Individual Library Members Research Articles

Growth amplification in ultrahigh-throughput microdroplet screening increases sensitivity of clonal enzyme assays and minimizes phenotypic variation.

Microfluidic ultrahigh-throughput screening of enzyme activities provides information on libraries with millions of variants in a day. Each individual library member is represented by a recombinant single cell, compartmentalised in an emulsion droplet, in which an activity assay is carried out. Key to the success of this approach is the precision and sensitivity of the assay. Assay quality is most profoundly challenged when initially weak, promiscuous activities are to be enhanced in early rounds of directed evolution or when entirely novel catalysts are to be identified from metagenomic sources. Implementation of measures to widen the dynamic range of clonal assays would increase the chances of finding and generating new biocatalysts. Here, we demonstrate that the assay sensitivity and DNA recovery can be improved by orders of magnitude by growth of initially singly compartmentalised cells in microdroplets. Homogeneous cell growth is achieved by continuous oxygenation and recombinant protein expression is regulated by diffusion of an inducer from the oil phase. Reaction conditions are adjusted by directed droplet coalescence to enable full control of buffer composition and kinetic incubation time, creating level playing field conditions for library selections. The clonal amplification multiplies the product readout because more enzyme is produced per compartment. At the same time, phenotypic variation is reduced by measuring monoclonal populations rather than single cells and recovery efficiency is increased. Consequently, this workflow increases the efficiency of lysate-based microfluidic enzyme assays and will make it easier for protein engineers to identify or evolve new enzymes for applications in synthetic and chemical biology.

Analysis of Current DNA Encoded Library Screening Data Indicates Higher False Negative Rates for Numerically Larger Libraries.

To optimize future DNA-encoded library design, we have attempted to quantify the library size at which the signal becomes undetectable. To accomplish this we (i) have calculated that percent yields of individual library members following a screen range from 0.002 to 1%, (ii) extrapolated that ∼1 million copies per library member are required at the outset of a screen, and (iii) from this extrapolation predict that false negative rates will begin to outweigh the benefit of increased diversity at library sizes >108. The above analysis is based upon a large internal data set comprising multiple screens, targets, and libraries; we also augmented our internal data with all currently available literature data. In theory, high false negative rates may be overcome by employing larger amounts of library; however, we argue that using more than currently reported amounts of library (≫10 nmoles) is impractical. The above conclusions may be generally applicable to other DNA encoded library platforms, particularly those platforms that do not allow for library amplification.

Universal strategies for the DNA-encoding of libraries of small molecules using the chemical ligation of oligonucleotide tags.

The affinity-mediated selection of large libraries of DNA-encoded small molecules is increasingly being used to initiate drug discovery programs. We present universal methods for the encoding of such libraries using the chemical ligation of oligonucleotides. These methods may be used to record the chemical history of individual library members during combinatorial synthesis processes. We demonstrate three different chemical ligation methods as examples of information recording processes (writing) for such libraries and two different cDNA-generation methods as examples of information retrieval processes (reading) from such libraries. The example writing methods include uncatalyzed and Cu(I)-catalyzed alkyne-azide cycloadditions and a novel photochemical thymidine-psoralen cycloaddition. The first reading method “relay primer-dependent bypass” utilizes a relay primer that hybridizes across a chemical ligation junction embedded in a fixed-sequence and is extended at its 3′-terminus prior to ligation to adjacent oligonucleotides. The second reading method “repeat-dependent bypass” utilizes chemical ligation junctions that are flanked by repeated sequences. The upstream repeat is copied prior to a rearrangement event during which the 3′-terminus of the cDNA hybridizes to the downstream repeat and polymerization continues. In principle these reading methods may be used with any ligation chemistry and offer universal strategies for the encoding (writing) and interpretation (reading) of DNA-encoded chemical libraries.

