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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.

Femtosecond (fs) laser irradiation techniques are emerging tools for inactivating viruses that do not involve ionizing radiation. In this work, the inactivation of two bacteriophages representing protective capsids with different geometric constraints, that is, the near-spherical MS2 (with a diameter of 27 nm) and the filamentous M13 (with a length of 880 nm) is compared using energetic visible and near-infrared fs laser pulses with various energies, pulse durations, and exposure times. Intriguingly, the results show that inactivation using 400 nm lasers is substantially more efficient for MS2 compared to M13. In contrast, using 800 nm lasers, M13 was slightly more efficiently inactivated. For both viruses, the genome was exposed to a harmful environment upon fs-laser irradiation. However, in addition to the protection of the genome, the metastable capsids differ in many properties required for stepwise cell entry that may explain their dissimilar behavior after (partial) disassembly. For MS2, the dominant mechanism of fs-laser inactivation was the aggregation of the viral capsid proteins, whereas aggregation did not affect M13 inactivation, suggesting that the dominant mechanism of M13 inactivation was related to breaking of secondary protein links.

Aqueous two-phase systems (ATPS) have been widely exploited for the recovery and partial purification of biological compounds. Recently our research group characterized the primary recovery and partial purification of bacteriophage M13 using polymer-salt and ionic liquid-salt ATPS. From such study, it was concluded that PEG 400-potassium phosphate ATPS with a volume ratio (VR ) of 1 and 25% w/w TLL were the best suitable for the primary recovery of bacteriophage M13 from a crude extract, achieving a recovery yield of 83.3%. Although such system parameters were proven to be adequate for the recovery of the product of interest, it was concluded that further optimization was desirable and attainable by studying the effect of additional system parameters such as VR , concentration of neutral salt (M) and sample load (% w/w). This research work presents an optimization of a previously reported process for the recovery of bacteriophage M13 directly from a crude extract using ATPS. The increase in VR and sample load showed a positive effect in the recovery of M13 indicating an improved performance of the proposed ATPS. According to the results presented here, a system composed of PEG 400 17.2% (w/w), potassium phosphate 15.5% (w/w) and a sample load of 30% (w/w) allowed the recovery of M13 directly from a crude extract with a top phase recovery of 80.1%, representing an increase of 4.8 times in the final concentration and a reduction of 2.65 times in the processing costs. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018 © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1177-1184, 2018.

Immobilized antibodies are extensively employed for medical diagnostics, such as in enzyme-linked immunosorbent assays. Despite their widespread use, the ability to control the orientation of immobilized antibodies on surfaces is very limited. Herein, we report a method for the covalent and orientation-selective immobilization of antibodies in designed cavities in 2D and 3D DNA origami structures. Two tris(NTA)-modified strands are inserted into the cavity to form NTA-metal complexes with histidine clusters on the Fc domain. Subsequent covalent linkage to the antibody was achieved by coupling to lysine residues. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) confirmed the efficient immobilization of the antibodies in the origami structures. This increased control over the orientation of antibodies in nanostructures and on surfaces has the potential to direct the interactions between antibodies and targets and to provide more regular surface assemblies of antibodies.

Biomacromolecules are likely to undergo self-assembly and show specific collective behavior concentrated in the medium. Although the assembly procedures have been studied for unraveling their mysteries, there are few cases to directly demonstrate the collective behavior and phase transition process in dynamic systems. In the contribution, the drying process of M13 droplet is investigated, and can be successfully simulated by a doctor blade coating method. The morphologies in the deposited film are measured by atomic force microscopy and the liquid crystal phase development is captured in real time using polarized optical microscope. Collective behaviors near the contact line are characterized by the shape of meniscus curve and particle movement velocity. With considering rheological properties and flow, the resultant chiral film is used to align gold nanorods, and this approach can suggest a way to use M13 bacteriophage as a scaffold for the multi-functional chiral structures.

Antiangiogenesis is a promising approach to cancer therapy but is limited by the lack of tumor-homing capability of the current antiangiogenic agents. Angiogenin, a protein overexpressed and secreted by tumors to trigger angiogenesis for their growth, has never been explored as an antiangiogenic target in cancer therapy. Here it is shown that filamentous fd phage, as a biomolecular biocompatible nanofiber, can be engineered to become capable of first homing to orthotopic breast tumors and then capturing angiogenin to prevent tumor angiogenesis, resulting in targeted cancer therapy without side effects. The phage is genetically engineered to display many copies of an identified angiogenin-binding peptide on its side wall and multiple copies of a breast-tumor-homing peptide at its tip. Since the tumor-homing peptide can be discovered and customized virtually toward any specific cancer by phage display, the angiogenin-binding phages are thus universal "plug-and-play" tumor-homing cancer therapeutics.

Nondestructive assembly of a nanostructured enzyme platform is developed in combination of the specific biomolecular attraction and electrostatic coupling for highly efficient direct electron transfer (DET) of enzymes with unprecedented applicability and versatility. The biologically assembled conductive nanomesh enzyme platform enables DET-based flexible integrated biosensors and DET of eight different enzyme with various catalytic activities.

