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Biomedical subjects

A D Ellington

Publications and source records attributed to A D Ellington.

At least 19 recordsLinked to original sources
PubMed2001

A combined in vitro/in vivo selection for polymerases with novel promoter specificities.

BACKGROUND: The DNA-dependent RNA polymerase from T7 bacteriophage (T7 RNAP) has been extensively characterized, and like other phage RNA polymerases it is highly specific for its promoter. A combined in vitro/in vivo selection method has been developed for the evolution of T7 RNA polymerases with altered promoter specificities. Large (10(3)-10(6)) polymerase libraries were made and cloned downstream of variant promoters. Those polymerase variants that can recognize variant promoters self-amplify both themselves and their attendant mRNAs in vivo. Following RT / PCR amplification in vitro, the most numerous polymerase genes are preferentially cloned and carried into subsequent rounds of selection. RESULTS AND CONCLUSIONS: A T7 RNA polymerase library that was randomized at three positions was cloned adjacent to a T3-like promoter sequence, and a 'specialist' T7 RNA polymerase was identified. A library that was randomized at a different set of positions was cloned adjacent to a promoter library in which four positions had been randomized, and 'generalist' polymerases that could utilize a variety of T7 promoters were identified, including at least one polymerase with an apparently novel promoter specificity. This method may have applications for evolving other polymerase variants with novel phenotypes, such as the ability to incorporate modified nucleotides.

Amino Acid Substitution↗
PubMed2001

In vitro evolution of beta-glucuronidase into a beta-galactosidase proceeds through non-specific intermediates.

The Escherichia coli beta-glucuronidase (GUS) was evolved in vitro to catalyze the hydrolysis of a beta-galactoside substrate 500 times more efficiently (k(cat)/K(m)) than the wild-type, with a 52 million-fold inversion in specificity. The amino acid substitutions that recurred among 32 clones isolated in three rounds of DNA shuffling and screening were mapped to the active site. The functional consequences of these mutations were investigated by introducing them individually or in combination into otherwise wild-type gusA genes. The kinetic behavior of the purified mutant proteins in reactions with a series of substrate analogues show that four mutations account for the changes in substrate specificity, and that they are synergistic. An evolutionary intermediate, unlike the wild-type and evolved forms, exhibits broadened specificity for substrates dissimilar to either glucuronides or galactosides. These results are consistent with the "patchwork" hypothesis, which postulates that modern enzymes diverged from ancestors with broad specificity.

Directed Molecular Evolution↗
PubMed2001

Automated selection of anti-protein aptamers.

The in vitro selection of nucleic acid binding species (aptamers) is frequently repetitive, time-consuming, and poorly adapted to high-throughput applications. We have adapted automated workstations to select anti-protein aptamers; as an example, we demonstrated the selection of anti-lysozyme aptamers that function as efficient inhibitors of cell lysis. The increases in throughput brought about by automation should potentiate the application of aptamer technology to the rapidly growing field of proteomics.

Animals↗
PubMed2001

Selection of deoxyribozyme ligases that catalyze the formation of an unnatural internucleotide linkage.

The chemical ligation of DNA molecules can be mediated by terminal phosphorothioate displacement of a 5' iodine. We have selected deoxyribozymes that can catalyze the formation of such phosphorothioester internucleotide linkages. The selected deoxyribozymes enhance the rate of ligation in part through the provision of a template that aligns the ligation junction and do not appear to require metal ions for catalysis.

Base Sequence↗
PubMed2001

Elucidation of the initial step of oligonucleotide fragmentation in matrix-assisted laser desorption/ionization using modified nucleic acids.

To probe the mechanism of gas-phase oligonucleotide ion fragmentation, modified oligonucleotides were studied using matrix-assisted laser desorption/ionization. The oligonucleotides were of the form 5'-TTTTXTTTTT, where X was a modified nucleotide. Modifications included substitution of hydroxy, methoxy, amino, and allyl groups at the 2'-position of the deoxyribose. The modified ribose contained adenine, guanine, cytosine, or uracil bases. For comparison, we studied oligomers where X was an unmodified adenosine, guanosine, cytidine, thymidine, or uridine deoxyribonucleotide. We found a very strong dependence of the matrix-to-analyte ratio on fragmentation for these oligomers. Analysis of these modifications suggests that the initial fragmentation step in MALDI-MS involves a two-step (E1) elimination of the base.

