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Biomedical subjects

Andrew D Griffiths

Publications and source records attributed to Andrew D Griffiths.

16 recordsLinked to original sources
PubMed2007

Droplets as microreactors for high-throughput biology.

Inspired by the principles of biological evolution, biologists--and others--have in recent decades harnessed the power of "natural" selection to sift through huge libraries of genes and find those with desirable properties. At the same time, the demand for high-throughput biochemical and genetic assays and screens has driven the development of increasingly miniaturised assay systems. An exciting synergy is now emerging between these two fields, whereby the tools of ultrahigh-throughput screening promise to open up new directions in molecular engineering.

Bacteria↗
PubMed2006

Miniaturising the laboratory in emulsion droplets.

Biochemical and genetic assays can be both miniaturized and parallelized by compartmentalization in living cells. In vitro compartmentalization (IVC) offers an alternative strategy based on partitioning reactions in water droplets dispersed to form microscopic compartments in water-in-oil emulsions. The cell-like volumes of these compartments (as low as one femtolitre), the ability to freely determine and regulate their content and the large number of compartments (>10(10) per millilitre emulsion) have provided the basis for a range of new, ultra-high-throughput, cell-free technologies. This review describes the scope and potential of IVC in areas such as in vitro evolution of proteins and RNAs, cell-free cloning and sequencing, genetics, genomics, and proteomics.

Cell-Free System↗
PubMed2006

Analogues with fluorescent leaving groups for screening and selection of enzymes that efficiently hydrolyze organophosphorus nerve agents.

Enzymes that efficiently hydrolyze highly toxic organophosphorus nerve agents could potentially be used as medical countermeasures. As sufficiently active enzymes are currently unknown, we synthesized twelve fluorogenic analogues of organophosphorus nerve agents with the 3-chloro-7-oxy-4-methylcoumarin leaving group as probes for high-throughput enzyme screening. This set included analogues of the pesticides paraoxon, parathion, and dimefox, and the nerve agents DFP, tabun, sarin, cyclosarin, soman, VX, and Russian-VX. Data from inhibition of acetylcholinesterase, in vivo toxicity tests of a representative analogue (cyclosarin), and kinetic studies with phosphotriesterase (PTE) from Pseudomonas diminuta, and a mammalian serum paraoxonase (PON1), confirmed that the analogues mimic the parent nerve agents effectively. They are suitable tools for high-throughput screens for the directed evolution of efficient nerve agent organophosphatases.

Acetylcholinesterase↗
PubMed2005

Selection of ribozymes that catalyse multiple-turnover Diels-Alder cycloadditions by using in vitro compartmentalization.

In vitro compartmentalization (IVC) has previously been used to evolve protein enzymes. Here, we demonstrate how IVC can be applied to select RNA enzymes (ribozymes) for a property that has previously been unselectable: true intermolecular catalysis. Libraries containing 10(11) ribozyme genes are compartmentalized in the aqueous droplets of a water-in-oil emulsion, such that most droplets contain no more than one gene, and transcribed in situ. By coencapsulating the gene, RNA, and the substrates/products of the catalyzed reaction, ribozymes can be selected for all enzymatic properties: substrate recognition, product formation, rate acceleration, and turnover. Here we exploit the complementarity of IVC with systematic evolution of ligands by exponential enrichment (SELEX), which allows selection of larger libraries (>/=10(15)) and for very small rate accelerations (k(cat)/k(uncat)) but only selects for intramolecular single-turnover reactions. We selected approximately 10(14) random RNAs for Diels-Alderase activity with five rounds of SELEX, then six to nine rounds with IVC. All selected ribozymes catalyzed the Diels-Alder reaction in a truly bimolecular fashion and with multiple turnover. Nearly all ribozymes selected by using eleven rounds of SELEX alone contain a common catalytic motif. Selecting with SELEX then IVC gave ribozymes with significant sequence variations in this catalytic motif and ribozymes with completely novel motifs. Interestingly, the catalytic properties of all of the selected ribozymes were quite similar. The ribozymes are strongly product inhibited, consistent with the Diels-Alder transition state closely resembling the product. More efficient Diels-Alderases may need to catalyze a second reaction that transforms the product and prevents product inhibition.

Alkenes↗
PubMed2005

High-throughput screens and selections of enzyme-encoding genes.

The availability of vast gene repertoires from both natural sources (genomic and cDNA libraries) and artificial sources (gene libraries) demands the development and application of novel technologies that enable the screening or selection of large libraries for a variety of enzymatic activities. We describe recent developments in the selection of enzyme-coding genes for directed evolution and functional genomics. We focus on HTS approaches that enable selection from large libraries (>10(6) gene variants) with relatively humble means (i.e. non-robotic systems), and on in vitro compartmentalization in particular.

