M13 Phage Research Articles

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 enabled 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 enabled 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.

Tumorigenic bacteria in colorectal cancer: mechanisms and treatments.

Colorectal cancer (CRC) is the third most common and the second most fatal cancer. In recent years, more attention has been directed toward the role of gut microbiota in the initiation and development of CRC. Some bacterial species, such as Fusobacterium nucleatum, Escherichia coli, Bacteroides fragilis, Enterococcus faecalis, and Salmonella sp. have been associated with CRC, based upon sequencing studies in CRC patients and functional studies in cell culture and animal models. These bacteria can cause host DNA damage by genotoxic substances, including colibactin secreted by pks + Escherichia coli, B. fragilis toxin (BFT) produced by Bacteroides fragilis, and typhoid toxin (TT) from Salmonella. These bacteria can also indirectly promote CRC by influencing host-signaling pathways, such as E-cadherin/β-catenin, TLR4/MYD88/NF-κB, and SMO/RAS/p38 MAPK. Moreover, some of these bacteria can contribute to CRC progression by helping tumor cells to evade the immune response by suppressing immune cell function, creating a pro-inflammatory environment, or influencing the autophagy process. Treatments with the classical antibacterial drugs, metronidazole or erythromycin, the antibacterial active ingredients, M13@ Ag (electrostatically assembled from inorganic silver nanoparticles and the protein capsid of bacteriophage M13), berberine, and zerumbone, were found to inhibit tumorigenic bacteria to different degrees. In this review, we described progress in elucidating the tumorigenic mechanisms of several CRC-associated bacteria, as well as progress in developing effective antibacterial therapies. Specific bacteria have been shown to be active in the oncogenesis and progression of CRC, and some antibacterial compounds have shown therapeutic potential in bacteria-induced CRC. These bacteria may be useful as biomarkers or therapeutic targets for CRC.

A blood circulation-prolonging peptide anchored biomimetic phage-platelet hybrid nanoparticle system for prolonged blood circulation and optimized anti-bacterial performance.

Phage therapy holds great promise for resolving the ever-worsening crisis of antibiotic resistance, but it also faces many challenges. One of the issues hampering phage therapy is the short blood residence time of bacteriophages. We have previously identified, through in vivo phage display, a blood circulation-prolonging peptide (BCP1) that was capable of significantly prolonging the blood retention time of a doxorubicin-loaded human ferritin nanocage, leading to enhanced therapeutic efficacy against tumors. Herein, we aimed to extend the application of BCP1 to anti-bacterial phage therapy.Methods: A genetically engineered M13 phage, BCP1-BGL, that displayed the BCP-1 peptide and expressed the restriction endonuclease Bgl II, was constructed. Taking advantage of the fact that BCP1 harbors an RGD motif (a three amino-acid sequence Arg-Gly-Asp with the ability to bind to integrins) and exerts its circulation-prolonging activity primarily through interaction with platelets, we further designed and fabricated a biomimetic phage-platelet hybrid nanoparticle (PPHN) via the physical binding of the BCP1-BGL phage to the platelet membrane nanoparticles derived via a repeated freeze-thaw procedure. A series of experiments in vitro and in vivo were conducted to reveal the long circulation and anti-bacterial capacities of BCP1-BGL phages and PPHNs.Results: The resulting PPHNs possessed a hydrodynamic size of 368 nm in deionized water, with each spherical membranous nanoparticle harboring approximately 12 rod-shaped phage particles stably bound to its surface. PPHNs, which were superior to the BCP1-BGL phages that displayed significantly prolonged anti-bacterial action in vivo against Escherichia coli infection, exhibited further extended blood retention time and optimal anti-bacterial performance in both the prophylactic and treatment approaches.Conclusion: Our work demonstrated a novel strategy in engineering biomimetic phage-based nanoparticles with improved blood retention and anti-bacterial performance and may have implications in phage therapy.

Selection of a d-Enantiomeric Peptide Specifically Binding to PHF6 for Inhibiting Tau Aggregation in Transgenic Mice.

