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PubMed2002

The application of multi-component reactions in drug discovery.

Multi-component reactions (MCRs) enable the facile, automated and high throughput generation of small organic molecules. MCRs have been used to create diversity oriented and biased combinatorial libraries, to accomplish the synthesis of highly complex natural products as well as for the large-scale production of drug candidates. This provides medicinal chemists with a powerful tool to create novel chemical diversity, matching the space of biological targets with relevant chemistry. The discovery of novel MCRs has become an increasingly active area of research, yielding novel chemical scaffolds for drug discovery efforts.

Animals↗
PubMed1999

Antigen sequence- and library-based mapping of linear and discontinuous protein-protein-interaction sites by spot synthesis.

The knowledge (antigen-derived peptide scans)- and library (de novo)-based mapping of linear and discontinuous antibody epitopes as well as protein-protein contact sites in general by spot synthesis now is a well established technique. Due to its automation, this technique also promises great potential for applications in functional genomics. It should help to elucidate the complex network of interacting protein molecules involved in signal transduction events (Adam-klages et al. 1996; Hoffmüller et al. 1999). Although little chemistry is involved in the preparation of peptide scans or libraries and the synthesis procedure is relatively simple, the laboratories of immunologists or molecular biologists are often not equipped to perform spot synthesis. In this case scans or libraries can be purchased from commercial suppliers.

Antigen-Antibody Reactions↗
PubMed1998

Molecular characterization of the mouse gene encoding cellular retinaldehyde-binding protein.

PURPOSE: To clone and characterize the mouse gene encoding cellular retinaldehyde-binding protein (CRALBP). CRALBP appears to modulate enzymatic generation and processing of 11-cis-retinol and regeneration of visual pigment in the vertebrate visual cycle. Mutations in human CRALBP segregate with autosomal recessive retinitis pigmentosa. METHODS: A genomic clone encompassing the 5' end of the CRALBP gene through exon 6 was isolated from a mouse 129/Sv genomic DNA library. Exons 7 and 8 were PCR amplified from mouse eye cDNA and 129/SvJ genomic DNA. The gene structure was determined by automated DNA sequence analysis. RESULTS: The sequence of 6855 nucleotides was determined, including all 8 exons, 3 introns plus 3932 and 629 bases from the 5'- and 3'-flanking regions, respectively. The lengths of introns 3-6 were determined by PCR amplification. Northern analysis identifies a approximately 2.1 kb transcript in mouse eye; Southern analysis supports a single copy gene. CONCLUSIONS: The mouse CRALBP gene is similar to the human gene; the coding sequence is approximately 87% identical, the non-coding sequence approximately 65% identical. In contrast to the human gene, the mouse gene contains a consensus TATA box. One of two photoreceptor consensus elements important for CRALBP expression in human retinal pigment epithelium is also present in the mouse gene. Additional conserved and species-specific consensus sequences are identified. The mouse CRALBP genomic clones and structure provide valuable tools for developing an in vivo model to study protein function and gene regulation.

Animals↗
PubMed2001

High-throughput generation and engineering of recombinant human antibodies.

The first version of the Human Combinatorial Antibody Library (HuCAL) is a single-chain Fv-based phage display library (HuCAL-scFv) with 2x10(9) members optimised for high-throughput generation and targeted engineering of human antibodies. 61% of the library genes code for functional scFv as judged by sequencing. We show here that since HuCAL-scFv antibodies are expressed in high levels in Escherichia coli, automated panning and screening in miniaturised settings (96- and 384-well format) have now become feasible. Additionally, the unique modular design of HuCAL-genes and -vectors allows the distinctly facilitated conversion of scFv into Fab, miniantibody and immunoglobulin formats, and the fusion with a variety of effector functions and tags not only convenient for therapeutic applications but also for high-throughput purification and detection. Thus, the HuCAL principle enables the rapid and high-throughput development of human antibodies by optimisation strategies proven useful in classical low molecular weight drug development. We demonstrate in this report that HuCAL is a very convenient source of human antibodies for various applications.

