Diversity assessment.

Several themes have been highlighted recently in both conferences and publications: the availability of product-focused and pharmacophore-based methods for the analysis and design of combinatorial libraries; the power of cell-based methods for molecular similarity, diversity and library design applications; methods for 'rational' diverse subset selection (with applicability to library design); the need for specialized optimization programs for the design of combinatorial libraries that maximize the use of common reagents; and the concept of 'drug-likeness' and its importance in the design of combinatorial libraries.     

Comparison of library screening techniques used in the development of dsDNA ligands

The gel retardation and FID (fluorescent intercalator displacement) techniques have been compared for the selection of dsDNA binding ligands out of library mixtures. The selection procedure involves the synthesis and screening of unnatural oligopeptide libraries based on an iterative deconvolution procedure. Both methods yield comparable selection results and binding constants for the selected compounds, meaning that they can be considered as complementary in the discovery process of new antigene compounds. Furthermore, a quinazolin-2,4-dione amino acid has been identified as possessing interesting properties for interaction with dsDNA.     

Affinity Selection and Mass Spectrometry-Based Strategies to Identify Lead Compounds in Combinatorial Libraries

The screening of diverse libraries of small molecules created by combinatorial synthetic methods is a recent development which has the potential to accelerate the identification of lead compounds in drug discovery. We have developed a direct and rapid method to identify lead compounds in libraries involving affinity selection and mass spectrometry. In our strategy, the receptor or target molecule of interest is used to isolate the active components from the library physically, followed by direct structural identification of the active compounds bound to the target molecule by mass spectrometry. In a drug design strategy, structurally diverse libraries can be used for the initial identification of lead compounds. Once lead compounds have been identified, libraries containing compounds chemically similar to the lead compound can be generated and used to optimize the binding characteristics. These strategies have also been adopted for more detailed studies of protein–ligand interactions.     

High-throughput screening for enhanced protein stability

High thermostability of proteins is a prerequisite for their implementation in biocatalytic processes and in the evolution of new functions. Various protein engineering methods have been applied to the evolution of increased thermostability, including the use of combinatorial design where a diverse library of proteins is generated and screened for variants with increased stability. Current trends are toward the use of data-driven methods that reduce the library size by using available data to choose areas of the protein to target, without specifying the precise changes. For example, the half-lives of subtilisin and a Bacillus subtilis lipase were increased 1500-fold and 300-fold, respectively, using a crystal structure to guide mutagenesis choices. Sequence homology based methods have also produced libraries where 50% of the variants have improved thermostability. Moreover, advances in the high-throughput measurement of denaturation curves and the application of selection methods to thermostability evolution have enabled the screening of larger libraries. The combination of these methods will lead to the rapid improvement of protein stability for biotechnological purposes.     

Phage display in pharmaceutical biotechnology

Over the past year, methods for the construction of M13 phage-display libraries have been significantly improved and new display formats have been developed. Phage-displayed peptide libraries have been used to isolate specific ligands for numerous protein targets. New phage antibody libraries have further expanded the practical applications of the technology and phage cDNA libraries have proven useful in defining natural binding interactions. In addition, phage-display methods have been developed for the rapid determination of binding energetics at protein-protein interfaces.     

Totally in vitro protein selection using mRNA-protein fusions and ribosome display.

Both chemists and biochemists have great interest in creating peptides and proteins with desired structure, recognition and catalytic properties. Recently, two techniques have been developed that afford functional selection of proteins entirely in vitro: mRNA-protein fusions, and ribosome display. Improvements in both methods have allowed peptide and protein libraries of unprecedented size (10(11)-10(13) different members) to be generated and screened for function.     

In vivo versus in vitro screening or selection for catalytic activity in enzymes and abzymes

The recent development of catalytic antibodies and the introduction of new techniques to generate huge libraries of random mutants of existing enzymes have created the need for powerful tools for finding in large populations of cells those producing the catalytically most active proteins. Several approaches have been developed and used to reach this goal. The screening techniques aim at easily detecting the clones producing active enzymes or abzymes; the selection techniques are designed to extract these clones from mixtures. These techniques have been applied both in vivo and in vitro. This review describes the advantages and limitations of the various methods in terms of ease of use, sensitivity, and convenience for handling large libraries. Examples are analyzed and tentative rules proposed. These techniques prove to be quite powerful to study the relationship between structure and function and to alter the properties of enzymes.     