UNLABELLED Transition metal oxide (TMO) thin films have been exploited as interlayers for charge extraction between electrodes and active layers in organic photovoltaic (OPV) devices. Additionally, graphene-electrode-based OPVs have received considerable attention as a means to enhance device stability. However, the film deposition process of a TMO thin-film layer onto the graphene electrode is highly restricted owing to the hydrophobic nature of the graphene surface; thus, the preparation of the device should rely on a vacuum process that is incompatible with solution processing. In this study, we present a novel means for creating a thin tungsten oxide (WO3 ) interlayer on a graphene electrode by employing an engineered biotemplate of M13 viruses, whereby nondestructive functionalization of the graphene and uniform synthesis of a WO3 thin interlayer are concurrently achieved. As a result, the incorporated virus-templated WO3 interlayer exhibited solar-conversion efficiency that was 20 % higher than that of conventional OPVs based on the use of a (3,4-ethylenedioxythiophene):poly(styrenesulfonate) ( PEDOT PSS) interlayer. Notably, bilayer-structured OPVs with synergistically integrated WO3 /PEDOT:PSS achieved >60 % enhancement in device performance.

Viral nanoparticles have attracted extensive research interests in diverse applications of diagnosis and therapy. In particular, filamentous M13 bacteriophages have shown great potential in biomedical applications. However, its pathways entering into cells still remain unclear, and this greatly hinders its further use as a drug or gene carrier. Here, a ratiometric M13 pH probe is designed by conjugating two fluorescent dyes onto the surface of M13. Since the intensity ratio is not influenced by probe concentration, ion strength, temperature, photobleaching, and optical path length, this ratiometric probe can be used to investigate the intracellular pH map of M13. More importantly, the internalization mechanism of M13 can be elucidated. It is found that this filamentous phage shows great cell-type dependence in interaction with cells and internalization mechanism. The phage tends to be bounded on the cell membrane of only epithelial cells, not endothelial cells. Furthermore, the M13 phage enters into cells through endocytosis with specific mechanism: clathrin-mediated endocytosis and macropinocytosis for HeLa; vesicular transport, clathrin-mediated endocytosis, and macropinocytosis for MCF-7; caveolae-mediated endocytosis for human dermal microvascular endothelial cell (HDMEC). This work provides key notes for cancer diagnosis and therapy based on filamentous bacteriophage, especially for design of pH-sensitive drug delivery systems.

A M13 virus based SERS nanoprobe is presented. Gold nanocubes closely aligned into chains along the length of the virus intensify Raman signals of various reporter molecules serving as specific labels. An antibody is expressed at one end to detect the analyte. This new SERS nanoprobe holds promise for infinitesimal and multiplexed detection of any antigen.

M13 bacteriophages are assembled via a covalent layer-by-layer process to form a highly nanoporous network capable of organizing nanoparticles and acting as a scaffold for templating metal-oxides. The morphological and optical properties of the film itself are presented as well as its ability to organize and disperse metal nanoparticles., Eni S.p.A. (Firm), MIT Energy Initiative (Eni-MIT Energy Fellowship), Natural Sciences and Engineering Research Council of Canada (Postgraduate Scholarship)), United States. Army Research Office. Institute for Soldier Nanotechnologies (Contract W911NF-13-D-0001), National Science Foundation (U.S.) (award number DMR-0819762)

Furthermore, as shown by Zhang and co-workers, where longrange PCR was used for the production of single-stranded scaffolds of up to 26 kb, [ 17 ] alternative scaffold sources enable the assembly of larger single structures while avoiding often low-yield hierarchical assembly procedures. [ 18–20 ] All these methods extend the DNA origami technique considerably but also rely on various time consuming and tedious modifi cation steps of either naturally occurring or synthetic template DNA such as several purifi cation steps, PCR, strand separation, enzymatic digestion and/or modifi cation. In addition, for many promising applications of the DNA origami technique – such as nanomedicine or drug delivery – a large quantity of the material is needed. Scaling up the assembly process and maximizing the assembly yield while reducing both labor and cost remains a major challenge for the future development of the fi eld. [ 21 ]

Hierarchically assembled nanomaterials can find a variety of applications in medicine, energy, and electronics. Here, an automatically controlled dip-pulling method is developed and optimized to generate an unprecedented ordered nano-to-micro hierarchical nanoridge-in-microridge (NiM) structure from a bacteria-specific human-safe virus, the filamentous phage with or without genetically displaying a foreign peptide. The NiM structure is pictured as a window blind with each lath (the microridge) made of parallel phage bundles (the nanoridges). It is independent of the substrate materials supporting it. Surprisingly, it can induce the bidirectional differentiation of stem cells into neurons and astrocytes within a short timeframe (only 8 d) not seen before, which is highly desired because both neurons and astrocytes are needed simultaneously in treating neurodegenerative diseases. Since phages can direct tissue regeneration, template materials formation, sense molecules, and build electrodes, the NiM structures displaying different peptides and on varying materials hold promise in many technologically important fields.

The growth of the biopharmaceutical industry has created a demand for new technologies for the purification of genetically engineered proteins.The efficiency of large-scale, high-gradient magnetic fishing could be improved if magnetic particles offering higher binding capacity and magnetization were available. This article describes several strategies for synthesizing microbeads that are composed of a M13 bacteriophage layer assembled on a superparamagnetic core. Chemical cross-linking of the pVIII proteins to a carboxyl-functionalized bead produces highly responsive superparamagnetic particles (SPM) with a side-on oriented, adherent virus monolayer. Also, the genetic manipulation of the pIII proteins with a His(6) peptide sequence allows reversible assembly of the bacteriophage on a nitrilotriacetic-acid-functionalized core in an end-on configuration. These phage-magnetic particles are successfully used to separate antibodies from high-protein concentration solutions in a single step with a90% purity. The dense magnetic core of these particles makes them five times more responsive to magnetic fields than commercial materials composed of polymer-(iron oxide) composites and a monolayer of phage could produce a 1000 fold higher antibody binding capacity. These new bionanomaterials appear to be well-suited to large-scale high-gradient magnetic fishing separation and promise to be cost effective as a result of the self-assembling and self-replicating properties of genetically engineered M13 bacteriophage.

