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.

Parallel molecular computation on digital data stored in DNA.

DNA is an incredibly dense storage medium for digital data. However, computing on the stored information is expensive and slow, requiring rounds of sequencing, in silico computation, and DNA synthesis. Prior work on accessing and modifying data using DNA hybridization or enzymatic reactions had limited computation capabilities. Inspired by the computational power of "DNA strand displacement," we augment DNA storage with "in-memory" molecular computation using strand displacement reactions to algorithmically modify data in a parallel manner. We show programs for binary counting and Turing universal cellular automaton Rule 110, the latter of which is, in principle, capable of implementing any computer algorithm. Information is stored in the nicks of DNA, and a secondary sequence-level encoding allows high-throughput sequencing-based readout. We conducted multiple rounds of computation on 4-bit data registers, as well as random access of data (selective access and erasure). We demonstrate that large strand displacement cascades with 244 distinct strand exchanges (sequential and in parallel) can use naturally occurring DNA sequence from M13 bacteriophage without stringent sequence design, which has the potential to improve the scale of computation and decrease cost. Our work merges DNA storage and DNA computing, setting the foundation of entirely molecular algorithms for parallel manipulation of digital information preserved in DNA.

Utility of protein-protein binding surfaces composed of anti-parallel alpha-helices and beta-sheets selected by phage display.

Over the past 3 decades, a diverse collection of small protein domains have been used as scaffolds to generate general purpose protein-binding reagents using a variety of protein display and enrichment technologies. To expand the repertoire of scaffolds and protein surfaces that might serve this purpose, we have explored the utility of (i) a pair of anti-parallel alpha-helices in a small highly disulfide-bonded 4-helix bundle, the CC4 domain from reversion-inducing Cysteine-rich Protein with Kazal Motifs and (ii) a concave beta-sheet surface and two adjacent loops in the human FN3 domain, the scaffold for the widely used monobody platform. Using M13 phage display and next generation sequencing, we observe that, in both systems, libraries of ∼30 million variants contain binding proteins with affinities in the low µM range for baits corresponding to the extracellular domains of multiple mammalian proteins. CC4- and FN3-based binding proteins were fused to the N- and/or C-termini of Fc domains and used for immunostaining of transfected cells. Additionally, FN3-based binding proteins were inserted into VP1 of AAV to direct AAV infection to cells expressing a defined surface receptor. Finally, FN3-based binding proteins were inserted into the Pvc13 tail fiber protein of an extracellular contractile injection system particle to direct protein cargo delivery to cells expressing a defined surface receptor. These experiments support the utility of CC4 helices B and C and of FN3 beta-strands C, D, and F together with adjacent loops CD and FG as surfaces for engineering general purpose protein-binding reagents.

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.

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.

Direct interaction between fd phage pilot protein pIII and the TolQ-TolR proton-dependent motor provides new insights into the import of filamentous phages.

Filamentous phages are one of the simplest examples of viruses with a protein capsid that protects a circular single-stranded DNA genome. The infection is very specific, nonlytic, and can strongly affect the physiology or provide new pathogenic factors to its bacterial host. The infection process is proposed to rely on a pore-forming mechanism similar to that of certain nonenveloped eukaryotic viruses. The Ff coliphages (including M13, fd, and f1) have been intensively studied and were used to establish the sequence of events taking place for efficient crossing of the host envelope structure. However, the mechanism involved in the penetration of the cell inner membrane is not well understood. Here, we identify new host players involved in the phage translocation mechanism. Interaction studies by a combination of in vivo biochemical methods demonstrate that the adhesion protein pIII located at the tip of the phage binds to TolQ and TolR, two proteins that form a conserved proton-dependent molecular motor in the inner membrane of the host cell. Moreover, in vivo cysteine cross-linking studies reveal that the interactions between the pIII and TolQ or TolR occur between their transmembrane helix domains and may be responding to the proton motive force status of the cell. These results allow us to propose a model for the late stage of filamentous phage translocation mediated by multiple interactions with each individual component of the host TolQRA complex.

Evaluation of bacteriophages for the alleviation of potential bacterial contamination risks in developmental engineering.