Improved Biocatalysts from a Synthetic Circular Permutation Library of the Flavin-Dependent Oxidoreductase Old Yellow Enzyme

Members of the old yellow enzyme (OYE) family are widely used, effective biocatalysts for the stereoselective trans-hydrogenation of activated alkenes. To further expand their substrate scope and improve catalytic performance, we have applied a protein engineering strategy called circular permutation (CP) to enhance the function of OYE1 from Saccharomyces pastorianus. CP can influence a biocatalyst's function by altering protein backbone flexibility and active site accessibility, both critical performance features because the catalytic cycle for OYE1 is thought to involve rate-limiting conformational changes. To explore the impact of CP throughout the OYE1 protein sequence, we implemented a highly efficient approach for cell-free cpOYE library preparation by combining whole-gene synthesis with in vitro transcription/translation. The versatility of such an ex vivo system was further demonstrated by the rapid and reliable functional evaluation of library members under variable environmental conditions with three reference substrates ketoisophorone, cinnamaldehyde, and (S)-carvone. Library analysis identified over 70 functional OYE1 variants with several biocatalysts exhibiting over an order of magnitude improved catalytic activity. Although catalytic gains of individual cpOYE library members vary by substrate, the locations of new protein termini in functional variants for all tested substates fall within the same four distinct loop/lid regions near the active site. Our findings demonstrate the importance of these structural elements in enzyme function and support the hypothesis of conformational flexibility as a limiting factor for catalysis in wild type OYE.

Picoliter Cell Lysate Assays in Microfluidic Droplet Compartments for Directed Enzyme Evolution

We demonstrate the utility of a microfluidic platform in which water-in-oil droplet compartments serve to miniaturize cell lysate assays by a million-fold for directed enzyme evolution. Screening hydrolytic activities of a promiscuous sulfatase demonstrates that this extreme miniaturization to the single-cell level does not come at a high price in signal quality. Moreover, the quantitative readout delivers a level of precision previously limited to screening methodologies with restricted throughput. The sorting of 3 × 10(7) monodisperse droplets per round of evolution leads to the enrichment of clones with improvements in activity (6-fold) and expression (6-fold). The detection of subtle differences in a larger number of screened clones provides the combination of high sensitivity and high-throughput needed to rescue a stalled directed evolution experiment and make it viable.

An arrayed human genomic library constructed in the PAC shuttle vector pJCPAC-Mam2 for genome-wide association studies

Genomic libraries constructed in the original Bacterial Artificial Chromosome (BAC) and P1 Artificial Chromosome (PAC) cloning systems were very useful for completion of the Human Genome Project. Libraries constructed in these vector systems were used to generate physical maps of all twenty three-chromosome pairs and served as the templates for DNA sequencing. The PAC vector pJCPAC-Mam2 is a 23 kilobase (kb) shuttle vector that is highly versatile and useful for functional studies in human tissue culture cell lines. pJCPAC-Mam2 contains the P1 single-copy replicon for low copy expression and a multi-copy lytic replicon under the control of the lac repressor for high copy expression in Escherichia coli, wild type and mutant loxP sites for generation of bidirectional nested deletions in any clone of interest and the Epstein Barr Virus (EBV) latent replication origin oriP, the Epstein Barr Nuclear Antigen 1 (EBNA1) gene, and a puromycin-resistance gene for propagation in mammalian cells. We constructed an arrayed 115,000-member human genomic library in pJCPAC-Mam2 that is housed in twelve hundred 96 well dishes. Subsequent pooling using a column and rows strategy reduced the library to 19 final pools for Polymerase Chain Reaction (PCR) screening. We estimate that the average insert size is approximately 57 kb based on NotI digestion and Field Inversion Gel Electrophoresis (FIGE) analysis of 473 random PAC clones. The library appears to represent a two- to three-fold coverage of the human genome based on screening of the library with PCR primers representing nine genes. This library should serve as a valuable resource to validate potential disease genes identified by genome-wide association studies (GWAS) in human tissue culture cell lines and in animal models. Also, individual library members could potentially be used for gene therapy trials for a variety of recessive disorders.