A synthetic method of using genetically engineered M13 virus to mineralize perovskite nanomaterials, particularly strontium titanate (STO) and bismuth ferrite (BFO), is presented. Genetically engineered viruses provide effective templates for perovskite nanomaterials. The virus-templated nanocrystals are small in size, highly crystalline, and show photocatalytic and photovoltaic properties.

Phage display is a high-throughput technology used to identify ligands for a given target. A drawback of the approach is the absence of PTMs in phage-displayed peptides. The applicability of phage display could be broadened considerably by the implementation of PTMs in this system. The aim of this study was to investigate the possible application of citrullination, a PTM of an arginine into a citrulline amino acid, in filamentous (M13) and lytic (T7) phage display. After in vitro citrullination of T7 and M13 phages, citrullination was confirmed and the infectivity of both citrullinated and non-citrullinated phage was compared by titer determination. We demonstrated the successful in vitro citrullination of T7 and M13 phage-displayed peptides. This in vitro modification did not affect the viability or infectivity of the T7 virions, a necessary prerequisite for the implementation of this approach in T7 phage display. For M13 phage, however, the infecting phage titer decreased five-fold upon citrullination, limiting the use of this modification in M13 phage display. In conclusion, in vitro citrullination can be applied in T7 phage display giving rise to a high-throughput and sensitive approach to identify citrulline-containing ligands by the use of the strengths of phage display technology.

Split inteins link their fused peptide or protein sequences with a peptide bond in an autocatalytic reaction called protein trans-splicing. This reaction is becoming increasingly important for a variety of applications in protein semisynthesis, polypeptide circularisation, construction of biosensors, or segmental isotopic labelling of proteins. However, split inteins exhibit greatly varying solubility, efficiency and tolerance towards the nature of the fused sequences as well as reaction conditions. We envisioned that phage display as an in vitro selection technique would provide a powerful tool for the directed evolution of split inteins with improved properties. As a first step towards this goal, we show that presentation of active split inteins on an M13 bacteriophage is feasible. Two different C-terminal intein fragments of the Ssp DnaB intein, artificially split at amino acid positions 104 and 11, were encoded in a phagemid vector in fusion to a truncated gpIII protein. For efficient production of hybrid phages, the presence of a soluble domain tag at their N-termini was necessary. Immunoblot analysis revealed that the hybrid phages supported protein trans-splicing with a protein or a synthetic peptide, respectively, containing the complementary intein fragment. Incorporation of biotin or desthiobiotin by this reaction provides a straightforward strategy for future enrichment of desired mutants from randomised libraries of the C-terminal intein fragments on streptavidin beads. Protein semisynthesis on a phage could also be exploited for the selection of chemically modified proteins with unique properties. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.

The fabrication of single virus arrays is herein demonstrated using the direct printing of unmodified anti-M13 bacteriophage antibodies on silicon with nanometer resolution, widely variable feature pitch, and flow alignment of the viruses. Organization of virus-based systems into functional, addressable arrays has many technological applications, including micro-array technology and bottom-up nano-assemblies.

Liposomes have been a center of research for many years due to their numerous applications in chemistry, biology, medicine, and nanotechnology. They can be used to entrap materials such as drugs either within the central aqueous compartment if they are water soluble, or within the hydrophobic domain of the lipid bilayer if they are oil soluble. In addition, their surface can bemodified to realize targeted delivery. For example, in biomedicine, liposomes are used as vehicles to deliver drugs and genes to specific parts of the body. When used in the delivery of certain anticancer drugs, liposomes help to shield healthy cells from the drug toxicity and prevent concentration in vulnerable tissues. In addition, the liposomes allowmuch smaller doses of drug to be used, thus reducing the side effects of that drug. On the other hand, zinc phthalocyanine (ZnPc) is a potential drug that is being tested as a photosensitizer for photodynamic therapy (PDT). PDT involves the systemic administration of a photosensitizer followed by illumination with light of an appropriate wavelength, which results in the formation of singlet oxygen (O2) for destroying cancer cells. [6] ZnPc has a high absorption coefficient at 650–700 nm with optimal tissue penetration. It has a long lifetime in the triplet excited state, thus resulting in the highly efficient production of O2 which is the main cytotoxic species in PDT. However, it is insoluble in water and must be incorporated into unilamellar liposomes (Figure 1a). The main drawback of liposomes is their instability in biological media, as well as their sensitivity to many external parameters, such as temperature or osmotic pressure. This instability problem affects the application of liposomes in drug/gene delivery and PDT. Attempts have been made to stabilize liposomes by adsorbing some

An Achilles heel inherent to all molecular display formats, background binding between target and display system introduces false positives into screens and selections. For example, the negatively charged surfaces of phage, mRNA, and ribosome display systems bind with unacceptably high nonspecificity to positively charged target molecules, which represent an estimated 35% of proteins in the human proteome. Here we report the first systematic attempt to understand why a broad class of molecular display selections fail, and then solve the underlying problem for both phage and RNA display. Firstly, a genetic strategy was used to introduce a short, charge-neutralizing peptide into the solvent-exposed, negatively charged phage coat. The modified phage (KO7(+)) reduced or eliminated nonspecific binding to the problematic high-pI proteins. In the second, chemical approach, nonspecific interactions were blocked by oligolysine wrappers in the cases of phage and total RNA. For phage display applications, the peptides Lys(n) (where n=16 to 24) emerged as optimal for wrapping the phage. Lys(8), however, provided effective wrappers for RNA binding in assays against the RNA binding protein HIV-1 Vif. The oligolysine peptides blocked nonspecific binding to allow successful selections, screens, and assays with five previously unworkable protein targets.

