong binding capability with dissociation constant as low as 10 pM. Comparing experimental data and computational analysis of the M13mp18 chain structural features, we found that the capturing performance of a specific probe is directly related to the multiplicity of binding sites on the genomic strand, and poorly connected with the predicted secondary and tertiary structure. We show that a model of weak cooperativity of transient bonds is compatible with the measured binding kinetics and accounts for the enhancement of probe capturing observed when more than 20 partial pairings with binding free energy lower than -10 kcal mol-1 are present. This mechanism provides a specific pattern of response of a genomic strand on a panel of properly selected oligomer probe sequences.

A gradient phage-assisted continuous evolution method for screening suppressor tRNAs in Escherichia coli.

Suppressor tRNAs, notable for their capability of reading through the stop codon while maintaining normal peptide synthesis, are promising in treating diseases caused by premature termination codons (PTC). However, the lack of effective engineering methods for suppressor tRNAs has curtailed their application potential. Here, we introduce a directed evolution technology that employs phage-assisted continuous evolution (PACE), combined with gradient biosensors featuring various PTCs in the M13 gene III. Utilizing this novel methodology, we have successfully evolved tRNATrp (UGG) reading through the UGA stop codon in Escherichia coli. Massively parallel sequencing revealed that these mutations predominantly occurred in the anticodon loop. Finally, two suppressor tRNATrp (UGA) mutants exhibited over fivefold increases in readthrough efficiency.

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.