An arrayed human genomic library constructed in the PAC shuttle vector pJCPAC-Mam2 for genome-wide association studies and gene therapy

The various iterations of the HapMap Project and many genome-wide association studies (GWAS) have identified hundreds of potential genes involved in monogenic and multifactorial traits. We constructed an arrayed 115,000-member human genomic library in the PAC shuttle vector pJCPAC-Mam2 that can be propagated in both bacterial and human cells. The library appears to represent a two-fold coverage of the human genome. Transient transfection of a p53-containing PAC clone into p53-null Saos-2 human osteosarcoma cells demonstrated that both p53 mRNA and protein were produced. Additionally, expression of the p53 protein triggered apoptosis in a subset of the Saos-2 cells. This library should serve as a valuable resource to validate potential disease genes identified by GWAS in human cell lines and in animal models. Also, individual library members could potentially be used for gene therapy trials for a variety of recessive disorders.

Comparison of different methods to analyze a DNA computing library using the polymerase chain reaction

Screening and analysis of collections of DNA molecules is a standard aspect of many DNA computing approaches. We describe the use of three different polymerase chain reaction (PCR) detection methods to screen specific members of a 5-site, 2-variable DNA computing library previously created using parallel overlap assembly of unique sequences generated from the SynDCode program. The three PCR methods (conventional gel-based PCR, SYBR Green real-time PCR and TaqMan real-time PCR) could all successfully identify individual library members separately or in a mixture. The TaqMan approach was also able to identify members in the original library we had not yet sequenced, providing more evidence supporting our hypothesis that the DNA library we generated may be complete. We expect these three approaches will be useful in future screening of other DNA computing libraries and structures.

Balancing Act for the Future: How the Academic Library Engages in Collection Development at the Local and Consortial Levels

ABSTRACT Collection development remains a major focus and concern of current interest for all types of libraries. Consortia began as a means of sharing resources and of leveraging purchasing power for its members. How does a library balance its individual collection needs, both present and future, when it is a member of a consortium? Do the expectations of the consortium outweigh the needs of the individual library member? This article will address how an academic library engages in collection development at the local and consortia levels, addresses the benefits and challenges of consortia memberships, and responds to its local patrons in light of its obligations and commitment to a consortium.

Host–Guest Binding Constants Can Be Estimated Directly from the Product Distributions of Dynamic Combinatorial Libraries

In the last decade, dynamic combinatorial chemistry has developed into a powerful method for the discovery of new sensor systems, catalysts, ligands for biomolecules, and synthetic receptors. Until now, determining the binding strength for individual library members has required their separation from the library and subsequent analysis. We now show that it is possible to obtain most host–guest binding affinities, even in 30+ component libraries, directly from product distributions by using a numerical data-fitting procedure. We have verified our method by using a threecomponent experimental system and showing that the binding constants computed from the behavior of the dynamic mixture agree well with those obtained independently by binding studies on the isolated library members. Dynamic combinatorial libraries (DCLs) of interconverting oligomers are made by mixing a set of building blocks that can reversibly combine with each other to give a dynamic equilibrium. The composition of the DCL is determined by the relative stability of all its members, so it is responsive to external stimuli that can affect these stabilities. Thus, addition of a guest molecule to a library of potential receptors should result in the stabilization and amplification of those library members that can form favorable interactions with the guest molecule. Ideally, the good receptors are amplified at the expense of the other oligomers. Quantifying their binding strength thus far entailed painstaking isolation of the selected compounds from the library. This process often requires switching off the exchange reaction through a substantial change in the pH value, or even covalent modification so that the protonation state or structure of the isolated species may no longer correspond to that of the species that was selected in the library. Also, under certain experimental conditions, the intuitive correlation between amplification and binding affinity can break down. Thus, there is a strong incentive to develop a method that allows the determination of binding affinities directly from the behavior of DCLs without requiring studies on isolated library members. We reasoned that the response of a DCL of potential receptors to changes in the concentrations of building blocks and guest molecules should contain enough information about the underlying host–guest binding constants that it should be possible to extract these by using a multivariable fitting procedure. We first tested this hypothesis on a simple set of seven libraries by using building block 1 (2 mm ; Figure 1) in the