The exposure of phosphatidylserine (PS) molecules from the inner to the outer leaflet of the plasma membrane has been recognized as a well-defined molecular epitope of cells undergoing apoptosis. Examination and monitoring of PS exposure is an extensively used molecular marker in non-invasive apoptosis imaging under a variety of clinical conditions, including the assessment of therapeutic anti-cancer agents and myocardial infarction. Herein, we report the identification of a PS-recognizing peptide which was identified by the screening of an M13 phage display peptide library onto PS-coated ELISA plates. Repeated biopanning for a total of four rounds revealed a predominant enrichment of the phage clone displaying peptide sequence, CLSYYPSYC (46%). The identified phage clone evidenced enhanced binding to a number of apoptotic cells over non-apoptotic cells, and this binding was inhibited by both annexin V and synthesized peptide displayed on the phage. The binding of the fluorescein-labelled CLSYYPSYC peptide to apoptotic versus normal cells was assessed by both FACS analysis and fluorescence microscopy. Optical imaging after the systemic administration of fluorescein-labelled CLSYYPSYC peptide to tumour-bearing nude mice (H460 cells xenograft model) treated with a single dose of an anticancer drug (camp-tothecin) indicated peptide homing to the tumour. The histological examination of tumour tissues showed intense staining of the tumour vasculature and apoptotic tumour cells. With these results, the CLSYYPSYC peptide is recognized as a novel PS-recognizing moiety which may possibly be developed into a molecular probe for the imaging of apoptosis in vivo. This application would clearly be relevant to assessments of the efficacy of anticancer therapy in tumours.

Borrelia burgdorferi, the agent of Lyme disease, disseminates from the site of deposition by Ixodes ticks to cause systemic infection. Dissemination occurs through the circulation and through tissue matrices, but the B. burgdorferi molecules that mediate interactions with the endothelium in vivo have not yet been identified. In vivo selection of filamentous phage expressing B. burgdorferi protein fragments on the phage surface identified several new candidate adhesins, and verified the activity of one adhesin that had been previously characterized in vitro. P66, a B. burgdorferi ligand for beta(3)-chain integrins, OspC, a protein that is essential for the establishment of infection in mammals, and Vls, a protein that undergoes antigenic variation in the mammal, were all selected for binding to the murine endothelium in vivo. Additional B. burgdorferi proteins for which no functions have been identified, including all four members of the OspF family and BmpD, were identified as candidate adhesins. The use of in vivo phage display is one approach to the identification of adhesins in pathogenic bacteria that are not easily grown in the laboratory, or for which genetic manipulations are not straightforward.

We investigated whether the T7 system of phage display could produce peptide libraries of greater diversity than the M13 system of phage display due to the differing processes of lytic and filamentous phage morphogenesis. Using a bioinformatics-assisted computational approach, collections of random peptide sequences obtained from a T7 12-mer library (X(12)) and a T7 7-mer disulfide-constrained library (CX(7)C) were analyzed and compared with peptide populations obtained from New England BioLabs' M13 Ph.D.-12 and Ph.D.-C7C libraries. Based on this analysis, peptide libraries constructed with the T7 system have fewer amino acid biases, increased peptide diversity, and more normal distributions of peptide net charge and hydropathy than the M13 libraries. The greater diversity of T7-displayed libraries provides a potential resource of novel binding peptides for new as well as previously studied molecular targets. To demonstrate their utility, several of the T7-displayed peptide libraries were screened for streptavidin- and neutravidin-binding phage. Novel binding motifs were identified for each protein.

BACKGROUND SB-236057 is a potent skeletal teratogen in rodents and rabbits, producing axial and posterior somite malformations in cultured rat embryos. The compound shares some structural similarity to cyclopamine. METHODS M13 phage display was used to identify amino acid motifs with binding affinity to SB-236057. A 10 μM SB-236057 solution was administered to cultured day 9 postcoitus rat embryos and real-time PCR was conducted at 6 hr posttreatment to evaluate early transcriptional response of axial development genes. Whole-mount in situ hybridization of selected transcripts was conducted on embryos at 48 hr post-compound administration. The rat-enhancer of split protein 1 (r-esp1) expression-functional characterization was done by transcriptional expression and morpholino antisense approaches. RESULTS We identified several amino acid motifs that had high binding affinity to SB-236057-biotin conjugates, one with 100% sequence homology to a region of r-esp1, one of the Groucho homologs transcribed by the enhancer of split complex (En[spl]C). SB-236057 repressed expression of r-esp1 and members of the Notch-En[spl]C pathway. Goosecoid and HNF3-β, both suspected to associate with Groucho proteins, were also responsive, although expression of another putative binding protein, engrailed-1 (en-1), and other en-1 pathway members was not affected. R-esp1 mRNA was localized along the axis and antisense inhibition produced similar somite malformations as SB-236057 did. At 48 hr post–SB-236057 or post–r-esp1 antisense administration, affected embryos demonstrated unchanged sonic hedgehog (shh) expression, however HNF3-β expression was either absent, altered, or reduced. CONCLUSIONS We present experimental evidence that the mechanism of SB-236057 teratogenicity includes transcriptional alterations to the Notch1-En[spl] pathway. In addition, alterations in HNF3-β expression were similar to those induced by cyclopamine. The relationships between r-esp1 with Notch1 and shh signaling pathways and potential mechanisms of SB-236057 teratogenicity are also discussed. Birth Defects Research (Part A), 2004. © 2004 Wiley-Liss, Inc.