Tauopathies refer to a group of neurodegenerative disorders caused by the accumulation of insoluble hyperphosphorylated Tau protein in the brain. The inhibition and interruption of Tau aggregation are considered important strategies to ameliorate the neurodegenerative process. Previous work has shown that hexapeptide 306VQIVYK311 (PHF6) located in the repeat domain 3 of Tau protein drives Tau aggregation and itself forms a β-sheet structure similar to those of Tau-oligomers and neurofibrillary tangles (NFTs). In this study, a mirror image phage display technology was used to screen protease-resistant and low-immunogenic d-enantiomeric peptides for their capacity to inhibit Tau aggregation. Following the preparation of d-enantiomeric PHF6 fibrils and M13 phage peptide library biopanning, 7 sets of high specificity peptides were obtained. Through ELISA and competition inhibition assays, we chose a highly specific peptide p-NH with the sequence N-I-T-M-N-S-R-R-R-R-N-H. The molecular docking results showed that p-NH interacted with PHF6 fibrils mainly through van der Waals forces and hydrogen bonding and could inhibit PHF6 aggregation in a d-configuration and concentration-dependent manner. In vitro, p-NH prohibited the formation of PHF6 fibrils and was able to enter into mouse neuroblastoma N2a cells (N2a cells) to inhibit Tau hyperphosphorylation and aggregation. Intranasal administration of p-NH reduced NFTs and improved the cognitive ability of TauP301S transgenic mice. These findings represent a straightforward methodology to find therapeutic peptides with potential applications in tauopathies.

Rapid detection of bacterial pathogens in blood through engineered phages-beads and integrated Real-Time PCR into MicroChip

The invasion of pathogens into the bloodstream can produce a systemic inflammatory response syndrome that, if not promptly treated, rapidly evolves in acute life-threatening dysfunction of remote organs (septic shock). Current conventional diagnostic tests are microbial blood cultures that need days for results. Therefore, it is crucial the availability of rapid and reliable pathogens detection for prompt diagnosis. In this work, we developed a new method for fast and sensitive detection of bacteria in blood based on the integration of pathogen capture through engineered M13 phages and molecular detection via PCR in miniaturized silicon microchip. Engineered M13 phage-clones exposing specific peptides able to bind Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and a novel identified phage clone, 9IIIB5, for Staphylococcus epidermidis, were used to functionalize magnetic beads and capturing/concentrate the pathogens. Due to the stability of beads-phage-bacteria complex, PCR inhibitors present in the blood were easily removed without detachment of captured bacteria. The complex was used as target for direct amplification into miniaturized silicon chip by a multiplex PCR followed by Real-Time PCR. The procedure was very fast (less than 3 hs) and sensitive (10 cells/reaction) enabling molecular detection inside to the critical period for clinical decision in sepsis. It could be applied to detect pathogens present in very few numbers or in a metabolically inactive state. The results here presented pay the way to future development of portable genetic Point-of-Care in the rapid identification of microorganisms present in the blood stream or also in different matrices, such as waters and foods.

Nanogenerators facilitated piezoelectric and flexoelectric characterizations for bioinspired energy harvesting materials

Abstract Deformation-derived electric polarization has been adopted as a core technology for electromechanics devices. Now, as the device experiences the multiple degrees of deformation, such as compressing, bending and stretching, upon the development of technology, the characterization techniques for devices need to be retrofitted to account for the different mechanistic origins of electric polarization. Here, we report the nanogenerators that enable a facile characterization of piezoelectricity and flexoelectricity in the device scale. The quadrant electrode NG along with nematically organized M13 bacteriophage provides the comprehensive piezoelectric coefficients while the flexoelectric coefficient was extracted from sandwich electrode NG. This approach offers a perspective on how to separate the piezoelectric and flexoelectric effects from the electric outputs. Such characterization will not only allow us an understanding of fundamental flexoelectric mechanisms but also help us to establish design rules for flexible electromechanical devices.

Investigation of intestinal transportation of peptide‐displaying bacteriophage particles using phage display method

To investigate whether peptide sequences with specific translocation across the gastrointestinal barrier can be identified as drug delivery vehicles, in vivo phage display was conducted. For this purpose, a random library of 12-mer peptides displayed on M13 bacteriophage was orally administered to mice followed by recovery of the phage particles from the blood samples after three consecutive biopanning rounds. The obtained peptide sequences were analyzed using bioinformatics tools and software. The results demonstrated that M13 bacteriophage bearing peptides translocate nonspecifically across the mice intestinal mucosal barrier deduced from random distribution of amino acids in different positions of the identified peptide sequences. The most probable reason for entering the phage particles into systemic circulation after oral administration of the peptide library can be related to the nanoscale nature of their structures which provides a satisfying platform for the purpose of designing nanocarriers in pharmaceutical applications.

Sites of vulnerability on ricin B chain revealed through epitope mapping of toxin-neutralizing monoclonal antibodies.