Comparisons of PCR-based genome amplification systems using CpG island microarrays

The characterization of complex DNA libraries by high-throughput sequencing technologies provides a powerful approach toward finding mutations and genetic variation in the human genome. However, the value of these analyses is highly dependent upon the quality of the DNA libraries themselves. For example, the sequence composition of libraries made using PCR-based procedures can be skewed due to biases in the amplification efficiency of individual library members. Here, we used CpG island microarrays to evaluate the biases incurred in PCR-based genome amplification using three different DNA polymerase mixtures designed to efficiently amplify problematic sequence tracts. Based on hybridization properties of dye-labeled DNA libraries to these microarrays, we quantified the overall and specific trends in the PCR efficiency of more than 1,400 sequences with high GC-content, which generally amplify with low efficiency using conventional PCR protocols. Overall, all three DNA polymerase mixtures produced libraries that show substantial increases in the representation of CpG island segments that poorly amplify with Taq DNA polymerase. However, the effects of these DNA polymerases were quite specific since they did not alter the relative representation of segments that efficiently amplify with Taq DNA polymerase. Furthermore, we demonstrate that DNA microarrays provide a robust platform for rapidly evaluating the ability of different PCR systems to amplify difficult genomic regions targeted for mutational, resequencing, and genotyping analyses.

Fourier transform analysis for periodic combinatorial arrays

Earlier we introduced a combinatorial synthetic method that employs one-dimensional supports ranging from cotton threads to optical fibres. This method affords parallel synthesis and availability of the complete library history ideally yielding identification of all library members. Moreover, the synthesized compound library will be arrayed periodically on the linear support, leading to assay data that also reflect this periodicity. This fact invites an analysis using the Fourier transform. Here we demonstrate how this approach presents n-dimensional data in a comprehensible manner and facilitates the identification of trends within the library. Carrying out an inverse Fourier transform on subsets of the data allows for the assignment of fitness profiles for each reactant and combination of reactants in the library. The tools should assist in drawing conclusions based on the diversity of library response as opposed to individual library members.

A Route to Diverse Combinatorial Libraries of Electroactive Nickel Hexacyanoferrate

Electroactive nickel hexacyanoferrate (NiHCF) thin films are of interest for their ion sensing and ion intercalation properties; these properties depend on the material's stoichiometry and structure. A small combinatorial library of NiHCF thin films was synthesized by microdispensing 2-μL droplets of reagent with varied proportions of a divalent nickel source (nickel sulfate), a ferricyanide source (potassium ferricyanide), and water on Pt substrates. Two different mixing orders were used (Ni II /H 2 O/Fe III and Fe III /H 2 O/Ni II ) and 13 discrete compositions on the ternary reagent diagram were chosen. These 26 library members were electrochemically intercalated/deintercalated with K + ions by cyclic voltammetry. Raman spectroscopy was used to screen individual library members for electroactivity; this step eliminated 11 inactive members, most of which resulted from Ni II -rich growth conditions. Energy-dispersive X-ray spectroscopy (EDS) was used to determine the number of intercalated K + ions in the oxidized unit cell (N K,ox ) for the remaining 15 members. The quantity N K,ox is a sensitive measure of NiHCF matrix stoichiometry, with a perfectly stoichiometric matrix possessing N K,ox = 4. N K,ox for this library was found to vary from 0 to ∼3 (maximum possible variability is 0-4), with smaller N K,ox from the Ni II /H 2 O/Fe III mixing order and larger N K,ox from the Fe III /H 2 O/Ni II order. The methods used to synthesize this small, but diverse, library are easily scalable to much larger libraries.