Animals↗
PubMed2005

High-throughput screening of enzyme libraries: in vitro evolution of a beta-galactosidase by fluorescence-activated sorting of double emulsions.

We describe a completely in vitro high-throughput screening system for directed evolution of enzymes based on in vitro compartmentalization (IVC). Single genes are transcribed and translated inside the aqueous droplets of a water-in-oil emulsion. Enzyme activity generates a fluorescent product and, after conversion into a water-in-oil-in-water double emulsion, fluorescent droplets are sorted using a fluorescence-activated cell sorter (FACS). Earlier in vivo studies have demonstrated that Ebg, a protein of unknown function, can evolve to allow Escherichia coli lacking the lacZ beta-galactosidase gene to grow on lactose. Here we demonstrate that we can evolve Ebg into an enzyme with significant beta-galactosidase activity in vitro. Only two specific mutations were ever seen to provide this improvement in Ebg beta-galactosidase activity in vivo. In contrast, nearly all the improved beta-galactosidases selected in vitro resulted from different mutations.

Directed Molecular Evolution↗
PubMed2005

New genotype-phenotype linkages for directed evolution of functional proteins.

The key practical consideration in directed evolution of functional biomolecules is the linkage of genotype and phenotype. In vitro selections offer the potential to select from libraries with up to 10(10)-10(14) members, with fewer constraints than current cell-based selections. New approaches such as mRNA display, ribosome display and in vitro compartmentalisation have complementary areas of application in selections for binding or catalysis.

Directed Molecular Evolution↗
PubMed2004

Determinants of cofactor binding to DNA methyltransferases: insights from a systematic series of structural variants of S-adenosylhomocysteine.

S-Adenosylmethionine (AdoMet) is a commonly used cofactor, second only to ATP in the variety of reactions in which it participates. It is the methyl donor in the majority of methyl transfer reactions, including methylation of DNA, RNA, proteins and small molecules. Almost all structurally characterised methyltransferases share a conserved AdoMet-dependent methyltransferase fold, in which AdoMet is bound in the same orientation. Although potential interactions between the cofactor and methyltransferases have been inferred from crystal structures, there has not been a systematic study of the contributions of each functional group to binding. To explore the binding interaction we synthesised a series of seven analogues of the methyltransferase inhibitor S-adenosylhomocysteine (AdoHcy), each containing a single modification, and tested them for the ability to inhibit methylation by HhaI and HaeIII DNA methyltransferase. Comparison of the Ki values highlights the structural determinants for cofactor binding, and indicates which nucleoside and amino acid functional groups contribute significantly to AdoMet binding. An understanding of the binding of AdoHyc to methyltransferases will greatly assist the design of AdoMet inhibitors.

Amino Acids↗
PubMed2004

In vitro compartmentalization by double emulsions: sorting and gene enrichment by fluorescence activated cell sorting.

Water-in-oil (w/o) emulsions can be used to compartmentalize and select large gene libraries for a predetermined function. The aqueous droplets of the w/o emulsion function as cell-like compartments in each of which a single gene is transcribed and translated to give multiple copies of the protein (e.g., an enzyme) it encodes. While compartmentalization ensures that the gene, the protein it encodes, and the products of the activity of this protein remain linked, it does not directly afford a way of selecting for the desired activity. Here we show that re-emulsification of w/o emulsions gives water-in-oil-in-water (w/o/w) emulsions with an external (continuous) water phase through which droplets containing fluorescent markers can be isolated by fluorescence-activated cell sorting (FACS). These w/o/w emulsions can be sorted by FACS, while the content of the aqueous droplets of the primary w/o emulsion remains intact. Consequently, genes embedded in these water droplets together with a fluorescent marker can be isolated and enriched from an excess of genes embedded in water droplets without a fluorescent marker. The ability of FACS instruments to sort up to 40000 events per second may endow this technology a wide potential in the area of high-throughput screening and the directed evolution of enzymes.

DNA Modification Methylases↗
PubMed2004

Altering the sequence specificity of HaeIII methyltransferase by directed evolution using in vitro compartmentalization.

Engineering the specificity of DNA-modifying enzymes has proven extremely challenging, as sequence recognition by these enzymes is poorly understood. Here we used directed evolution to generate a variant of HaeIII methyltransferase that efficiently methylates a novel target site. M.HaeIII methylates the internal cytosine of the canonical sequence GGCC, but there is promiscuous methylation of a variety of non-canonical sites, notably AGCC, at a reduced rate. Using in vitro compartmentalization (IVC), libraries of M.HaeIII genes were selected for the ability to efficiently methylate AGCC. A two-step mutagenesis strategy, involving initial randomization of DNA-contacting residues followed by randomization of the loop that lies behind these residues, yielded a mutant with a 670-fold improvement in catalytic efficiency (k(cat)/K(m)(DNA)) using AGCC and a preference for AGCC over GGCC. The mutant methylates three sites efficiently (AGCC, CGCC and GGCC). Indeed, it methylates CGCC slightly more efficiently than AGCC. However, the mutant discriminates against other non-canonical sites, including TGCC, as effectively as the wild-type enzyme. This study provides a rare example of a laboratory-evolved enzyme whose catalytic efficiency surpasses that of the wild-type enzyme with the principal substrate.