The use of EST expression matrixes for the quality control of gene expression data

EST expression profiling provides an attractive tool for studying differential gene expression, but cDNA libraries' origins and EST data quality are not always known or reported. Libraries may originate from pooled or mixed tissues; EST clustering, EST counts, library annotations and analysis algorithms may contain errors. Traditional data analysis methods, including research into tissue-specific gene expression, assume EST counts to be correct and libraries to be correctly annotated, which is not always the case. Therefore, a method capable of assessing the quality of expression data based on that data alone would be invaluable for assessing the quality of EST data and determining their suitability for mRNA expression analysis. Here we report an approach to the selection of a small generic subset of 244 UniGene clusters suitable for identification of the tissue of origin for EST libraries and quality control of the expression data using EST expression information alone. We created a small expression matrix of UniGene IDs using two rounds of selection followed by two rounds of optimisation. Our selection procedures differ from traditional approaches to finding "tissue-specific" genes and our matrix yields consistency high positive correlation values for libraries with confirmed tissues of origin and can be applied for tissue typing and quality control of libraries as small as just a few hundred total ESTs. Furthermore, we can pick up tissue correlations between related tissues e.g. brain and peripheral nervous tissue, heart and muscle tissues and identify tissue origins for a few libraries of uncharacterised tissue identity. It was possible to confirm tissue identity for some libraries which have been derived from cancer tissues or have been normalised. Tissue matching is affected strongly by cancer progression or library normalisation and our approach may potentially be applied for elucidating the stage of normalisation in normalised libraries or for cancer staging.     

DNA display II. Genetic manipulation of combinatorial chemistry libraries for small-molecule evolution

Biological in vitro selection techniques, such as RNA aptamer methods and mRNA display, have proven to be powerful approaches for engineering molecules with novel functions. These techniques are based on iterative amplification of biopolymer libraries, interposed by selection for a desired functional property. Rare, promising compounds are enriched over multiple generations of a constantly replicating molecular population, and subsequently identified. The restriction of such methods to DNA, RNA, and polypeptides precludes their use for small-molecule discovery. To overcome this limitation, we have directed the synthesis of combinatorial chemistry libraries with DNA "genes," making possible iterative amplification of a nonbiological molecular species. By differential hybridization during the course of a traditional split-and-pool combinatorial synthesis, the DNA sequence of each gene is read out and translated into a unique small-molecule structure. This "chemical translation" provides practical access to synthetic compound populations 1 million-fold more complex than state-of-the-art combinatorial libraries. We carried out an in vitro selection experiment (iterated chemical translation, selection, and amplification) on a library of 10(6) nonnatural peptides. The library converged over three generations to a high-affinity protein ligand. The ability to genetically encode diverse classes of synthetic transformations enables the in vitro selection and potential evolution of an essentially limitless collection of compound families, opening new avenues to drug discovery, catalyst design, and the development of a materials science "biology."     

Dynamic diversity and small-molecule evolution: a new paradigm for ligand identification.

A longstanding goal of organic, medicinal and bioorganic chemists has been the discovery of efficient methods for designing or identifying biologically active compounds. Recently, several groups have reported using the directed evolution of combinatorial libraries as a new method of identifying compounds capable of binding tightly to a target molecule. Although significant development remains to be done, the initial results suggest that dynamic diversity and associated selection methods will prove to be valuable additions to the drug-discovery process.     