A random peptide library of heptamers displayed on the surface of M13 bacteriophage was used to identify specific epitopes of antibodies in pooled sera of swine naturally infected by Nipah virus. The selected heptapeptides were aligned with protein sequences of Nipah virus and several putative epitopes were identified within the nucleocapsid protein. A total of 41 of 60 (68%) selected phage clones had inserts resembling a region with the sequence SNRTQGE, located at the C-terminal end (amino acids 503-509) of the nucleocapsid protein. The binding of antibodies in the swine and human antisera to the phage clone was inhibited by a synthetic peptide corresponding to this region. Epitopes identified by phage display are consistent with the predicted antigenic sites for the Nipah virus nucleocapsid protein. The selected phage clone used as a coating antigen discriminated swine and human Nipah virus sera-positive from sera-negative samples exhibiting characteristics, which might be attractive for diagnostic tests.

The family of three-dimensional molecular structures of the major coat protein from the M13 bacteriophage, which was determined in detergent micelles by NMR methods, has been analyzed by constrained geometry optimization in a phospholipid environment. A single-layer solvation shell of dioleoyl phosphatidylcholine lipids was built around the protein, after replacing single residues by cysteines with a covalently attached maleimide spin label. Both the residues substituted and the phospholipid were chosen for comparison with site-directed spin labeling EPR measurements of distance and local mobility made previously on membranous assemblies of the M13 coat protein purified from viable mutants. The main criteria for identifying promising candidate structures, out of the 300 single-residue mutant models generated for the membranous state, were 1) lack of steric conflicts with the phospholipid bilayer, 2) good match of the positions of spin-labeled residues along the membrane normal with EPR measurements, and 3) a good match between the sequence profiles of local rotational freedom and a structural restriction parameter for the spin-labeled residues obtained from the model. A single subclass of structure has been identified that best satisfies these criteria simultaneously. The model presented here is useful for the interpretation of future experimental data on membranous M13 coat protein systems. It is also a good starting point for full-scale molecular dynamics simulations and for the design of further site-specific spectroscopic experiments.

UVM is an SOS-independent inducible response characterized by elevated mutagenesis at a site-specific 3, N4-ethenocytosine (epsilonC) residue borne on M13 single-stranded DNA transfected into Escherichia coli cells pretreated with DNA-damaging agents. By constructing and using E. coli strain AM124 (polA polB umuDC dinB lexA1[Ind-]), we show here that the UVM response is manifested in cells deficient for SOS induction, as well as for all four of the 'non-replicative' DNA polymerases, namely DNA polymerase I (polA), II (polB), IV (dinB) and V (umuDC). These results confirm that UVM represents a novel, previously unidentified cellular response to DNA-damaging agents. To address the question as to whether the UVM response is accompanied by an error-prone DNA replication activity, we applied a newly developed in vitro replication assay coupled to an in vitro mutation analysis system. In the assay, circular M13 single-stranded DNA bearing a site-specific lesion is converted to circular double-stranded replicative-form DNA in the presence of cell extracts and nucleotide precursors under conditions that closely mimic M13 replication in vivo. The newly synthesized (minus) DNA strand is selectively amplified by ligation-mediated polymerase chain reaction (LM-PCR), followed by a multiplex sequence analysis to determine the frequency and specificity of mutations. Replication of DNA bearing a site-specific epsilonC lesion by cell extracts from uninduced E. coli AM124 cells results in a mutation frequency of about 13%. Mutation frequency is elevated fivefold (to 58%) in cell extracts from UVM-induced AM124 cells, with C --A mutations predominating over C --T mutations, a specificity similar to that observed in vivo. These results, together with previously reported data, suggest that the UVM response is mediated through the induction of a transient error-prone DNA replication activity and that a modification of DNA polymerase III or the expression of a previously unidentified DNA polymerase may account for the UVM phenotype.

An unusual nucleotide sequence, called H10, was previously isolated by biopanning with a random peptide library on filamentous phage. The sequence encoded a peptide that bound to the growth hormone binding protein. Despite the fact that the H10 sequence can be expressed in Escherichia coli as a fusion to the gene III minor coat protein of the M13 phage, the sequence contained two TGA stop codons in the zero frame. Several mutant derivatives of the H10 sequence carried not only a stop codon, but also showed frameshifts, either +1 or -1 in individual isolates, between the H10 start and the gene III sequences. In this work, we have subcloned the H10 sequence and three of its derivatives (one requiring a +1 reading frameshift for expression, one requiring a -1 reading frameshift, and one open reading frame) in gene fusions to a reporter beta-galactosidase gene. These sequences have been cloned in all three reading frames relative to the reporter. The non-open reading frame constructs gave (surprisingly) high expression of the reporter (10-40% of control vector expression levels) in two out of the three frames. A site-directed mutant of the TGA stop codon (to TTA) in the +1 shifter greatly reduced the frameshift and gave expression primarily in the zero frame. By contrast, a site-directed mutant of the TGA in the -1 shifter had little effect on the pattern of expression, and alteration of the first TGA (of two) in H10 itself paradoxically reduced expression by half. We believe these phenomena to reflect a translational recoding mechanism in which ribosomes switch reading frames or read past stop codons upon encountering a signal encoded in the nucleotide sequence of the mRNA, because both the open reading frame derivative (which has six nucleotide changes from parental H10) and the site-directed mutant of the +1 shifter, primarily expressed the reporter only in the zero frame.