Ricin toxin's B subunit (RTB) is a multifunctional galactose (Gal)-/N-acetylgalactosamine (GalNac)-specific lectin that promotes uptake and intracellular trafficking of ricin's ribosome-inactivating subunit (RTA) into mammalian cells. Structurally, RTB consists of two globular domains (RTB-D1, RTB-D2), each divided into three homologous sub-domains (α, β, γ). The two carbohydrate recognition domains (CRDs) are situated on opposite sides of RTB (sub-domains 1α and 2γ) and function non-cooperatively. Previous studies have revealed two distinct classes of toxin-neutralizing, anti-RTB monoclonal antibodies (mAbs). Type I mAbs, exemplified by SylH3, inhibit (~90%) toxin attachment to cell surfaces, while type II mAbs, epitomized by 24B11, interfere with intracellular toxin transport between the plasma membrane and the trans-Golgi network (TGN). Localizing the epitopes recognized by these two classes of mAbs has proven difficult, in part because of RTB's duplicative structure. To circumvent this problem, RTB-D1 and RTB-D2 were expressed as pIII fusion proteins on the surface of filamentous phage M13 and subsequently used as "bait" in mAb capture assays. We found that SylH3 captured RTB-D1 (but not RTB-D2) in a dose-dependent manner, while 24B11 captured RTB-D2 (but not RTB-D1) in a dose-dependent manner. We confirmed these domain assignments by competition studies with an additional 8 RTB-specific mAbs along with a dozen a single chain antibodies (VHHs). Collectively, these results demonstrate that type I and type II mAbs segregate on the basis of domain specificity and suggest that RTB's two domains may contribute to distinct steps in the intoxication pathway.

Improvements in the production of purified M13 bacteriophage bio-nanoparticle

M13 bacteriophage is a well-established versatile nano-building block, which can be employed to produce novel self-assembled functional materials and devices. Sufficient production and scalability of the M13, often require a large quantity of the virus and thus, improved propagation methods characterised by high capacity and degree of purity are essential. Currently, the ‘gold-standard’ is represented by infecting Escherichia coli cultures, followed by precipitation with polyethylene glycol (PEG). However, this is considerably flawed by the accumulation of contaminant PEG inside the freshly produced stocks, potentially hampering the reactivity of the individual M13 filaments. Our study demonstrates the effectiveness of implementing an isoelectric precipitation procedure to reduce the residual PEG along with FT-IR spectroscopy as a rapid, convenient and effective analytic validation method to detect the presence of this contaminant in freshly prepared M13 stocks.

Screening polymeric ionic liquids for chromatography-based purification of bacteriophage M13

M13 bacteriophage is a key instrument in phage display applications, as well as a possible antibacterial therapeutic agent due to its highly restrictive bacterial pathogenesis, and other applications. The traditional phage purification process is usually achieved by gradient ultracentrifugation or a combination of precipitation, centrifugation and microfiltration. These approaches easily lead to long process times, high operational costs, phage aggregation and consequent product loss (approximately 60%). This work is thus focused on an alternative potential large-scale process to achieve high yield and purity while minimizing the operational costs.Electrostatic-based separation processes are also common biomolecules purification techniques. Although anion exchange chromatography has been used before to purify several viral particles, this technique has been poorly reported for the purification of M13 phage. In a recent work, our group has demonstrated the use of a predominant anion exchange process, where a polymeric ionic liquid (PIL) was used as an alternative separation matrix for M13 bacteriophage. In this work, a variety of system parameters was studied, including chemical structure of the cation and the anion, the crosslinker nature and its concentration, either in batch adsorption/elution or chromatographic operation mode. The PIL-based chromatographic operation mode revealed to be a suitable separation process for M13 from directly filtered E. coli supernatant, reaching over 70% M13 recovery and 4.6 purification factor in a single step. To our knowledge, this is the first time that PILs have been reported as separation agents for bioproducts from complex mixtures.

Viruses Masquerading as Antibodies in Biosensors: The Development of the Virus BioResistor.

