Liquid phase combinatorial synthesis of benzylpiperazines

A novel method for soluble, inexpensive polymer-supported synthesis of piperazine and piperidine libraries on the basis of nucleophilic benzylic substitution and N-acylation is reported. Disubstituted piperazine and piperidine derivatives, that are potential drug candidates, can be isolated in high yields and excellent purity by simple precipitation and washing. This liquid phase method proves to be a useful tool for constructing combinatorial libraries containing diamine moieties.     

Selection of streptavidin binders from a DNA-encoded chemical library

DNA-encoded libraries of small organic molecules facilitate the construction of large, encoded self-assembling chemical libraries for the identification of high-affinity binders to protein targets. We have constructed a library of 477 chemical compounds, coupled to 48mer-oligonucleotides, each containing a unique six-base sequence serving as "bar-code" for the identification of the chemical moiety. The functionality of the library was confirmed by selection and amplification of both high- and low-affinity binding molecules specific to streptavidin.     

Analysis of the diversity of diazotrophic bacteria in peat soil by cloning of the <Emphasis Type="Italic">nifH</Emphasis> gene

The diversity of nitrogen-fixing microorganisms in the soil of an oligotrophic Sphagnum peat bog was studied by molecular cloning of fragments of the nifH gene encoding one of the main components of the nitrogenase complex. The fragments were amplified from the DNA isolated from the peat samples collected at the same site in January (library I) and November (library II), 2005. Analysis of the nifH sequence libraries revealed high diversity of diazotrophic bacteria in peat soil: the first library consisted of 237 clones and 55 unique sequence types, the second one included 171 clones and 52 sequence types. Comparison of the two clone libraries showed that the composition and population structure of the nitrogen-fixing community depended greatly on the sampling time; they shared only 11 phylotypes. The sequences of representatives of the class Alphaproteobacteria prevailed in both libraries (27% and 57% of clones in libraries I and II, respectively). Representatives of the classes Deltaproteobacteria and Chlorobea were minor components of library I (6% and 7% of clones, respectively), whereas they prevailed in library II (18% and 24% of clones, respectively). Members of the class Chloroflexi were present only in library I, while members of the classes Bacilli, Clostridia, and Methanomicrobia were present only in library II. Our studies demonstrated that, for complete evaluation of the diversity of natural nitrogen-fixing communities, nifH libraries should consist of at least 200–300 clones.     

Principles and application of antibody libraries for infectious diseases

Antibodies have been used efficiently for the treatment and diagnosis of many diseases. Recombinant antibody technology allows the generation of fully human antibodies. Phage display is the gold standard for the production of human antibodies in vitro. To generate monoclonal antibodies by phage display, the generation of antibody libraries is crucial. Antibody libraries are classified according to the source where the antibody gene sequences were obtained. The most useful library for infectious diseases is the immunized library. Immunized libraries would allow better and selective enrichment of antibodies against disease antigens. The antibodies generated from these libraries can be translated for both diagnostic and therapeutic applications. This review focuses on the generation of immunized antibody libraries and the potential applications of the antibodies derived from these libraries.     

Label-free primary screening and affinity ranking of fragment libraries using parallel analysis of protein panels

The authors present fragment screening data obtained using a label-free parallel analysis approach where the binding of fragment library compounds to 4 different target proteins can be screened simultaneously using surface plasmon resonance detection. They suggest this method as a first step in fragment screening to identify and select binders, reducing the demanding requirements on subsequent X-ray or nuclear magnetic resonance studies, and as a valuable "clean-up" tool to eliminate unwanted promiscuous binders from libraries. A small directed fragment library of known thrombin binders and a general 500-compound fragment library were used in this study. Thrombin, blocked thrombin, carbonic anhydrase, and glutathione-S-transferase were immobilized on the sensor chip surface, and the direct binding of the fragments was studied in real time. Only 12 microg of each protein is needed for screening of a 3000-compound fragment library. For screening, a binding site-blocked target as reference facilitates the identification of binding site-selective hits and the signals from other reference proteins for the elimination of false positives. The scope and limitations of this screening approach are discussed for both target-directed and general fragment libraries.     