Display of active subtilisin 309 on phage: analysis of parameters influencing the selection of subtilisin variants with changed substrate specificity from libraries using phosphonylating inhibitors

Many attempts have been made to endow enzymes with new catalytic activities. One general strategy involves the creation of random combinatorial libraries of mutants associated with an efficient screening or selection scheme. Phage display has been shown to greatly facilitate the selection of polypeptides with desired properties by establishing a close link between the polypeptide and the gene that encodes it. Selection of phage displayed enzymes for new catalytic activities remains a challenge. The aim of this study was to display the serine protease subtilisin 309 (savinase) from Bacillus lentus on the surface of filamentous fd phage and to develop selection schemes that allow the extraction of subtilisin variants with a changed substrate specificity from libraries. Subtilisins are produced as secreted preproenzyme that mature in active enzyme autocatalytically. They have a broad substrate specificity but exhibit a significant preference for hydrophobic residues and very limited reactivity toward charged residues at the P4 site in the substrate. Here, we show that savinase can be functionally displayed on phage in the presence of the proteic inhibitor CI2. The free enzyme is released from its complex with CI2 upon addition of the anionic detergent LAS. The phage-enzyme can be panned on streptavidin beads after labelling by reaction with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-l-Ala-l-Ala-l-P ro-Phe(P)-diphenyl ester. Reactions of libraries, in which residues 104 and 107 forming part of the S4 pocket have been randomised, with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-alpha-l-Lys-l-A la-l-Pro-Phe(P)-diphenylester allowed us to select enzymes with increased specific activity for a substrate containing a lysine in P4. Parameters influencing the selection as for instance the efficiency of maturation of mutant enzymes in libraries have been investigated.     

mRNA display for the selection and evolution of enzymes from in vitro-translated protein libraries

The mRNA display technology enables the in vitro selection and directed evolution of functional proteins from libraries of more than 10(12) different mutants in a single test tube. The size of these libraries is well beyond the limit of screening technologies and of most in vivo and in vitro selection methods. The mRNA display technology has been used to select peptides and proteins that bind to a specific ligand, as well as novel enzymes. This protocol details the procedure to produce mRNA-displayed proteins (3 d) and to subject them to a selection and evolution of enzymes for bond-forming reactions (4-10 weeks). This method is demonstrated by the generation of new RNA ligase enzymes.     

Phage display: Concept,innovations, applications and future

Phage display is the technology that allows expression of exogenous (poly)peptides on the surface of phage particles. The concept is simple in principle: a library of phage particles expressing a wide diversity of peptides is used to select those that bind the desired target. The filamentous phage M13 is the most commonly used vector to create random peptide display libraries. Several methods including recombinant techniques have been developed to increase the diversity of the library. On the other extreme, libraries with various biases can be created for specific purposes. For instance, when the sequence of the peptide that binds the target is known, its affinity and selectivity can be increased by screening libraries created with limited mutagenesis of the peptide. Phage libraries are screened for binding to synthetic or native targets. The initial screening of library by basic biopanning has been extended to column chromatography including negative screening and competition between selected phage clones to identify high affinity ligands with greater target specificity. The rapid isolation of specific ligands by phage display is advantageous in many applications including selection of inhibitors for the active and allosteric sites of the enzymes, receptor agonists and antagonists, and G-protein binding modulatory peptides. Phage display has been used in epitope mapping and analysis of protein-protein interactions. The specific ligands isolated from phage libraries can be used in therapeutic target validation, drug design and vaccine development. Phage display can also be used in conjunction with other methods. The past innovations and those to come promise a bright future for this field.     

Synthetic antibodies: Concepts, potential and practical considerations

The last 100years of enquiry into the fundamental basis of humoral immunity has resulted in the identification of antibodies as key molecular sentinels responsible for the in vivo surveillance, neutralization and clearance of foreign substances. Intense efforts aimed at understanding and exploiting their exquisite molecular specificity have positioned antibodies as a cornerstone supporting basic research, diagnostics and therapeutic applications [1]. More recently, efforts have aimed to circumvent the limitations of developing antibodies in animals by developing wholly in vitro techniques for designing antibodies of tailored specificity. This has been realized with the advent of synthetic antibody libraries that possess diversity outside the scope of natural immune repertoires and are thus capable of yielding specificities not otherwise attainable. This review examines the convergence of technologies that have contributed to the development of combinatorial phage-displayed antibody libraries. It further explores the practical concepts that underlie phage display, antibody diversity and the methods used in the generation of and selection from phage-displayed synthetic antibody libraries, highlighting specific applications in which design approaches gave rise to specificities that could not easily be obtained with libraries based upon natural immune repertories.     

