viruses Review Landscape Phage: Evolution from Phage Display to Nanobiotechnology Valery A. Petrenko Department of Pathobiology, Auburn University, Auburn, AL 36849-5519, USA; [email protected]; Tel.: +1-334-844-2897 Received: 11 May 2018; Accepted: 5 June 2018; Published: 7 June 2018 Abstract: The development of phage engineering technology has led to the construction of a novel type of phage display library—a collection of nanofiber materials with diverse molecular landscapes accommodated on the surface of phage particles. These new nanomaterials, called the “landscape phage”, serve as a huge resource of diagnostic/detection probes and versatile construction materials for the preparation of phage-functionalized biosensors and phage-targeted nanomedicines. Landscape-phage-derived probes interact with biological threat agents and generate detectable signals as a part of robust and inexpensive molecular recognition interfaces introduced in mobile detection devices. The use of landscape-phage-based interfaces may greatly improve the sensitivity, selectivity, robustness, and longevity of these devices. In another area of bioengineering, landscape-phage technology has facilitated the development and testing of targeted nanomedicines. The development of high-throughput phage selection methods resulted in the discovery of a variety of cancer cell-associated phages and phage proteins demonstrating natural proficiency to self-assemble into various drug- and gene-targeting nanovehicles. The application of this new “phage-programmed-nanomedicines” concept led to the development of a number of cancer cell-targeting nanomedicine platforms, which demonstrated anticancer efficacy in both in vitro and in vivo experiments. This review was prepared to attract the attention of chemical scientists and bioengineers seeking to develop functionalized nanomaterials and use them in different areas of bioscience, medicine, and engineering. Keywords: phage display; landscape phage; major coat protein; nanomedicine; diagnostics; biosensors 1. Introduction Filamentous bacteriophages (shortly phages), remarkably tolerant of dramatic structural alteration, are in every respect the perfect material for bioengineering. The structure of phage capsids is encoded in small single-stranded circular DNA and can be reengineered using common DNA techniques. Their capsids have a well-defined architecture, consistently adaptable for nanofabrication. The precise structures of filamentous phages and their coat proteins have been determined, allowing for the engineering of variants with precise arrangements of fused functional peptides and desired shapes and functions of phage-originated nanocomposites. The development of phage-functionalized biosensors and phage-targeted nanomedicines, which is the focus of this review, has been closely linked to the advances in structural phage biology and phage display technology. For a complete summary of Ff phage structure and biology, one can be referred to comprehensive reviews [1,2]. This review focuses on Ff phage display vectors, as precursors of landscape phages and as major workhorses in phage nanobiotechnology. It considers also those aspects of the structure and life cycle of Ff phages that are of direct practical interest to chemical scientists and bioengineers seeking to develop phage virions Viruses 2018, 10, 311; doi:10.3390/v10060311 www.mdpi.com/journal/viruses Viruses 2018, 10, 311 2 of 17 Viruses 2018, 10, x FOR PEER REVIEW 2 of 17 nanoparticlesas specifically and functionalized use them nanoparticlesin different andareas use of thembioscience, in different medicine, areas ofmaterial bioscience, science medicine,, and engineering,material science, as summarized and engineering, in [3]. as summarized in [3]. 2.2. The The History History of of Filamentous Filamentous Phage Phagess TheThe story of bacteriophage discovery discovery is is told told by by Stent in his book Molecular Biology of Bacterial Viruses [4][4].. After After French French microbiologist microbiologist Félix Félix d’Hérelle d’Hérelle introduced introduced phages phages as as antibacterial antibacterial therapeutic therapeutic agents,agents, phage phage therapy therapy soon soon became became a a boom boom and and brought about great optimism in biomedical scientistsscientists [5] [5].. After After the the discovery discovery of of penicillin penicillin in in 1 1928928 [6] [6] and and its its triumphal triumphal introduction introduction in in the the 1940s, 1940s, thethe interest interest in in the the practical practical applications applications of of phages phages as as antibacterial antibacterial agents agents faded, faded, along along with with incre increasingasing interestinterest in in phages as tools for fundamentalfundamental