The 2018 Nobel Prize in Chemistry recognized in vitro evolution, including the development by George Smith and Gregory Winter of phage display, a technology for engineering the functional capabilities of antibodies into viruses. Such bacteriophages solve inherent problems with antibodies, including their high cost, thermal lability, and propensity to aggregate. While phage display accelerated the discovery of peptide and protein motifs for recognition and binding to proteins in a variety of applications, the development of biosensors using intact phage particles was largely unexplored in the early 2000s. Virus particles, 16.5 MDa in size and assembled from thousands of proteins, could not simply be substituted for antibodies in any existing biosensor architectures.Incorporating viruses into biosensors required us to answer several questions: What process will allow the incorporation of viruses into a functional bioaffinity layer? How can the binding of a protein disease marker to a virus particle be electrically transduced to produce a signal? Will the variable salt concentration of a bodily fluid interfere with electrical transduction? A completely new biosensor architecture and a new scheme for electrical transduction of the binding of molecules to viruses were required.This Account describes the highlights of a research program launched in 2006 that answered these questions. These efforts culminated in 2018 in the invention of a biosensor specifically designed to interface with virus particles: the Virus BioResistor (VBR). The VBR is a resistor consisting of a conductive polymer matrix in which M13 virus particles are entrained. The electrical impedance of this resistor, measured across 4 orders of magnitude in frequency, simultaneously measures the concentration of a target protein and the ionic conductivity of the medium in which the resistor is immersed. Large signal amplitudes coupled with the inherent simplicity of the VBR sensor design result in high signal-to-noise ratio (S/N > 100) and excellent sensor-to-sensor reproducibility. Using this new device, we have measured the urinary bladder cancer biomarker nucleic acid deglycase (DJ-1) in urine samples. This optimized VBR is characterized by extremely low sensor-to-sensor coefficients of variation in the range of 3-7% across the DJ-1 binding curve down to a limit of quantitation of 30 pM, encompassing 4 orders of magnitude in concentration.

High quantum efficiency and stability of biohybrid quantum dots nanojunctions in bacteriophage-constructed perovskite

Forming heterojunctions is a traditional approach for improving the performance of semiconducting nanomaterials. However, using a biological nanostructure to achieve such a goal has rarely been studied. Here we showcase a novel biohybrid junction comprising a functional nanobiomaterial (bacteriophage) and a semiconductor nanomaterial (perovskite quantum dots). We found that M13 bacteriophage, genetically modified to bear increased negative charges, could assist the growth of cubic cesium lead bromide (CsPbBr3) perovskite quantum dots with a size of 13.25 ± 2.69 nm. The M13 bacteriophage further functioned as a scaffold for the assembly of the formed CsPbBr3 quantum dots into a bacteriophage-perovskite biohybrid with a photoluminescence quantum yield of 40.1%, 2.99-fold higher than that of conventional CsPbBr3 quantum dots (13.4%). In addition, compared to the conventional perovskite quantum dots, the perovskite quantum dots in the bacteriophage-based biohybrids exhibited a decreased full width at half maximum (FWHM) of the photoluminescence, indicating that the biohybrids are a better color gamut for light-emitting device applications. Such high optical performance arose from the ordered arrangement of the quantum dots by the bacteriophage, which further affected the charge density due to the interaction between the bacteriophage and CsPbBr3 surface. The materials lifetime of the M13 bacteriophage-assisted perovskite quantum dots was also 1.76 times greater than that of the conventional counterparts without the assistance of bacteriophages due to the reduced valence energy level in the biohybrids. The experiment using bacteriophage with artificially increased surface charge density through genetic manipulation proved our assumption that the surface charge density of the bacteriophage contributes to the stabilization of perovskite quantum dots. The light-emitting diode (LED) with perovskite quantum dots assembled and functionalized by bacteriophage exhibited a 17-fold higher maximum luminance than that of conventional CsPbBr3 quantum dots. This work demonstrates a novel nanobiotechnological approach to the production and assembly of perovskite quantum dots for light-emitting applications.

Nanopore sequencing of native adeno-associated virus (AAV) single-stranded DNA using a transposase-based rapid protocol.

Next-generation sequencing of single-stranded DNA (ssDNA) enables transgene characterization of gene therapy vectors such as adeno-associated virus (AAV), but current library generation uses complicated and potentially biased second-strand synthesis. We report that libraries for nanopore sequencing of ssDNA can be conveniently created without second-strand synthesis using a transposase-based protocol. We show for bacteriophage M13 ssDNA that the MuA transposase has unexpected residual activity on ssDNA, explained in part by transposase action on transient double-stranded hairpins. In case of AAV, library creation is additionally aided by genome hybridization. We demonstrate the power of direct sequencing combined with nanopore long reads by characterizing AAV vector transgenes. Sequencing yielded reads up to full genome length, including GC-rich inverted terminal repeats. Unlike short-read techniques, single reads covered genome-genome and genome-contaminant fusions and other recombination events, whilst additionally providing information on epigenetic methylation. Single-nucleotide variants across the transgene cassette were revealed and secondary genome packaging signals were readily identified. Moreover, comparison of sequence abundance with quantitative polymerase chain reaction results demonstrated the technique's future potential for quantification of DNA impurities in AAV vector stocks. The findings promote direct nanopore sequencing as a fast and versatile platform for ssDNA characterization, such as AAV ssDNA in research and clinical settings.