Dynamic covalent chemistry on self-templating peptides: formation of a disulfide-linked beta-hairpin mimic.

Dynamic covalent chemistry on self-templating peptides: formation of a disulfide-linked beta-hairpin mimic.

Construction of a PAC vector system for the propagation of genomic DNA in bacterial and mammalian cells and subsequent generation of nested deletions in individual library members

The BAC and PAC cloning systems allow investigators to propagate large genomic DNA fragments up to 300 kb in size in E. colicells.We describe a new PAC shuttle vector that can be propagated in both bacterial and human cells. Specifically, the P1 cloning vector pAd10sacBII was modified by the insertion of a puromycin-resistance gene ( pac), the Epstein-Barr Virus (EBV) latent replication origin oriP,and the EBV EBNA1 gene. Transfection studies in HEK 293 cells demonstrated that the modified vector was stably maintained as an episome for at least 30 generations. And since pJCPAC-Mam1 contains a loxP site, genomic DNA cloned into this vector can be subjected to loxP-Cre -mediated deletion events. The transposon vector pTnPGKpuro/loxP was modified to make this system amenable to propagation in human cells by inserting pac, oriP, and EBNA1 elements into the vector (Chatterjee, P.K., Coren, J.C., 1997. Isolating large nested deletions in PACs and BACs by in vivo selection of P1 headful-packaged products of Cre-catalyzed recombination between the loxP site in PAC and BAC and one introduced in transposition. NAR 25, 2205–2212.). pTnPGKpuro/loxP-EBV was then used to generate deletions in an individual library member to demonstrate that all of the deletions still contain the required eukaryotic elements and that they were nested. All library members constructed in pJCPAC-Mam1 can be directly transformed into human cells to assess function. And the deletion technology can be used to aid in delineating the boundaries of genes and other cis-acting elements.

Cytotoxic hammerhead ribozymes.

Small catalytic RNA molecules of the hammerhead ribozyme type were found to have cytotoxic effects unrelated to their intended activity. An expression library of ribozyme sequence variants was constructed in a recA-deficient strain of Escherichia coli such that individual library members differed in regions designed to form base pairs with human immunodeficiency virus-1 (HIV-1) tat mRNA. The parental ribozyme and many variants exhibited a bacteriostatic effect. One variant studied in detail was also bactericidal. When its expression was induced, ribozyme-dependent inhibition of bacterial growth was not observed in recA+ or recA+ lexA3 (Ind-) cells, suggesting that the recombination function of the RecA protein, not the absence of the SOS response, is sufficient to alleviate the cytotoxic effect. These data document the need for careful testing for toxic effects during intracellular studies of ribozyme action.

Solution Phase Combinatorial Synthesis of Biaryl Libraries Employing Heterogeneous Conditions for Catalysis and Isolation with Size Exclusion Chromatography for Purification

A convenient and effective approach to the solution phase synthesis of biaryl libraries from iodoarenes is described enlisting 10% Pd−C as an inexpensive and readily available supported catalyst. Isolation and purification of small library mixtures, individual library members, and intermediates in the multistep sequence by a combination of liquid−liquid and liquid−solid extractions and size exclusion chromatography are detailed.