Species richness in soil bacterial communities: a proposed approach to overcome sample size bias

Estimates of species richness based on 16S rRNA gene clone libraries are increasingly utilized to gauge the level of bacterial diversity within various ecosystems. However, previous studies have indicated that regardless of the utilized approach, species richness estimates obtained are dependent on the size of the analyzed clone libraries. We here propose an approach to overcome sample size bias in species richness estimates in complex microbial communities. Parametric (Maximum likelihood-based and rarefaction curve-based) and non-parametric approaches were used to estimate species richness in a library of 13,001 near full-length 16S rRNA clones derived from soil, as well as in multiple subsets of the original library. Species richness estimates obtained increased with the increase in library size. To obtain a sample size-unbiased estimate of species richness, we calculated the theoretical clone library sizes required to encounter the estimated species richness at various clone library sizes, used curve fitting to determine the theoretical clone library size required to encounter the "true" species richness, and subsequently determined the corresponding sample size-unbiased species richness value. Using this approach, sample size-unbiased estimates of 17,230, 15,571, and 33,912 were obtained for the ML-based, rarefaction curve-based, and ACE-1 estimators, respectively, compared to bias-uncorrected values of 15,009, 11,913, and 20,909.     

Comparison of large-insert,small-insert and pyrosequencing libraries for metagenomic analysis

The development of DNA sequencing methods for characterizing microbial communities has evolved rapidly over the past decades. To evaluate more traditional, as well as newer methodologies for DNA library preparation and sequencing, we compared fosmid, short-insert shotgun and 454 pyrosequencing libraries prepared from the same metagenomic DNA samples. GC content was elevated in all fosmid libraries, compared with shotgun and 454 libraries. Taxonomic composition of the different libraries suggested that this was caused by a relative underrepresentation of dominant taxonomic groups with low GC content, notably Prochlorales and the SAR11 cluster, in fosmid libraries. While these abundant taxa had a large impact on library representation, we also observed a positive correlation between taxon GC content and fosmid library representation in other low-GC taxa, suggesting a general trend. Analysis of gene category representation in different libraries indicated that the functional composition of a library was largely a reflection of its taxonomic composition, and no additional systematic biases against particular functional categories were detected at the level of sequencing depth in our samples. Another important but less predictable factor influencing the apparent taxonomic and functional library composition was the read length afforded by the different sequencing technologies. Our comparisons and analyses provide a detailed perspective on the influence of library type on the recovery of microbial taxa in metagenomic libraries and underscore the different uses and utilities of more traditional, as well as contemporary ‘next-generation'' DNA library construction and sequencing technologies for exploring the genomics of the natural microbial world.     

Compartmentalized linkage of genes encoding interacting protein pairs

Emulsion technology has been successfully applied to the fields of next-generation high-throughput sequencing, protein engineering and clinical diagnostics. Here, we extend its scope to proteomics research by developing and characterizing a method, termed iCLIP (in vitro compartmentalized linkage of interacting partners), which enables genes encoding interacting protein pairs to be linked in a single segment of DNA. This will facilitate archiving of the interactomes from library versus library two-hybrid screens as libraries of linked DNAs. We further demonstrate the ability to interrogate a model yeast two-hybrid iCLIP library for interactants by "PCR-pulldown," using a primer specific to a gene of interest along with a universal primer. iCLIP libraries may also be subjected to high-throughput sequencing to generate interactome information. The applicability of the technique is also demonstrated in the related context of the bacterial two-hybrid system.     

Construction and characterization of a normalized yeast two-hybrid library derived from a human protein-coding clone collection

The nuclear yeast two-hybrid (Y2H) system is the most widely used technology for detecting interactions between proteins. A common approach is to screen specific test proteins (baits) against large compilations of randomly cloned proteins (prey libraries). For eukaryotic organisms, libraries have traditionally been generated using messenger RNA (mRNA) extracted from various tissues and cells. Here we present a library construction strategy made possible by ongoing public efforts to establish collections of full-length protein encoding clones. Our approach generates libraries that are essentially normalized and contain both randomly fragmented as well as full-length inserts. We refer to this type of protein-coding clone-derived library as random and full-length (RAFL) Y2H library. The library described here is based on clones from the Mammalian Gene Collection, but our strategy is compatible with the use of any protein-coding clone collection from any organism in any vector and does not require inserts to be devoid of untranslated regions. We tested our prototype human RAFL library against a set of baits that had previously been searched against multiple cDNA libraries. These Y2H searches yielded a combination of novel as well as expected interactions, indicating that the RAFL library constitutes a valuable complement to Y2H cDNA libraries.     