Animals↗
PubMed2003

Approaches towards the automated interpretation and prediction of electrospray tandem mass spectra of non-peptidic combinatorial compounds.

Combinatorial chemistry is widely used within the pharmaceutical industry as a means of rapid identification of potential drugs. With the growth of combinatorial libraries, mass spectrometry (MS) became the key analytical technique because of its speed of analysis, sensitivity, accuracy and ability to be coupled with other analytical techniques. In the majority of cases, electrospray mass spectrometry (ES-MS) has become the default ionisation technique. However, due to the absence of fragment ions in the resulting spectra, tandem mass spectrometry (MS/MS) is required to provide structural information for the identification of an unknown analyte. This work discusses the first steps of an investigation into the fragmentation pathways taking place in electrospray tandem mass spectrometry. The ultimate goal for this project is to set general fragmentation rules for non-peptidic, pharmaceutical, combinatorial compounds. As an aid, an artificial intelligence (AI) software package is used to facilitate interpretation of the spectra. This initial study has focused on determining the fragmentation rules for some classes of compound types that fit the remit as outlined above. Based on studies carried out on several combinatorial libraries of these compounds, it was established that different classes of drug molecules follow unique fragmentation pathways. In addition to these general observations, the specific ionisation processes and the fragmentation pathways involved in the electrospray mass spectra of these systems were explored. The ultimate goal will be to incorporate our findings into the computer program and allow identification of an unknown, non-peptidic compound following insertion of its ES-MS/MS spectrum into the AI package. The work herein demonstrates the potential benefit of such an approach in addressing the issue of high-throughput, automated MS/MS data interpretation.

Automation↗
PubMed1994

Solid-phase cloning to create sublibraries suitable for DNA sequencing.

A solid-phase method is described to create subclones, suitable for DNA sequencing, from lambda or cosmid libraries. The purified target DNA is sonicated and two linkers, with one oligonucleotide biotinylated in the 5'-end, are ligated to the ends of the fragments produced by sonication. After size separation, the fragments are immobilised onto a solid support and the non-biotinylated strand of each immobilised fragment is eluted. In this way, a library of single-stranded fragments is obtained. All fragments contain 'universal' flanking sequences of 22 bases introduced by the linker ligation. These flanking sequences can subsequently be used for solid-phase cloning into a single-stranded vector containing the complementary sequences. Thus, cloning can be achieved without the use of ligase or restriction enzymes. The resulting subclones are used for direct solid-phase sequencing and the immobilised strand can be used to selectively remove homologous DNA from the library of single-stranded fragments. Thus, a sublibrary of non-sequenced fragments can be created. Here, we show that a library of clones, suitable for direct solid-phase sequencing, can be obtained starting with lambda DNA. The efficiency of selective hybridisation of homologous and non-homologous fragments was investigated. The possibility of using this approach for automated cloning strategies for large-scale genomic and cDNA sequencing is discussed.

Base Sequence↗
PubMed2001

Folded-back solution structure of monomeric factor H of human complement by synchrotron X-ray and neutron scattering, analytical ultracentrifugation and constrained molecular modelling.