Allyl Compounds↗
PubMed2001

Optimization and optimality of a short ribozyme ligase that joins non-Watson-Crick base pairings.

A small ribozyme ligase (L1) selected from a random sequence population appears to utilize non-Watson-Crick base pairs at its ligation junction. Mutational and selection analyses confirmed the presence of these base pairings. Randomization of the L1 core and selection of active ligases yielded highly active variants whose rates were on the order of 1 min(-1). Base-pairing covariations confirmed the general secondary structure of the ligase, and the most active ligases contained a novel pentuple sequence covariation. The optimized L1 ligases may be optimal within their sequence spaces, and minimal ligases that span less than 60 nt in length have been engineered based on these results.

Base Pairing↗
PubMed2001

In vitro selection of nucleoprotein enzymes.

Natural nucleic acids frequently rely on proteins for stabilization or catalytic activity. In contrast, nucleic acids selected in vitro can catalyze a wide range of reactions even in the absence of proteins. To augment selected nucleic acids with protein functionalities, we have developed a technique for the selection of protein-dependent ribozyme ligases. After randomizing a previously selected ribozyme ligase, L1, we selected variants that required one of two protein cofactors, a tyrosyl transfer RNA (tRNA) synthetase (Cyt18) or hen egg white lysozyme. The resulting nucleoprotein enzymes were activated several thousand fold by their cognate protein effectors, and could specifically recognize the structures of the native proteins. Protein-dependent ribozymes can potentially be adapted to novel assays for detecting target proteins, and the selection method's generality may allow the high-throughput identification of ribozymes capable of recognizing a sizable fraction of a proteome.

Base Sequence↗
PubMed2001

Selection and characterization of Escherichia coli variants capable of growth on an otherwise toxic tryptophan analogue.

Escherichia coli isolates that were tolerant of incorporation of high proportions of 4-fluorotryptophan were evolved by serial growth. The resultant strain still preferred tryptophan for growth but showed improved growth relative to the parental strain on other tryptophan analogues. Evolved clones fully substituted fluorotryptophan for tryptophan in their proteomes within the limits of mass spectral and amino acid analyses. Of the genes sequenced, many genes were found to be unaltered in the evolved strain; however, three genes encoding enzymes involved in tryptophan uptake and utilization were altered: the aromatic amino acid permease (aroP) and tryptophanyl-tRNA synthetase (trpS) contained several amino acid substitutions, and the tyrosine repressor (tyrR) had a nonsense mutation. While kinetic analysis of the tryptophanyl-tRNA synthetase suggests discrimination against 4-fluorotryptophan, an analysis of the incorporation and growth patterns of the evolved bacteria suggest that other mutations also aid in the adaptation to the tryptophan analogue. These results suggest that the incorporation of unnatural amino acids into organismal proteomes may be possible but that extensive evolution may be required to reoptimize proteins and metabolism to accommodate such analogues.

Amino Acid Transport Systems↗
PubMed2001

Toward automated nucleic acid enzyme selection.

Methods for automation of nucleic acid selections are being developed. The selection of aptamers has been successfully automated using a Biomek 2000 workstation. Several binding species with nanomolar affinities were isolated from diverse populations. Automation of a deoxyribozyme ligase selection is in progress. The process requires eleven times more robotic manipulations than an aptamer selection. The random sequence pool contained a 5' iodine residue and the ligation substrate contained a 3' phosphorothioate. Initially, a manual deoxyribozyme ligase selection was performed. Thirteen rounds of selection yielded ligators with a 400-fold increase in activity over the initial pool. Several difficulties were encountered during the automation of DNA catalyst selection, including effectively washing bead-bound DNA, pipetting 50% glycerol solutions, purifying single strand DNA, and monitoring the progress of the selection as it is performed. Nonetheless, automated selection experiments for deoxyribozyme ligases were carried out starting from either a naive pool or round eight of the manually selected pool. In both instances, the first round of selection revealed an increase in ligase activity. However, this activity was lost in subsequent rounds. A possible cause could be mispriming during the unmonitored PCR reactions. Potential solutions include pool redesign, fewer PCR cycles, and integration of a fluorescence microtiter plate reader to allow robotic 'observation' of the selections as they progress.