A base substitution mutation (mutA) in the Escherichia coli glyV tRNA gene potentiates asp --> gly mistranslation and confers a strong mutator phenotype that is SOS independent, but requires recA, recB and recC genes. Here, we demonstrate that mutA cells express an error-prone DNA polymerase by using an in vitro experimental system based on the conversion of phage M13 single-stranded viral DNA bearing a model mutagenic lesion to the double-stranded replicative form. Amplification of the newly synthesized strand followed by multiplex DNA sequence analysis revealed that mutation fixation at 3, N4-ethenocytosine (varepsilonC) was approximately 3% when the DNA was replicated by normal cell extracts, approximately 48% when replicated by mutA cell extracts and approximately 3% when replicated by mutA recA double mutant cell extracts, in complete agreement with previous in vivo results. Mutagenesis at undamaged DNA sites was significantly elevated by mutA cell-free extracts in the M13 lacZ(alpha) forward mutagenesis system. Neither polA (DNA polymerase I) nor polB (DNA polymerase II) genes are required for the mutA phenotype, suggesting that the phenotype is mediated through a modification of DNA polymerase III or the activation of a previously unidentified DNA polymerase. These findings define the major features of a novel mutagenic pathway and imply the existence of previously unrecognized links between translation, recombination and replication.

Urease is an important virulence factor for Helicobacter pylori and is critical for bacterial colonization of the human gastric mucosa. Specific inhibition of urease activity has been proposed as a possible strategy to fight this bacteria which infects billions of individual throughout the world and can lead to severe pathological conditions in a limited number of cases. We have selected peptides which specifically bind and inhibit H. pylori urease from libraries of random peptides displayed on filamentous phage in the context of pIII coat protein. Screening of a highly diverse 25-mer combinatorial library and two newly constructed random 6-mer peptide libraries on solid phase H. pylori urease holoenzyme allowed the identification of two peptides, 24-mer TFLPQPRCSALLRYLSEDGVIVPS and 6-mer YDFYWW that can bind and inhibit the activity of urease purified from H. pylori. These two peptides were chemically synthesized and their inhibition constants (Ki) were found to be 47 microM for the 24-mer and 30 microM for the 6-mer peptide. Both peptides specifically inhibited the activity of H. pylori urease but not that of Bacillus pasteurii.

We have investigated the mechanism of virus photoinactivation with methylene blue (MB) by conducting deuterium oxide (D2O), azide ion (N3-) and oxygen-dependent, studies. Inactivation of M13 bacteriophage and singlet oxygen (1O2) generation by MB photosensitization were irradiation dose dependent. Inactivation of M13 was enhanced by D2O and inhibited by N3-, suggesting that 1O2 participates in M13 inactivation by MB photosensitization. However, N3- did not inhibit M13 inactivation completely. On the other hand, deoxygenating the reaction solution still caused 52-67% of M13 inactivation observed in the presence of oxygen. These results suggest that 102-mediated (Type II) and sensitizer-mediated (Type I) reactions may both play roles in M13 inactivation by MB photosensitization.

We report here on the identification of phosphopeptide ligands which interact with the Src-homology 2 (SH2) domain of the adapter protein Grb2 by screening a random peptide library established on phage. Phage were phosphorylated in vitro at an invariant tyrosine residue by a mixture of phosphotyrosine kinases c-Src, Blk and Syk. Selection of binding motifs was carried out by interaction of the library with the recombinant SH2 domain of Grb2 expressed as a glutathione S-transferase (GST) fusion protein. Several subsequent cycles of selection led to the enrichment of phage which bound to the GST-Grb2 SH2 domain only when previously phosphorylated. Sequence analysis revealed that all of the selected phage displayed peptides with the consensus motif Y*M/ENW (Y* denotes phosphotyrosine). One of these peptides, bearing the Y*ENW motif, bound the Grb2 SH2 domain with a threefold higher affinity than the peptide motif Y*VNV derived from the natural ligand She. Thus, phage display can be employed to rapidly identify high affinity ligands to SH2 domains.

International audience; Three monoclonal IgG2a anti-DNA polyreactive autoantibodies, derived from lupus-prone mice (NZB x NZW)F1, were studied by surface plasmon resonance (BIAcore) analysis using three different synthetic double-stranded (ds) oligonucleotides of 25, 30, and 25 base pairs (bp). These monoclonal antibodies (mAb) exhibited dissociation rate constants (k(off)), ranging from 0.0001 (mAb F14.6 and F4.1) to 0.01/s (mAb J20.8) and k(on) ranging from 2 x 10(5) to 2 x 10(6) /M/s. The screening of a constrained random peptide library displayed on M13 bacteriophages on these mAb allowed the determination of the specific consensus motifs (mimotopes) for mAb F14.6 and J20.8, but not for mAb F4.1. No cross-reaction was observed between F14.6- and J20.8-specific peptides (and vice versa). Binding of all phages selected on F14.6 was inhibited with 700 ng/ml soluble DNA. The binding of one group of peptides selected on J20.8 was inhibited by 400 ng/ml soluble DNA, of a second group by 2500 ng/ml, while binding of a third group could not be inhibited. The determined consensus sequences do not match with known sequences. Peptides specific for F14.6 share negative charges and aromatic rings that may mimic a DNA backbone, while peptides selected with J20.8 do not bear any negative charge, implying a different kind of molecular recognition, for example hydrogen or salt bonds. The peptides selected on J20.8 also bind serum antibodies from human patients with systemic lupus erythematosus. In addition, BALB/c mice immunized with some of the selected phages exhibit high serum titers of IgG3 anti-dsDNA antibodies, further supporting the hypothesis that peptide epitopes may mimic an oligonucleotide structure.