Factor H (FH) is a regulatory cofactor for the protease factor I in the breakdown of C3b in the complement system of immune defence, and binds to heparin and other polyanionic substrates. FH is composed of 20 short consensus/complement repeat (SCR) domains, for which the overall arrangement in solution is unknown. As previous studies had shown that FH can form monomeric or dimeric structures, X-ray and neutron scattering was accordingly performed with FH in the concentration range between 0.7 and 14 mg ml(-1). The radius of gyration of FH was determined to be 11.1-11.3 nm by both methods, and the radii of gyration of the cross-section were 4.4 nm and 1.7 nm. The distance distribution function P(r) showed that the overall length of FH was 38 nm. The neutron data showed that FH was monomeric with a molecular mass of 165,000(+/-17,000) Da. Analytical ultracentrifugation data confirmed this, where sedimentation equilibrium curve fits gave a mean molecular mass of 155,000(+/-3,000) Da. Sedimentation velocity experiments using the g*(s) derivative method showed that FH was monodisperse and had a sedimentation coefficient of 5.3(+/-0.1) S. In order to construct a full model of FH for scattering curve and sedimentation coefficient fits, homology models were constructed for 17 of the 20 SCR domains using knowledge of the NMR structures for FH SCR-5, SCR-15 and SCR-16, and vaccinia coat protein SCR-3 and SCR-4. Molecular dynamics simulations were used to generate a large conformational library for each of the 19 SCR-SCR linker peptides. Peptides from these libraries were combined with the 20 SCR structures in order to generate stereochemically complete models for the FH structure. Using an automated constrained fit procedure, the analysis of 16,752 possible FH models showed that only those models in which the 20 SCR domains were bent back upon themselves were able to account for the scattering and sedimentation data. The best-fit models showed that FH had an overall length of 38 nm and is flexible. This length is significantly less than a predicted length of 73 nm if the 20 SCR structures had been arranged in an extended arrangement. This outcome is attributed to several long linker sequences. These bent-back domain structures may correspond to conformational flexibility in FH and enable the multiple FH binding sites for C3 and heparin to come into close proximity.

Amino Acid Sequence↗
PubMed1994

The 5'-sequence of the murine Hox-b3 (Hox-2.7) gene and its intron contain multiple transcription-regulatory elements.

We sought to clone and characterize the murine Hox-b3 gene. In Xenopus embryos, the homologous gene has been shown to be responsive to retinoic acid, an agent which has profound effects on tissue growth and development. By plaque hybridization, using a partial, murine Hox-b3 cDNA as a probe, we screened a genomic library and isolated a series of overlapping clones. Restriction fragments from positive clones were sequenced by the dideoxy method on an automated DNA sequencer. We report the genomic sequence of the murine Hox-b3 gene. The sequence has been submitted to the GenBank database (accession number U02278). Our sequence extends from the P1 promoter through the coding sequence of the gene to the 3'-untranslated region. In common with other homeobox genes, there is an intron between the conserved hexapeptide and the homeobox. It is 866 bp long and has 3'- and 5'-splice sites very similar to the consensus, a long polypyrimidine tract and a potential branch point near the 3'-splice site. We have analyzed the sequence 5' to the initiation codon and the intron for putative control elements, and have identified a series of putative transcription factor binding sites in the P1 promoter and intron, including two for the retinoid X receptor-beta. Their possible significance is discussed. The sequences we have identified may be responsible for the observed pattern of expression of the gene. This sequence and the clones from which it is derived will enable a molecular dissection of the P1 promoter region.

Amino Acid Sequence↗
PubMed2002

The Criticism Management System for the Cochrane Library.

In April 1997, the Cochrane Collaboration implemented the Criticism Management System for The Cochrane Library, allowing readers to electronically submit criticisms on Cochrane reviews and protocols. As a result of resource constraints and user feedback, the Criticism Management System evolved through three different versions. Major problems have included difficult access to the system, a lack of automation within the system, a low number of submissions (171 criticisms submitted per 1,388 reviews), and a low response rate to these criticisms by authors of reviews (26 criticisms have received published responses). These low figures suggest that users of The Cochrane Library are not commenting on the published reviews or not using the Criticism Management System to make their criticisms known to the authors. An easier-to-use system and better ways of encouraging feedback from readers and responses from reviewers are needed in the future.

Databases, Bibliographic↗
PubMed2002

Automated main-chain model building by template matching and iterative fragment extension.