Nanomechanics of graphene oxide-bacteriophage based self-assembled porous composites

Graphene oxide, integrated with the filamentous bacteriophage M13, forms a 3D large-scale multifunctional porous structure by self-assembly, with considerable potential for applications. We performed Raman spectroscopy under pressure on this porous composite to understand its fundamental mechanics. The results show that at low applied pressure, the sp^2 bonds of graphene oxide stiffen very little with increasing pressure, suggesting a complicated behaviour of water intercalated between the graphene layers. The key message of this paper is that water in a confined space can have a significant impact on the nanostructure that hosts it. We introduced carbon nanotubes during the self-assembly of graphene oxide and M13, and a similar porous macro-structure was observed. However, in the presence of carbon nanotubes, pressure is transmitted to the sp^2 bonds of graphene oxide straightforwardly as in graphite. The electrical conductivity of the composite containing carbon nanotubes is improved by about 30 times at a bias voltage of 10 V. This observation suggests that the porous structure has potential in applications where good electrical conductivity is desired, such as sensors and batteries.

M13 phage-based nanoprobe for SERS detection and inactivation of Staphylococcus aureus

Rapid and sensitive diagnosis of bacterial infections at early stage is of great significance for food safety monitoring as well as clinical treatment. Herein, we construct a surface-enhanced Raman scattering (SERS) nanoprobe based on M13 phages for the selective detection and inactivation of Staphylococcus aureus (S. aureus). M13 phage with specific S. aureus-binding heptapeptide displayed on the N-terminal of pIII protein is selected from phage display peptide library. The S. aureus-specific SERS probe is thus constructed by in situ growth of gold nanoparticles (AuNPs) on M13 phage surface, followed by modification with 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) as SERS active molecule. Upon the addition of this SERS probe, M13 phage selectively binds with S. aureus to induce anchoring of AuNPs on S. aureus surface, and the SERS probe-labeled S. aureus cells are collected by centrifugation for SERS detection. For the quantification of S. aureus, a linear range of 10-106 cfu mL−1 is achieved in aqueous medium. It is further demonstrated by spiking recovery in soft drinks. Furthermore, this SERS probe exhibits bactericidal capabilities towards S. aureus, which shows promising potential to serve as a multifunctional platform for simultaneous detection and inactivation of S. aureus.

A novel approach to nonsurgical sterilization; application of menadione-modified gonocyte-targeting M13 bacteriophage for germ cell ablation in utero.

There remains a compelling need for the development of nonsurgical sterilizing agents to expand the fertility management options for both domestic and feral animal species. We hypothesize that an efficacious sterilization approach would be to selectively ablate nonrenewable cell types that are essential for reproduction, such as the undifferentiated gonocytes within the embryonic gonad. Here, we report a novel strategy to achieve this goal centered on the use of a chemically modified M13 bacteriophage to effect the targeted delivery of menadione, a redox‐cycling naphthoquinone, to mouse gonocytes. Panning of the M13 random peptide ‘phage display library proved effective in the isolation of gonocyte‐specific targeting clones. One such clone was modified via N‐succinimidyl‐S‐acetylthioacetate (SATA) linkage to the N‐terminus of the major PVIII capsid protein. Subsequent deacetylation of the SATA was undertaken to expose a thiol group capable of reacting with menadione through Michael addition. This chemical modification was confirmed using UV spectrophotometry. In proof‐of‐concept experiments we applied the modified ‘phage to primary cultures of fetal germ cells and induced, an approximately, 60% reduction in the viability of the target cell population. These studies pave the way for in vivo application of chemically modified M13 bacteriophage in order to achieve the selective ablation of nonrenewable cell types in the reproductive system, thereby providing a novel nonsurgical approach the regulation of fertility in target species.