Automation↗
PubMed2000

Design and optimization of effector-activated ribozyme ligases.

A selected ribozyme ligase, L1, has been engineered to respond to small organic effectors. Residues important for ribozyme catalysis were mapped to a compact core structure. Aptamers that bound adenosine and theophylline were appended to the core structure, and the resultant aptazymes proved to be responsive to their cognate effectors. Rational sequence substitutions in the joining region between the aptamer and the ribozyme yielded aptazymes whose activities were enhanced from 800-1600-fold in the presence of 1 mM ATP or theophylline, respectively. However, when an anti-flavin aptamer was appended to the core ribozyme structure flavin-responsivity was minimal. The joining region between the aptamer and the ribozyme core was randomized and a series of negative and positive selection steps yielded aptazymes that were activated by up to 260-fold in the presence of 100 microM FMN. The selected joining regions proved to be 'communication modules' that could be used to join other aptamers to the ribozyme core to form aptazymes. These results show that ribozyme ligases can be readily engineered to function as allosteric enzymes, and reveal that many of the techniques and principles previously demonstrated during the development of hammerhead aptazymes may be generalizable.

Base Sequence↗
PubMed2000

In vitro selection of RNA molecules that inhibit the activity of ricin A-chain.

The cytotoxin ricin disables translation by depurinating a conserved site in eukaryotic rRNA. In vitro selection has been used to generate RNA ligands (aptamers) specific for the catalytic ricin A-chain (RTA). The anti-RTA aptamers bear no resemblance to the normal RTA substrate, the sarcin-ricin loop (SRL), and were not depurinated by RTA. An initial 80-nucleotide RNA ligand was minimized to a 31-nucleotide aptamer that contained all sequences and structures necessary for interacting with RTA. This minimal RNA formed high affinity complexes with RTA (K(d) = 7.3 nM) which could compete directly with the SRL for binding to RTA. The aptamer inhibited RTA depurination of the SRL and could partially protect translation from RTA inhibition. The IC(50) of the aptamer for RTA in an in vitro translation assay is 100 nM, roughly 3 orders of magnitude lower than a small molecule inhibitor of ricin, pteroic acid, and 2 orders of magnitude lower than the best known RNA inhibitor. The novel anti-RTA aptamers may find application as diagnostic reagents for a potential biological warfare agent and hold promise as scaffolds for the development of strong ricin inhibitors.

Base Sequence↗
PubMed2000

RNA aptamers that bind to and inhibit the ribosome-inactivating protein, pepocin.

Pepocin, isolated from Cucurbita pepo, is a ribosome-inactivating protein (RIP). RIPs site-specifically recognize and depurinate an adenosine at position 4324 in rat 28 S rRNA, rendering the ribosome incapable of interacting with essential elongation factors. Aptamers that target pepocin were isolated from a degenerate RNA pool by in vitro selection. A conserved hairpin motif, quite different from the sequence of the toxin-substrate domain in rat 28 S rRNA, was identified in the aptamer sequences. The aptamers selectively bind to pepocin with dissociation constants between 20 and 30 nM and inhibit the N-glycosidase activity of pepocin on rat liver 28 S rRNA. Competitive binding experiments using aptamer variants suggest that the conserved hairpin region in the anti-pepocin aptamer binds near the catalytic site on pepocin and prevents the interaction of pepocin and 28 S rRNA. Anti-RIP aptamers have potential use in diagnostic systems for the detection of pepocin or could be used as therapy to prevent the action of pepocin in mammalian cells.

Animals↗