Construction of a human chromosome 3 specific NotI linking library using a novel cloning procedure.

Two new diphasmid vectors (lambda SK17 and SK22) and a novel procedure to construct linking libraries are described. A partial filling-in reaction provides counter-selection against false linking clones in the library, and obviates the need for supF selection. The diphasmid vectors, in combination with the novel selection procedure, have been used to construct a chromosome 3 specific NotI linking library from a human chromosome 3/mouse microcell hybrid cell line. The application of the new vectors and the strong biochemical and biological selections resulted in a library of 60,000 NotI linking clones. As practically all of them are real NotI linking clones (no false recombinants) the library represents approximately 3,000 human recombinants (equal to 10-15 genomic equivalents of chromosome 3). Previously published methods for construction of linking libraries are compared with the procedure described in the present paper. The advantages of the new vectors and the novel protocol are discussed.     

Phage display as a tool for the directed evolution of enzymes

Since its introduction in 1985, phage display has had a tremendous impact on the discovery of peptides that bind to a variety of receptors, the generation of binding sites within predefined scaffolds, and the creation of high-affinity antibodies without immunization. Its application to enzymology has required the development of techniques that couple enzymatic activity to selection protocols based on affinity chromatography. Here, we describe both indirect methods, using transition-state analogues and suicide substrates, and direct methods, using the ability of active phage-enzymes to transform substrate into product. The methods have been applied to large libraries for mechanistic-based studies and to generate variants with new or improved properties. In addition, such techniques have been successfully used to select catalytic antibodies and improve their catalytic efficiency.     

Analytical applications of aptamers

So far, several bio-analytical methods have used nucleic acid probes to detect specific sequences in RNA or DNA targets through hybridisation. More recently, specific nucleic acids, aptamers, selected from random sequence pools, have been shown to bind non-nucleic acid targets, such as small molecules or proteins. The development of in vitro selection and amplification techniques has allowed the identification of specific aptamers, which bind to the target molecules with high affinity. Many small organic molecules with molecular weights from 100 to 10,000 Da have been shown to be good targets for selection. Moreover, aptamers can be selected against difficult target haptens, such as toxins or prions. The selected aptamers can bind to their targets with high affinity and even discriminate between closely related targets.

Aptamers can thus be considered as a valid alternative to antibodies or other bio-mimetic receptors, for the development of biosensors and other analytical methods. The production of aptamers is commonly performed by the SELEX (systematic evolution of ligands by exponential enrichment) process, which, starting from large libraries of oligonucleotides, allows the isolation of large amounts of functional nucleic acids by an iterative process of in vitro selection and subsequent amplification through polymerase chain reaction.

Aptamers are suitable for applications based on molecular recognition as analytical, diagnostic and therapeutic tools. In this review, the main analytical methods, which have been developed using aptamers, will be discussed together with an overview on the aptamer selection process.     

Scalable High Throughput Selection From Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.     

The phage display technique: advantages and recent patents

Phage display technology has advanced considerably since its creation, and the number of research projects using this technique is constantly increasing, generating numerous antibody and antigen libraries. These libraries, besides expediting library screening, improving selection methods and allowing evaluation of novel applications, have great potential for the development of new vaccines, drugs and diagnosis tests. Consequently, patent registries for the protection of these sequences are essential.     

Selection of proteins and peptides from libraries displayed on filamentous bacteriophage

This article attempts to review recent developments in the rapidly developing field of phage display libraries. The current state of peptide, antibody, and cDNA libraries, as well as current and future applications of phage display libraries are discussed. The main focus of the article is on the methods for selecting binding ligands against targets in a variety of different formats. These include solid phase and in-solution selection methods, and the strategies used to select for higher affinity, and binding ligands ampure and cellular target proteins.