— Although numerous photosensitizers have been used experimentally to decontaminate viruses in cellular blood components, little is known about their mechanisms of photoinactivation. Using M13 bacteriophage and vesicular stomatitis virus (VSV) as model viruses, we have investigated alteration of the viral genome, protein and envelope after phototreatment. Methylene blue (MB) and aluminum phthalocyanine tetrasulfonate (AlPcS4) phototreatment inactivated bacteriophage M13 and decreased the fraction of single-stranded circular genomic DNA (sc-DNA) by converting it to linear form. This conversion was enhanced by treating the extracted DNA with piperidine at 55°C. Piperidine-labile breaks were well correlated to phage survival (5.1% sc-DNA at 1.7% phage survival for MB) under conditions where only minor differences were seen in the relative abundance of M13 coat protein on sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS-PAGE). Neither aluminum phthalocyanine (AlPc) nor merocyanine 540 (MC540) inactivated M13 nor were there significant changes observed in DNA and coat protein. Methylene blue, AlPcS4 and AlPc inactivated VSV and inhibited fusion of the virus envelope to Vero cells at pH 5.7 (i.e. with plasma membrane). However, the degree of this inhibition was small compared to the extent of virus inactivation (43% inhibition vs 4.7 log10 or 99.998% inactivation, for MB). In contrast, an antibody to VSV G-spike protein inhibited fusion at pH 5.7 by 52% with a concomitant decline in VSV infectivity of 0.15 log10 (30%). Few changes were observed in the relative abundance of G protein for MB and AlPcS4 phototreated samples and no additional protein bands were observed on SDS-PAGE. Phototreatment did not appreciably change the relative fusion ability at pH 7.2 (via the endocytotic pathway). These results collectively suggest that nucleic acid may be an important target for photoinactivation of these model viruses by MB and AlPcS4.

BACKGROUND: Treatment with psoralens and UVA (PUVA) has been shown to be efficacious in eliminating the risk of virus transmission by platelet concentrates (PCs). It has previously been demonstrated that, during the inactivation of cell-free vesicular stomatitis virus (VSV) by aminomethyltrimethylpsoralen (AMT) and UVA in PCs, platelet function could be protected either by oxygen removal before irradiation or by inclusion of a type I free radical quencher, such as mannitol. STUDY DESIGN AND METHODS: Under previous PUVA treatment conditions for PCs (25 micrograms/mL AMT; 30 min UVA at 7 mW/cm2; 2 mM [2 mmol/L] mannitol), more than 6 log10 of added cell-free VSV was completely inactivated. In the current study, various PUVA conditions are evaluated for efficacy in inactivating other viral forms that could be present in PCs. Maintenance of platelet integrity (i.e., platelet number, solution pH, and aggregation response during initial storage after treatment) and kill of cell-associated VSV are examined. RESULTS: While cell-free viruses were inactivated efficiently under previous PUVA conditions, cell-associated VSV and the non-lipid-enveloped bacteriophage M13 were not. Effective inactivation of these viruses was achieved by raising the concentration of AMT to 50 micrograms per mL and extending the period of irradiation to 90 minutes (39 J/cm2). However, for maintenance of platelet integrity under these conditions, the prior removal of oxygen or the inclusion of compounds known to quench both type I and type II photoreactants (e.g., flavonoids such as rutin) was required. CONCLUSION: These findings suggest that the viral safety of PCs may be enhanced through treatment with AMT and UVA in the presence of flavonoids, and that flavonoid use may prove beneficial in other systems where oxygen-mediated damage occurs

Propylene oxide (PO) is a widely used industrial reagent which is mutagenic and carcinogenic. We have recently shown that a variety of aliphatic epoxides, including propylene oxide, can react with DNA to form hydroxyalkyl adducts at N-3 of cytosine which rapidly undergo hydrolytic deamination to produce uracil adducts. These 3-hydroxyalkyl uracil adducts are stable in DNA and are postulated to be an important class of potentially mutagenic lesions. Mutagenesis at cytosine residues due to PO modification of single-stranded M13mp2/C141 DNA was studied by transfection of modified DNA into SOS and non-SOS induced E. coli host cells. Mutations of the proline (CCC) codon at C141 which result in reversion of the lacZ phenotype (blue plaques) were scored. It was found that PO treatment of single-stranded DNA results in dose-dependent mutagenesis that is highly SOS dependent. The spectrum of base-substitution mutations found at this site differed when PO-modified DNA was transfected into E. coli with different DNA repair backgrounds. These results indicate that propylene oxide induced DNA adducts at template cytosine residues are mutagenic in E. coli and that this mutagenesis is greatly increased by SOS processing. They also show that these lesions may be repaired by one or more mechanisms.