An algorithm for the automated macromolecular model building of polypeptide backbones is described. The procedure is hierarchical. In the initial stages, many overlapping polypeptide fragments are built. In subsequent stages, the fragments are extended and then connected. Identification of the locations of helical and beta-strand regions is carried out by FFT-based template matching. Fragment libraries of helices and beta-strands from refined protein structures are then positioned at the potential locations of helices and strands and the longest segments that fit the electron-density map are chosen. The helices and strands are then extended using fragment libraries consisting of sequences three amino acids long derived from refined protein structures. The resulting segments of polypeptide chain are then connected by choosing those which overlap at two or more C(alpha) positions. The fully automated procedure has been implemented in RESOLVE and is capable of model building at resolutions as low as 3.5 A. The algorithm is useful for building a preliminary main-chain model that can serve as a basis for refinement and side-chain addition.

Algorithms↗
PubMed1994

DNA sequencing: modular primers for automated walking.

Here we describe our progress in the development of the technology of DNA sequencing by primer walking based on the modular primer approach, which eliminates the primer synthesis bottleneck. Modular primers are assembled from 5-mers, 6-mers, or 7-mers selected from a presynthesized library of as few as 1000 oligonucleotides. This technology is expected to reduce the time per walk by a factor of 20 to 50, and the cost of DNA sequencing by a factor of 5 to 15. Both time and expense will be saved not only on the primer synthesis per se but, more importantly, as a result of the closed-end automation of the complete cycle of walking sequencing, where no human intervention will be required between the walks. Such closed-end automation has until now been precluded due to the need to synthesize a new primer for each walk. As a part of the closed cycle automation development, this report deals with modular primers used for sequencing with fluorescent dye terminators. The success rate and the quality of automated sequencing with modular primers are found similar to those with the conventional 17-20-base-long primers. One of the advantages of the described technique is simple purification without any proteins that need to be removed.

Base Sequence↗
PubMed1993

Backbone-dependent rotamer library for proteins. Application to side-chain prediction.

A backbone-dependent rotamer library for amino acid side-chains is developed and used for constructing protein side-chain conformations from the main-chain co-ordinates. The rotamer library is obtained from 132 protein chains in the Brookhaven Protein Database. A grid of 20 degrees by 20 degrees blocks for the main-chain angles phi, psi is used in the rotamer library. Significant correlations are found between side-chain dihedral angle probabilities and backbone phi, psi values. These probabilities are used to place the side-chains on the known backbone in test applications for six proteins for which high-resolution crystal structures are available. A minimization scheme is used to reorient side-chains that conflict with the backbone or other side-chains after the initial placement. The initial placement yields 59% of both chi 1 and chi 2 values in the correct position (to within 40 degrees) for thermolysin to 81% for crambin. After refinement the values range from 61% (lysozyme) to 89% (crambin). It is evident from the results that a single protein does not adequately test a prediction scheme. The computation time required by the method scales linearly with the number of side-chains. An initial prediction from the library takes only a few seconds of computer time, while the iterative refinement takes on the order of hours. The method is automated and can easily be applied to aid experimental side-chain determinations and homology modeling. The high degree of correlation between backbone and side-chain conformations may introduce a simplification in the protein folding process by reducing the available conformational space.

Amino Acid Sequence↗
PubMed1989

Sequencing of megabase plus DNA by hybridization: theory of the method.

A mismatch-free hybridization of oligonucleotides containing from 11 to 20 monomers to unknown DNA represents, in essence, a sequencing of a complementary target. Realizing this, we have used probability calculations and, in part, computer simulations to estimate the types and numbers of oligonucleotides that would have to be synthesized in order to sequence a megabase plus segment of DNA. We estimate that 95,000 specific mixes of 11-mers, mainly of the 5'(A,T,C,G)(A,T,C,G)N8(A,T,C,G)3' type, hybridized consecutively to dot blots of cloned genomic DNA fragments would provide primary data for the sequence assembly. An optimal mixture of representative libraries in M13 vector, having inserts of (i) 7 kb, (ii) 0.5 kb genomic fragments randomly ligated in up to 10-kb inserts, and (iii) tandem "jumping" fragments 100 kb apart in the genome, will be needed. To sequence each million base pairs of DNA, one would need hybridization data from about 2100 separate hybridization sample dots. Inevitable gaps and uncertainties in alignment of sequenced fragments arising from nonrandom or repetitive sequence organization of complex genomes and difficulties in cloning "poisonous" sequences in Escherichia coli, inherent to large sequencing by any method, have been considered and minimized by choice of libraries and number of subclones used for hybridization. Because it is based on simpler biochemical procedures, our method is inherently easier to automate than existing sequencing methods. The sequence can be derived from simple primary data only by extensive computing. Phased experimental tests and computer simulations increasing in complexity are needed before accurate estimates can be made in terms of cost and speed of sequencing by the new approach. Nevertheless, sequencing by hybridization should show advantages over existing methods because of the inherent redundancy and parallelism in its data gathering.