Amino Acid Sequence↗
PubMed2003

Directed evolution of an extremely fast phosphotriesterase by in vitro compartmentalization.

We describe the selection of a phosphotriesterase with a very fast k(cat) (over 10(5) s(-1)), 63 times higher than the already very efficient wild-type enzyme. The enzyme was selected from a library of 3.4 x 10(7) mutated phosphotriesterase genes using a novel strategy based on linking genotype and phenotype by in vitro compartmentalization (IVC) using water-in-oil emulsions. First, microbeads, each displaying a single gene and multiple copies of the encoded protein, are formed by compartmentalized in vitro translation. These microbeads can then be selected for catalysis or binding. To select for catalysis the microbeads are re-emulsified in a reaction buffer of choice with a soluble substrate. The product and any unreacted substrate are coupled to the beads when the reaction is finished. Product-coated beads, displaying active enzymes and the genes that encode them, are detected with anti-product antibodies and selected using flow cytometry. This completely in vitro process selects for all enzymatic features simultaneously (substrate recognition, product formation, rate acceleration and turnover) and single enzyme molecules can be detected.

Amino Acid Sequence↗
PubMed2002

Microbead display by in vitro compartmentalisation: selection for binding using flow cytometry.

In vitro compartmentalisation in an emulsion was used to physically link proteins to the DNA that encodes them via microbeads. These microbeads can be selected for catalysis, or, as demonstrated here, for binding. Genes encoding a peptide containing an epitope (haemagglutinin) were enriched to near purity from a 10(6)-fold excess of genes encoding a different peptide by two rounds of selection using flow cytometry, indicating approximately 1000-fold enrichment per round. Single beads can be isolated using flow sorting and the single gene on the bead amplified by polymerase chain reaction. Hence, the entire process can be performed completely in vitro.

Amino Acid Sequence↗
PubMed2002

Investigating the target recognition of DNA cytosine-5 methyltransferase HhaI by library selection using in vitro compartmentalisation.

In vitro compartmentalisation (IVC), a technique for selecting genes encoding enzymes based on compartmentalising gene translation and enzymatic reactions in emulsions, was used to investigate the interaction of the DNA cytosine-5 methyltransferase M.HhaI with its target DNA (5'-GCGC-3'). Crystallography shows that the active site loop from the large domain of M.HhaI interacts with a flipped-out cytosine (the target for methylation) and two target recognition loops (loops I and II) from the small domain make almost all the other base-specific interactions. A library of M.HhaI genes was created by randomising all the loop II residues thought to make base-specific interactions and directly determine target specificity. The library was selected for 5'-GCGC-3' methylation. Interestingly, in 11 selected active clones, 10 different sequences were found and none were wild-type. At two of the positions mutated (Ser252 and Tyr254) a number of different amino acids could be tolerated. At the third position, however, all active mutants had a glycine, as in wild-type M.HhaI, suggesting that Gly257 is crucial for DNA recognition and enzyme activity. Our results suggest that recognition of base pairs 3 and 4 of the target site either relies entirely on main chain interactions or that different residues from those identified in the crystal structure contribute to DNA recognition.

Amino Acids↗
PubMed2002

Promiscuous methylation of non-canonical DNA sites by HaeIII methyltransferase.

The cytosine C5 methyltransferase M.HaeIII recognises and methylates the central cytosine of its canonical site GGCC. Here we report that M.HaeIII can also, with lower efficiency, methylate cytosines located in a wide range of non-canonical sequences. Using bisulphite sequencing we mapped the methyl- cytosine residues in DNA methylated in vitro and in vivo by M.HaeIII. Methyl-cytosine residues were observed in multiple sequence contexts, most commonly, but not exclusively, at star sites (sites differing by a single base from the canonical sequence). The most frequently used star sites had changes at positions 1 and 4, but there is little or no methylation at star sites changed at position 2. The rate of methylation of non-canonical sites can be quite significant: a DNA substrate lacking a canonical site was methylated by M.HaeIII in vitro at a rate only an order of magnitude slower than an otherwise identical substrate containing the canonical site. In vivo methylation of non-canonical sites may therefore be significant and may have provided the starting point for the evolution of restriction-modification systems with novel sequence specificities.

Base Sequence↗