Scrutiny of NOE data available for the protein encoded by gene V of the filamentous phage IKe (IKe GVP), resulted in the elucidation of a beta-sheet structure which is partly five stranded. The DNA-binding domain of IKe GVP was investigated using a spin-labeled deoxytrinucleotide. The paramagnetic-relaxation effects observed in the 1H-NMR spectrum of IKe GVP, upon binding of this DNA fragment, could be visualized using two-dimensional difference spectroscopy. In this way, the residues present in the DNA-binding domain of IKe GVP can be located in the structure of the protein. They exhibit a high degree of identity with residues in the gene V protein encoded by the distantly related phage M13 (M13 GVP), for which similar spectral perturbations are induced by such a spin-labeled oligonucleotide. Binding studies with negatively charged lanthanide-1,4,7,10-tetraazacyclodecanetrayl-1,4,7-10- tetrakis(methylene)tetrakisphosphonic acid (DOTP) complexes, showed that these complexes bind to IKe and M13 GVP at two spatially remote sites whose affinities have different pH dependencies. Above pH 7, there is one high-affinity binding site for Gd(DOTP)5-/M13 GVP monomer, which coincides with the single-stranded DNA-binding domain as mapped with the aid of spin-labeled oligonucleotide fragments. The results show that single-stranded DNA binds to conserved (phosphate binding) electropositive clusters at the surface of M13 and IKe GVP. These positive patches are interspersed with conserved or conservatively replaced hydrophobic residues. At pH 5, a second Gd(DOTP)(5-)-binding site becomes apparent. The corresponding pattern of spectral perturbations indicates the accommodation of patches of conserved, or conservatively replaced, hydrophobic residues in the cores of the M13 and IKe dimers.

While human parvovirus B19 is associated with fetal damage and chronic suppression of bone marrow in patients with leukemia, much less is known of the genomic characteristics of B19 isolated from damaged human fetuses and leukemia patients. B19 genome DNAs from human fetal organs and fluids and sera from leukemia patients were amplified by the polymerase chain reaction. The nucleotide sequences of amplified products in the region between nucleotide (nt) 3141 and nt3411 were determined following molecular cloning in M13 phage. The genome types of B19 from the fetuses and leukemia patients were similar to the types from patients with aplastic crisis and an asymptomatic individual. Wide diversity of the nucleotide sequence, i.e., six or more (6-11) substitutions, were evident in ten M13 phage clones of each DNA source from fetal materials, while substitutions in sera from patients with leukemia and aplastic crisis and of an asymptomatic individual numbered four or fewer (0-4). This wide diversity of B19 viruses in the fetus, revealed after many rounds of DNA replication, probably depends on a persistent state of infection.

The structure in solution of a β-loop in mutant Y41H of the single-stranded DNA binding protein encoded by gene-V of the filamentous phage M13 has been elucidated using 2-dimensional 1H-nuclear magnetic resonance techniques. Furthermore, these studies enabled us to demonstrate that an identical structural element is present in wild-type gene-V-protein and that this element intimately is involved in the binding of gene-V-protein to single-stranded DNA. It is shown that the structure of the DNA binding wing deviates from that proposed for the same amino acid sequence on the basis of X-ray diffraction data. The structure is, however, identical to that of the DNA binding wing present in the single-stranded DNA binding protein encoded by the genome of the evolutionary distantly related filamentous phage IKe. The latter observations support our current view that in the binding of these proteins to single-stranded DNA a common structural motif is involved.

This unit describes the process and analysis of affinity selecting bacteriophage M13 from libraries displaying combinatorial peptides fused to either a minor or major capsid protein. Direct affinity selection uses target protein bound to a microtiter plate followed by purification of selected phage by ELISA. Alternatively, there is a bead-based affinity selection method. These methods allow one to readily isolate peptide ligands that bind to a protein target of interest and use the consensus sequence to search proteomic databases for putative interacting proteins.

A DNA sequence can be specifically altered by synthesizing the desired sequence change within an oligonucleotide, and then converting this into a biologically active circular DNA strand by using the oligonucleotide to prime in vitro synthesis on a single-stranded circular template. This unit presents a protocol which uses a DNA template containing a small number of uracil residues in place of thymine. Use of the uracil-containing template allows rapid and efficient recovery of mutants; in principle this same template can be applied to most of the other mutagenesis protocols in use. The length of the oligonucleotide primer is highly variable and depends on the nature of the change being made.

This unit contains protocols for preparing DNA suitable for use as dideoxy sequencing templates and as material for end labeling and chemical sequencing. In all protocols, the starting material contains the recombinant molecule to be sequenced. DNA from M13mp-derived phage is easily prepared and is currently the most reliable source of template for large-scale dideoxy sequencing projects. Because it is occasionally necessary or convenient to use a lambda-derived phage as a source of DNA, a protocol for preparing lambda phage DNA from plate lysates is provided. Two protocols for minipreps of plasmid DNA are provided, one intended for dideoxy sequencing, the other for end labeling and chemical sequencing; they differ primarily in the way in which cellular RNA is removed. Alkali denaturation of double-stranded DNA (necessary prior to annealing) is described, and a final protocol describes the preparation of template for thermal cycle sequencing from a single phage plaque or bacterial colony.

To generate antibodies to defined cell-surface antigens, we used a large phage antibody fragment library to select on cell transfectants expressing one of three chosen receptors. First, in vitro panning procedures and phage antibody screening ELISAs were developed using whole live cells stably expressing the antigen of interest. When these methodologies were applied to Chinese hamster ovary (CHO) cells expressing one of the receptors for a neuropeptide, somatostatin, using either direct cell panning or a strategy of depletion or ligand-directed elution, many different pan-CHO-cell binders were selected, but none was receptor specific. However, when using direct panning on CHO-cells expressing the human membrane protein CD36, an extraordinary high frequency of antigen-specific phage antibodies was found. Panning on myoblasts expressing the rat homologue of CD36 revealed a similar selection dominance for anti-(CD36). Binding of all selected 20 different anti-(CD36) phage was surprisingly inhibited by one anti-(CD36) mAb CLB-IVC7, which recognizes a functional epitope that is also immunodominant in vivo. Similar inhibition was found for seven anti-(rat) CD36 that cross-reacted with human CD36. Our results show that, although cells can be used as antigen carriers to select and screen phage antibodies, the nature of the antigen target has a profound effect on the outcome of the selection.