Algorithms↗
PubMed2000

Identification of pediococci by ribotyping.

Pediococci are among the most prevalent microbial contaminants in breweries and they can cause ropiness and the accumulation of high levels of diacetyl in beer. The accurate identification of pediococci is important, because different species do not possess equal spoilage potential. In this study, 18 Pediococcus strains, mainly of brewery origin, were first identified using phenotypical characterization (API 50 CHL and SDS-PAGE profiling), and then ribotyped using a RiboPrinterR System. Six Pediococcus type strains and three other Pediococcus strains were used as references. Ribotyping showed higher discriminative capacity than phenotypical identification methods. Strains could be identified to species level and in many cases, differentiated even at strain level using this genetic fingerprinting method. The identifications performed by ribotyping were confirmed by 16S rDNA sequencing of selected strains. Automated ribotyping was found to be a rapid and reliable method for identifying pediococci, but requires the construction of a comprehensive fingerprint library.

Bacterial Typing Techniques↗
PubMed2024

Reflective Evaluation of Next-Generation Sequencing Data during Early Phase Detection of the Delta Variant.

During the SARS-CoV-2 pandemic, next-generation sequencing (NGS) technologies like the Ion Torrent S5 and Illumina MiSeq, alongside advanced software, improved genomic surveillance in South Africa. This study analysed anonymized samples from the Eastern Cape using Genome Detective and NextClade, showing Ion Torrent S5 and Illumina MiSeq success rates of 96% and 94%, respectively. The study focused on genomic coverage (above 80%) and mutation detection (below 100), with the Ion Torrent S5 achieving 99% coverage compared to Illumina MiSeq's 80%, likely due to different primers used in amplification. The Ion Torrent S5 was more effective in sequencing varied viral loads, whereas Illumina MiSeq had difficulties with lower loads. Both platforms were adept at identifying clades, successfully differentiating between Beta (<45%) and Delta variants (<30%), despite minor discrepancies in assignments due to Illumina MiSeq's lower coverage, leading to a failure rate of up to 6%. Manual library preparation showed similar sample processing and clade identification capabilities for both platforms. However, differences in sequencing duration (3.5 vs. 36 hours), automation level, genomic coverage (80% vs. 99%), and viral load compatibility were noted, highlighting each platform's unique advantages and challenges in SARS-CoV-2 genomic surveillance. In conclusion, the Illumina MiSeq and Ion Torrent S5 platforms are both efficacious in executing whole-genome sequencing (WGS) via amplicons, facilitating precise, accurate, and high-throughput examinations of SARS-CoV-2 viral genomes. However, it is important to note the existence of disparities in the quality of data produced by each platform. Each system offers unique benefits and limitations, rendering them viable choices for the genomic surveillance of SARS-CoV-2.

Illumina MiSeq↗
PubMed1999

Application of in situ PCR to yeast cells for screening YAC libraries.

We have used in situ PCR technology in yeast cells with the ultimate goal of cloning and screening genomic yeast artificial chromosome (YAC) libraries. The target sequences in YAC clones were amplified "in situ" in yeast cells by the same set of microsatellite primers used in solution-based PCR screening. The method is fast and sensitive and obviates the steps required for individual isolation of DNAs from hundreds to thousands of YAC clones and thus has an advantage over conventional solution-based PCR screening. This approach can conceivably be applied to the products of automated robotic workstations.

Animals↗