Construction and characterization of two rice bacterial artificial chromosome libraries from the parents of a permanent recombinant inbred mapping population

Rice is a leading grain crop and the staple food for over half of the world population. Rice is also an ideal species for genetic and biological studies of cereal crops and other monocotyledonous plants because of its small genome and well developed genetic system. To facilitate rice genome analysis leading to physical mapping, the identification of molecular markers closely linked to economic traits, and map-based cloning, we have constructed two rice bacterial artificial chromosome (BAC) libraries from the parents of a permanent mapping population (Lemont and Teqing) consisting of 400 F9 recombinant inbred lines (RILs). Lemont (japonica) and Teqing (indica) represent the two major genomes of cultivated rice, both are leading commercial varieties and widely used germplasm in rice breeding programs. The Lemont library contains 7296 clones with an average insert size of 150 kb, which represents 2.6 rice haploid genome equivalents. The Teqing library contains 14208 clones with an average insert size of 130 kb, which represents 4.4. rice haploid genome equivalents. Three single-copy DNA probes were used to screen the libraries and at least two overlapping BAC clones were isolated with each probe from each library, ranging from 45 to 260 kb in insert size. Hybridization of BAC clones with chloroplast DNA probes and fluorescent in situ hybridization using BAC DNA as probes demonstrated that both libraries contain very few clones of chloroplast DNA origin and are likely free of chimeric clones. These data indicate that both BAC libraries should be suitable for map-based cloning of rice genes and physical mapping of the rice genome.     

Increased Throughput by Parallelization of Library Preparation for Massive Sequencing

Background

Massively parallel sequencing systems continue to improve on data output, while leaving labor-intensive library preparations a potential bottleneck. Efforts are currently under way to relieve the crucial and time-consuming work to prepare DNA for high-throughput sequencing.

Methodology/Principal Findings

In this study, we demonstrate an automated parallel library preparation protocol using generic carboxylic acid-coated superparamagnetic beads and polyethylene glycol precipitation as a reproducible and flexible method for DNA fragment length separation. With this approach the library preparation for DNA sequencing can easily be adjusted to a desired fragment length. The automated protocol, here demonstrated using the GS FLX Titanium instrument, was compared to the standard manual library preparation, showing higher yield, throughput and great reproducibility. In addition, 12 libraries were prepared and uniquely tagged in parallel, and the distribution of sequence reads between these indexed samples could be improved using quantitative PCR-assisted pooling.

Conclusions/Significance

We present a novel automated procedure that makes it possible to prepare 36 indexed libraries per person and day, which can be increased to up to 96 libraries processed simultaneously. The yield, speed and robust performance of the protocol constitute a substantial improvement to present manual methods, without the need of extensive equipment investments. The described procedure enables a considerable efficiency increase for small to midsize sequencing centers.     

Synthesis and screening of a random dimeric peptide library using the one-bead-one-dimer combinatorial approach

Large combinatorial libraries of random peptides have been used for a variety of applications that include analysis of protein-protein interactions, epitope mapping, and drug targeting. The major obstacle in screening these libraries is the loss of specific but low affinity binding peptides during washing steps. Loss of these specific binders often results in isolation of peptides that bind nonspecifically to components used in the selection process. Previously, it has been demonstrated that dimerizing or multimerizing a peptide can remarkably improve its binding kinetics by 10- to 1000-fold due to an avidity effect. To take advantage of this observation, we constructed a random library of 12 amino acid dimeric peptides on polyethylene glycol acrylamide (PEGA) beads by modifying the 'one-bead-one-compound' approach. The chemical synthesis of 100,000 peptides as dimers can be problematic due to steric and aggregation effects and the presence of many peptide sequences that are difficult to synthesize. We have designed a method, described in detail here, to minimize the problems inherent in the synthesis of a dimeric library by modifying the existing 'split and pool' synthetic method. Using this approach the dimeric library was used to isolate a series of peptides that bound selectively to epithelial cancer cells. One peptide with the amino acid sequence QMARIPKRLARH bound as a dimer to prostate cancer cells spiked into the blood but did not bind to circulating hematopoeitic cells. The monomeric form of this peptide, however, did not bind well to the same LNCaP cell line. These data demonstrate that "hits" obtained from such a 'one-bead-one-dimer' library can be used directly for the final application or used as leads for construction of second generation libraries.     