This research aimed to address the potential bacterial contamination risks in developmental engineering (DE) using bacteriophages. To compare and contrast the exemplar Escherichia coli T4 and M13 bacteriophages, human dermal fibroblasts cultivated on culture plates, natural cellulosic scaffolds, and poly(methyl methacrylate) (PMMA) particles were utilized as two-dimensional (2D) cell, three-dimensional (3D) tissue, and modular tissue culture models, respectively. When directly introduced into these distinct culture systems, both phages survived, exhibited no significant effects on the cultured cells or tissues, yet displayed their potentials to alleviate the infections caused by corresponding bacterial host cells. Apart from direct addition into the culture medium, both phages were also coated on PMMA, polystyrene, poly(lactic acid) particles with different diameters (5, 10, 30, and 100 µm) and cellulosic scaffolds. The coated phages endured the coating processes and demonstrated their viabilities in plaque assays. Further testing indicated that the phages coated on the PMMA particles tolerated multiple deliberate rinses and centrifugations, but not thermal treatment at 60-80°C. In summary, T4 and M13 bacteriophages not only manifested their antibacterial functions in diverse 2D cell, 3D tissue, and modular tissue culture systems, but also demonstrated their potentials of coating modular scaffolds to alleviate the bacterial contamination risks in DE.

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.

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.

Advanced detection of cervical cancer biomarkers using engineered filamentous phage nanofibers.

Cervical cancer is a major global health concern, characterized by its high incidence and mortality rates. The detection of tumor markers is crucial for managing cancer, making treatment decisions, and monitoring disease progression. Vascular endothelial growth factor (VEGF) and programmed death-ligand 1 (PDL-1) are key targets in cervical cancer therapy and valuable biomarkers in predicting treatment response and prognosis. In this study, we found that combining the measurement of VEGF and soluble PDL-1 can be used for diagnosing and evaluating the progression of cervical cancer. To explore a more convenient approach for detecting and assessing cervical cancer, we designed and prepared an engineered fd bacteriophage, a human-safe viral nanofiber, equipped with two peptides targeting VEGF and PD-L1. The dual-display phage nanofiber specifically recognizes and binds to both proteins. Utilizing this nanofiber as a novel capture agent, we developed a new enzyme-linked immunosorbent assay (ELISA) method. This method shows significantly enhanced detection sensitivity compared to conventional ELISA methods, which use either anti-VEGF or anti-PD-L1 antibodies as capture agents. Therefore, the phage dual-display nanofiber presents significant potential in detecting cancer markers, evaluating medication efficacy, and advancing immunotherapy drug development. KEY POINTS: • The combined measurement of VEGF and soluble Programmed Death-Ligand 1(sPD-L1) demonstrates an additive effect in the diagnosis of cervical cancer. Fd phage nanofibers have been ingeniously engineered to display peptides that bind to VEGF and PD-L1, enabling the simultaneous detection of both proteins within a single assay • Genetically engineered phage nanofibers, adorned with two distinct peptides, can be utilized for the diagnosis and prognosis of cancer and can be mass-produced cost-effectively through bacterial infections • Employing dual-display fd phage nanofibers as capture probes, the phage ELISA method exhibited significantly enhanced detection sensitivity compared to traditional sandwich ELISA. Furthermore, phage ELISA facilitates the detection of a single protein or the simultaneous detection of multiple proteins, rendering them powerful tools for protein analysis and diagnosis across various fields, including cancer research.

Truncated M13 phage for smart detection of E. coli under dark field.