Solid-phase extraction on C18 silica as a purification strategy in the solution synthesis of a 1-thio-β-d-galactopyranoside library

A novel strategy for the purification of carbohydrate-based chemical libraries synthesized in solution was developed. Purification of reaction products was accomplished by means of solid-phase extraction enabled by protecting the 2-, 3-, 4-, and 6-hydroxyl groups of a galactose derivative as their hydrophobic O-laurates. The presence of multiple O-laurates allowed adsorption of reaction products onto C18 silica while reagents and by-products were washed away with MeOH. Products were quantitatively eluted with pentane. Purification of products using solid-phase extraction offers the combined advantages of solution synthesis (normal solution reactivity and ease of reaction monitoring) with those of solid-phase synthesis (facile product isolation permitting the use of large excesses of reagents). To demonstrate the utility of the hydrophobic recovery-procedure, tetra-O-lauroyl-β-d-galactopyranose-1-thiol was subjected to high-yielding reactions with a panel of Michael-acceptors and an α-chloro ketone. The resulting ketone adducts were then either reduced to the alcohols or reductively aminated with a selection of amino acids to give 30 different 1-thio-β-d-galactosides as mixtures of four diastereomers after removal of protecting groups. At each step, the product was separated from the reagents and their by-products by simple adsorption onto C18 silica, washing with MeOH and elution of product with pentane. After completion of the combinatorial chemistry sequence, the O-laurates were cleaved by methanolysis and the product methyl laurate in turn removed from the desired water-soluble products by C18 adsorption. Individual library members were thus conveniently produced on 10–30mg scales at purity levels of >90%. One of the 1-thio-β-d-galactosides thus produced was found to be a competitive inhibitor of the β-galactosidase from E. coli with Ki value of 1.7μM.

Soluble Supports Tailored for Organic Synthesis: Parallel Polymer SynthesisviaSequential Normal/Living Free Radical Processes

To expand the availability and solubility range of polymer supports for liquid-phase organic synthesis (LPOS) we have applied a sequence of normal and “living” free radical polymerization to generate a library of block copolymers possessing either block or graft architecture with initiators 1−4 and a diverse set of vinyl monomers 5−9. The structure, molecular weight, and polydispersity (PD) of the individual library members have been determined by size exclusion chromatography (SEC), 1H and 13C NMR, and as a function of the solubility of each polymer in a range of solvents. One copolymer, polyBS-DS (Mn = 17 000, PD = 1.54) derived from 4-tert-butylstyrene (6, BS), 3,4-dimethoxystyrene (7, DS), and initiator 1 has a solubility profile [soluble in toluene, tetrahydrofuran (THF), ether, acetone and methylene chloride (DCM), insoluble in methanol and water] that is different from the present polymer of choice for LPOS, poly(ethylene) glycol (PEG), and has been studied in some detail as a new support in LPOS. The α-nitrile groups of polyBS-DS are reduced smoothly with LiAlH4 in THF to give the amino functionalized copolymer 22 (0.14 mmol g-1 of amino groups based on a quantitative ninhydrin analysis). Kinetic studies have revealed that derivatization of the amino groups of 22 with 4-dimethylaminocinnamaldehyde 23 occurs at a comparable rate to a solution counterpart (kpoly22 = 0.49 L mol-1 h-1 vs kaminohexane = 0.69 L mol-1 h-1). Following reaction of poly22 with N-glutaroyl-(2S,4S)-4-diphenylphosphino-2-[(diphenylphosphino)methyl]pyrrolidine (26) and exchange of Rh(I), the resulting phosphine containing copolymer Rh(I)-27, catalyzes the enantioselective hydrogenation of 2-N-acetamidoacrylic acid (28) to N-acetylalanine (29) in THF. An 87% enantiomeric excess (ee) of (S)-29 is obtained, comparable to that observed with a homogeneous phosphine ligand. This work highlights the power of a parallel polymer synthesis strategy, from conception to application, for the generation of polymers possessing unique solubility profiles and functionality which can serve as novel supports in LPOS.

Chemically-tagged mitsunobu reagents for use in solution-phase chemical library synthesis

A general method for high-throughput product purification of Mitsunobu reactions is described. Tagged phosphine and azodicarboxylate reagents are used to synthesize individual library members in solution-phase. Workup and purification are easily accomplished by post-reaction sequestration of the tagged reagents and reagent byproducts by a complementary functionalized ion exchange resin. The reagents are utilized in a 3 step library synthesis.