Hierarchically Structured M13 Phage Aerogel for Enhanced Sound‐Absorption

AbstractAerogels are a novel type of open‐cell porous material. They are particularly suitable for passive sound‐absorption due to their high porosity and high‐surface areas of the internal structures, but with the drawbacks of limited absorption rate of low‐frequency sound and poor mechanical performance. Here, it is first demonstrated that the M13 phage aerogel(M13AG) with hierarchically porous and ribbing structures is beneficial to sound‐absorption and the local piezoelectric effect attributed to M13 phages can further improve the sound‐absorption ability in the low‐frequency. The sound‐absorption coefficient of the M13AG with thickness of 20 mm attains peak value of 0.992 at 1600 Hz. In addition, the M13AG is assembled on a metallic foam framework to enhance its mechanical performance. The resulting M13AG/nickel foam exhibits enhanced compressive yield strength up to 0.14 MPa. This study opens up a new opportunity for passive sound‐absorption.

M13 Bacteriophage-Assisted Morphological Engineering of Crack-Based Sensors for Highly Sensitive and Wide Linear Range Strain Sensing.

Despite their extraordinary mechanosensitivities, most channel-like crack-based strain sensors are limited by their poor levels of stretchability and linearity. This work presents a simple yet efficient way of modulating the cracking structure of thin metal films on elastomers to facilitate the development of high-performance wearable strain sensors. A net-shaped crack structure based on a thin platinum (Pt) film can be produced by coating an elastomer surface with M13 bacteriophages (phages) and consequently engineering the surface strain upon stretching. This process produces a Pt-on-phage (PoP) strain sensor that simultaneously exhibits high levels of stretchability (24%), sensitivity (maximum gauge factor ≈ 845.6 for 20-24%), and linearity (R2 ≈ 0.988 up to 20%). In addition, the sensor performance can be further modulated by either changing the phage coating volume or adding a silver nanowire coating to the PoP sensor film. The balanced strain-sensing performance, combined with fast response times and high levels of mechanical flexibility and operational stability, enables the devices to detect a wide range of human motions in real time after being attached to various body parts. Furthermore, PoP-based strain sensors can be usefully extended to detect more complex multidimensional strains through further strain engineering on a cross-patterned PoP film.

A screening study of high affinity peptide as molecular binder for AXL, tyrosine kinase receptor involving in Zika virus entry

The recent extensive spread of Zika virus has led to increased interest in the development of early diagnostic tests. To the best of our knowledge, this is the first study to demonstrate the successful use of phage display to identify affinity peptides for quantitative analysis of AXL, a tyrosine kinase receptor involved in Zika virus entry. Biopanning of M13 phage library successfully identified a high affinity peptide, with the sequence AHNHTPIKQKYL. To study the feasibility of using free peptides for molecular recognition, we synthesized a series of amino acid-substituted peptides and examined their binding affinity for AXL using electrochemical impedance spectroscopy and square wave voltammetry. Most synthetic peptides had non-identical random coil structures based on circular dichroism spectroscopy. Of the peptides tested, AXL BP1 exhibited nanomolar binding affinity for AXL. To verify whether AXL BP1 could be used as a peptide inhibitor at the cellular level, two functional tests were carried out: a WST assay for cell viability and qRT-PCR for quantification of RNA levels in Zika virus-infected Huh7 cells. The results showed that AXL BP1 had low cytotoxicity and could block Zika virus entry. These results indicate that newly identified affinity peptides could potentially be used for the development of Zika virus entry inhibitors.

Identification of novel paraben-binding peptides using phage display

Parabens are alkyl esters of 4-hydroxybenzoic acid, which is derived from a family of synthetic esters of p-hydroxybenzoic acid. Among all the kinds of paraben, two parabens (methyl paraben, MP; and n-propyl paraben, PP) are the most generally used as preservatives in personal care products, such as cosmetics, pharmaceuticals, and food also, and are often presented together. However, a number of studies have reported that the toxicity of parabens affects the water environment, and human as well. This study utilized M13 phage display technology to provide easy, efficient, and relatively inexpensive methods to identify peptides that bind to MP and PP, respectively, to remove in wastewater. At first, biopanning was performed, to sort MP and PP specific binding phages, and three cases of experiment, including negative control (NC), which could sort unspecific binding phage, were conducted at the same time. Phage binding affinity tests were substituted by concentration reduction using antibody conjugated magnetic beads, and paraben concentration was measured by HPLC. Analysis showed that the MP concentration reduction of 38% was the highest in M4 phage, while the PP concentration reduction of 44% was the highest in P3 phage. We successfully screened two peptides specific to MP and PP, namely, MP4 and PP3, respectively; the results showed that the MP concentration reduction in MP4 was the highest at 44%, and the PP concentration reduction in PP3 was the highest at 39%, and their specificity was measured by the capture rate between target and control. In conclusion, the phage display technique shows applicability to the removal of parabens in water; furthermore, it also shows the possibility of the detection or removal of other chemicals.