Non-human primate immune libraries combined with germline humanization: An (almost) new,and powerful approach for the isolation of therapeutic antibodies

Panning of libraries constructed from immunised non-human primates (NHP) has not been widely used, even though this has proven to be a successful approach for the isolation of human-like antibody fragments with affinities in the nanomolar to the picomolar range. As recently demonstrated, after initial isolation of antibodies with such high affinities, germline humanization may be applied to these Fabs or scFvs to increase the similarity of their framework regions with those encoded by human germline genes. ‘Germlinized’ antibody fragments may be converted to full size IgGs; indications are given that these IgGs could be better tolerated in clinical use than human antibodies. The use of the combination of NHP immune libraries and germline humanization thus may compete with use of libraries of human origin, whether naïve or immune, and with synthetic libraries. In this report, the various approaches will be compared, and advantages of the two-step NHP-based method, as well as corresponding intellectual property aspects, will be discussed.Key words: non-human primate, antibody, naïve library, synthetic library, immune library, affinity, epitope, tolerance, patent     

Construction of "small-intelligent" focused mutagenesis libraries using well-designed combinatorial degenerate primers

Site-saturation mutagenesis is a powerful tool for protein optimization due to its efficiency and simplicity. A degenerate codon NNN or NNS (K) is often used to encode the 20 standard amino acids, but this will produce redundant codons and cause uneven distribution of amino acids in the constructed library. Here we present a novel "small-intelligent" strategy to construct mutagenesis libraries that have a minimal gene library size without inherent amino acid biases, stop codons, or rare codons of Escherichia coli by coupling well-designed combinatorial degenerate primers with suitable PCR-based mutagenesis methods. The designed primer mixture contains exactly one codon per amino acid and thus allows the construction of small-intelligent mutagenesis libraries with one gene per protein. In addition, the software tool DC-Analyzer was developed to assist in primer design according to the user-defined randomization scheme for library construction. This small-intelligent strategy was successfully applied to the randomization of halohydrin dehalogenases with one or two randomized sites. With the help of DC-Analyzer, the strategy was proven to be as simple as NNS randomization and could serve as a general tool to efficiently randomize target genes at positions of interest.     

Mathematical expressions useful in the construction, description and evaluation of protein libraries

The creation of protein libraries by random mutagenesis and cassette mutagenesis has proven to be a successful method of protein engineering. Appropriate statistical analysis is important for the proper construction of these libraries and even more important for the interpretation of data from these libraries. We present simple mathematical expressions useful in the creation and evaluation of such libraries. These equations are useful in estimating the distribution of mutations, the degeneracy of the library and the frequency of a particular clone in the library. In addition, general equations addressing the probability that a particular clone is in a library, the probability that a library is complete, and as the consequences of retransformation of the library on these probabilities are presented.     

Towards the construction of high-quality mutagenesis libraries

Objectives

To improve the quality of mutagenesis libraries in directed evolution strategy.

Results

In the process of library transformation, transformants which have been shown to take up more than one plasmid might constitute more than 20% of the constructed library, thereby extensively impairing the quality of the library. We propose a practical transformation method to prevent the occurrence of multiple-plasmid transformants while maintaining high transformation efficiency. A visual library model containing plasmids expressing different fluorescent proteins was used. Multiple-plasmid transformants can be reduced through optimizing plasmid DNA amount used for transformation based on the positive correlation between the occurrence frequency of multiple-plasmid transformants and the logarithmic ratio of plasmid molecules to competent cells.

Conclusions

This method provides a simple solution for a seemingly common but often neglected problem, and should be valuable for improving the quality of mutagenesis libraries to enhance the efficiency of directed evolution strategies.

     

Factors important in evolutionary shaping of immunoglobulin gene loci

Background

The extraordinary diversity characterizing the antibody repertoire is generated by both evolution and lymphocyte development. Much of this diversity is due to the existence of immunoglobulin (Ig) variable region gene segment libraries, which were diversified during evolution and, in higher vertebrates, are used in generating the combinatorial diversity of antibody genes. The aim of the present study was to address the following questions: What evolutionary parameters affect the size and structure of gene libraries? Are the number of genes in libraries of contemporary species, and the corresponding gene locus structure, a random result of evolutionary history, or have these properties been optimized with respect to individual or population fitness? If a larger number of genes or different genome structures do not increase the fitness, then the current structure is probably optimized.

Results

We used a simulation of variable region gene library evolution. We measured the effect of different parameters on gene library size and diversity, and the corresponding fitness. We found compensating relationships between parameters, which optimized Ig library size and diversity.

Conclusions

We conclude that contemporary species' Ig libraries have been optimized by evolution in terms of Ig sequence lengths, the number and diversity of Ig genes, and antibody-antigen affinities.