BACKGROUND: The urgent need for affordable and rapid detection methodologies for foodborne pathogens, particularly Escherichia coli (E. coli), highlights the importance of developing efficient and widely accessible diagnostic systems. Dark field microscopy, although effective, requires specific isolation of the target bacteria which can be hindered by the high cost of producing specialized antibodies. Alternatively, M13 bacteriophage, which naturally targets E. coli, offers a cost-efficient option with well-established techniques for its display and modification. Nevertheless, its filamentous structure with a large length-diameter ratio contributes to nonspecific binding and low separation efficiency, posing significant challenges. Consequently, refining M13 phage methodologies and their integration with advanced microscopy techniques stands as a critical pathway to improve detection specificity and efficiency in food safety diagnostics. METHODS: We employed a dual-plasmid strategy to generate a truncated M13 phage (tM13). This engineered tM13 incorporates two key genetic modifications: a partial mutation at the N-terminus of pIII and biotinylation at the hydrophobic end of pVIII. These alterations enable efficient attachment of tM13 to diverse E. coli strains, facilitating rapid magnetic separation. For detection, we additionally implemented a convolutional neural network (CNN)-based algorithm for precise identification and quantification of bacterial cells using dark field microscopy. RESULTS: The results obtained from spike-in and clinical sample analyses demonstrated the accuracy, high sensitivity (with a detection limit of 10 CFU/µL), and time-saving nature (30 min) of our tM13-based immunomagnetic enrichment approach combined with AI-enabled analytics, thereby supporting its potential to facilitate the identification of diverse E. coli strains in complex samples. CONCLUSION: The study established a rapid and accurate detection strategy for E. coli utilizing truncated M13 phages as capture probes, along with a dark field microscopy detection platform that integrates an image processing model and convolutional neural network.

Engineered Living Bacteriophage-Enabled Self-Adjuvanting Hydrogel for Remodeling Tumor Microenvironment and Cancer Therapy.

Due to the complexity and heterogeneity in the tumor microenvironment, the efficacy of breast cancer treatment has been significantly impeded. Here, we established a living system using an engineered M13 bacteriophage through chemical cross-linking and biomineralization to remodel the tumor microenvironment. Chemically cross-linking of the engineered bacteriophage gel (M13 Gel) could in situ synthesize photothermal palladium nanoparticles (PdNPs) on the pVIII capsid protein to obtain M13@Pd Gel. In addition, NLG919 was further loaded into a gel to form (M13@Pd/NLG gel) for down-regulating the expression of tryptophan metabolic enzyme indoleamine 2,3-dioxygenase 1 (IDO1). Both in vitro and in vivo studies showed that the M13 bacteriophage served not only as a cargo-loaded device but also as a self-immune adjuvant, which induced the immunogenic death of tumor cells effectively and down-regulated IDO1 expression. Such a bioactive gel system constructed by natural living materials could reverse immunosuppression and significantly improve the anti-breast cancer response.

Engineered M13-Derived Bacteriophages Capable of Gold Nanoparticle Synthesis and Nanogold Manipulations.

For years, gold nanoparticles (AuNPs) have been widely used in medicine and industry. Although various experimental procedures have been reported for their preparation and manipulation, none of them is optimal for all purposes. In this work, we engineered the N-terminus of the pIII minor coat protein of bacteriophage (phage) M13 to expose a novel HLYLNTASTHLG peptide that effectively and specifically binds gold. In addition to binding gold, this engineered phage could synthesize spherical AuNPs of 20 nm and other sizes depending on the reaction conditions, aggregate them, and precipitate gold from a colloid, as revealed by transmission electron microscopy (TEM), atomic force microscopy (AFM), and scanning electron microscopy (SEM), as well as ultraviolet-visible (UV-vis) and Fourier-transform infrared (FTIR) spectroscopic methods. We demonstrated that the engineered phage exposing a foreign peptide selected from a phage-displayed library may serve as a sustainable molecular factory for both the synthesis of the peptide and the subsequent overnight preparation of AuNPs from gold ions at room temperature and neutral pH in the absence of strong reducing agents, such as commonly used NaBH4. Taken together, the results suggest the potential applicability of the engineered phage and the new, in vitro-identified gold-binding peptide in diverse biomimetic manipulations.

Atomic-Resolution Structure of the Protein Encoded by Gene V of fd Bacteriophage in Complex with Viral ssDNA Determined by Magic-Angle Spinning Solid-State NMR.