biological studies [[7]7].. On On this this new new wave wave of of “phage rerenaissance”,naissance”, in the early 1960s, 1960s, a a group group of of filamentous filamentous phages phages (M13, fd fd,, and and fl) fl) infecting infecting conjugative conjugative FF-pilus-positive-pilus-positive EscherichiaEscherichia coli coli strainsstrains were were isolated isolated from from sewage sewage systems systems in the in United the United States States and Europeand Europe [1]. These [1]. Thesesmall male small-specific male-specific filamentous filamentous coliphages coliphages incorporate incorporate a single- astranded single-stranded circular DNAcircular encapsulated DNA encapsulated in a rigid intubular a rigid capsid tubular (Figure capsid 1). (Figure Because1). genomes Because of genomes these phages of these code phages for a smalcodel fornumber a small of numberproteins, of reproduction proteins, reproduction of the phages of the is phagesmostly issupported mostly supported by host bacteria. by host bacteria.For this andFor thisother and reasons, other reasons,the small the DNA small phages DNA became phages very became popular very popularmodel systems model systemsin molecular in molecular biology andbiology versatile and versatiletools in genetic tools in engineering genetic engineering and biotechnology. and biotechnology. FigureFigure 1. ElectronElectron micro microscopyscopy image image of of filamentous filamentous phage phage ( (leftleft)) and and electron electron density density model model ( (rightright)) ofof filamentous filamentous phage M13 M13 ( (CourtesyCourtesy of of Lee Lee Makowski Makowski and and Gregory Gregory Kishchenko Kishchenko.. Adapted Adapted from from [8] [8]).). BlueBlue and and red red arrows depict the sharp and blunt ends of the phage capsid with attached minor coat proteins pIII/ pIII/pIVpIV and and pVII/p pVII/pIX,IX, respectively respectively (five (five copies copies each) each).. Major Major coat coat protein protein (~2700 (~2700 copies) copies) formsforms the the tubular tubular capsid capsid around viral single single-stranded-stranded DNA. 3. Phage Engineering 3. Phage Engineering In the mid-1970s, as a result of the development of the gene-splicing technique, the creation of In the mid-1970s, as a result of the development of the gene-splicing technique, the creation of molecular chimeras became a novel stirring area in molecular biology and biotechnology [9]. For a molecular chimeras became a novel stirring area in molecular biology and biotechnology [9]. For a comprehensive presentation of the most commonly used molecular cloning systems, the reader is comprehensive presentation of the most commonly used molecular cloning systems, the reader is referred to [10]. As oligonucleotide-directed mutagenesis and DNA sequencing grew into routine referred to [10]. As oligonucleotide-directed mutagenesis and DNA sequencing grew into routine genetic engineering techniques in the 1970s [9], filamentous phages emerged as unique DNA cloning genetic engineering techniques in the 1970s [9], filamentous phages emerged as unique DNA cloning vectors, very convenient for gene reconstruction and protein engineering (reviewed in [11]). These vectors, very convenient for gene reconstruction and protein engineering (reviewed in [11]). These vectors provide a researcher with one of the two strands of vector DNA in a very useful form—easily vectors provide a researcher with one of the two strands of vector DNA in a very useful form—easily purified phage particles. The extracted single-stranded DNA serves then as a template for sequencing purified phage particles. The extracted single-stranded DNA serves then as a template for sequencing and site-directed mutagenesis. The extendable tubular capsid of filamentous phages, oppositely to and site-directed mutagenesis. The extendable tubular capsid of filamentous phages, oppositely to spherical capsids, readily accommodate enclosed recombinant DNA of any length [2]. spherical capsids, readily accommodate enclosed recombinant DNA of any length [2]. In 1985, the emerged filamentous-phage recombinant DNA technique was adapted to design a novel type of viral chimera that inspired the development of phage display technology [12]. To construct this type of viral chimera, an alien DNA fragment is inserted into a gene encoding one of Viruses 2018, 10, 311 3 of 17 In 1985, the emerged filamentous-phage recombinant DNA technique was adapted to design a novel type of viral chimera that inspired the development of phage display technology [12]. To construct this type of viral chimera, an alien DNA fragment is inserted into a gene encoding one of the phage coat proteins in such a way that the encoded “foreign” peptide is genetically fused to a coat protein and, by