F-specific filamentous phages, elongated particles with circular single-stranded DNA encased in a symmetric protein capsid, undergo an intermediate step, where thousands of homodimers of a non-structural protein, gVp, bind to newly synthesized strands of DNA, preventing further DNA replication and preparing the circular genome in an elongated conformation for assembly of a new virion structure at the membrane. While the structure of the free homodimer is known, the ssDNA-bound conformation has yet to be determined. We report an atomic-resolution structure of the gVp monomer bound to ssDNA of fd phage in the nucleoprotein complex elucidated via magic-angle spinning solid-state NMR. The model presents significant conformational changes with respect to the free form. These modifications facilitate the binding mechanism and possibly promote cooperative binding in the assembly of the gVp-ssDNA complex.

Investigation of Peptides for Molecular Recognition of C-Reactive Protein-Theoretical and Experimental Studies.

We investigate the interactions between C-reactive protein (CRP) and new CRP-binding peptide materials using experimental (biological and physicochemical) methods with the support of theoretical simulations (computational modeling analysis). Three specific CRP-binding peptides (P2, P3, and P9) derived from an M13 bacteriophage have been identified using phage-display technology. The binding efficiency of the peptides exposed on phages toward the CRP protein was demonstrated via biological methods. Fibers of the selected phages/peptides interact differently due to different compositions of amino acid sequences on the exposed peptides, which was confirmed by transmission electron microscopy. Numerical and experimental studies consistently showed that the P3 peptide is the best CRP binder. A combination of theoretical and experimental methods demonstrates that identifying the best binder can be performed simply, cheaply, and fast. Such an approach has not been reported previously for peptide screening and demonstrates a new trend in science where calculations can replace or support laborious experimental techniques. Finally, the best CRP binder─the P3 peptide─was used for CRP recognition on silicate-modified indium tin oxide-coated glass electrodes. The obtained electrodes exhibit a wide range of operation (1.0-100 µg mL-1) with a detection limit (LOD = 3σ/S) of 0.34 µg mL-1. Moreover, the dissociation constant Kd of 4.2 ± 0.144 µg mL-1 (35 ± 1.2 nM) was evaluated from the change in the current. The selectivity of the obtained electrode was demonstrated in the presence of three interfering proteins. These results prove that the presented P3 peptide is a potential candidate as a receptor for CRP, which can replace specific antibodies.

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.

Genetically encoded multivalent liquid glycan array displayed on M13 bacteriophage.

The central dogma of biology does not allow for the study of glycans using DNA sequencing. We report a liquid glycan array (LiGA) platform comprising a library of DNA 'barcoded' M13 virions that display 30-1,500 copies of glycans per phage. A LiGA is synthesized by acylation of the phage pVIII protein with a dibenzocyclooctyne, followed by ligation of azido-modified glycans. Pulldown of the LiGA with lectins followed by deep sequencing of the barcodes in the bound phage decodes the optimal structure and density of the recognized glycans. The LiGA is target agnostic and can measure the glycan-binding profile of lectins, such as CD22, on cells in vitro and immune cells in a live mouse. From a mixture of multivalent glycan probes, LiGAs identify the glycoconjugates with optimal avidity necessary for binding to lectins on living cells in vitro and in vivo.

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.

M13 phage: a versatile building block for a highly specific analysis platform.

Viruses are changing the biosensing and biomedicine landscape due to their multivalency, orthogonal reactivities, and responsiveness to genetic modifications. As the most extensively studied phage model for constructing a phage display library, M13 phage has received much research attention as building blocks or viral scaffolds for various applications including isolation/separation, sensing/probing, and in vivo imaging. Through genetic engineering and chemical modification, M13 phages can be functionalized into a multifunctional analysis platform with various functional regions conducting their functionality without mutual disturbance. Its unique filamentous morphology and flexibility also promoted the analytical performance in terms of target affinity and signal amplification. In this review, we mainly focused on the application of M13 phage in the analytical field and the benefit it brings. We also introduced several genetic engineering and chemical modification approaches for endowing M13 with various functionalities, and summarized some representative applications using M13 phages to construct isolation sorbents, biosensors, cell imaging probes, and immunoassays. Finally, current issues and challenges remaining in this field were discussed and future perspectives were also proposed.