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Search Results: 1 - 10 of 206108 matches for " Andrew D Ellington "
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Man versus Machine versus Ribozyme
Andrew D. Ellington
PLOS Biology , 2012, DOI: 10.1371/journal.pbio.0060132
Man versus Machine versus Ribozyme
Andrew D Ellington
PLOS Biology , 2008, DOI: 10.1371/journal.pbio.0060132
Design Principles for Ligand-Sensing, Conformation-Switching Ribozymes
Xi Chen,Andrew D. Ellington
PLOS Computational Biology , 2009, DOI: 10.1371/journal.pcbi.1000620
Abstract: Nucleic acid sensor elements are proving increasingly useful in biotechnology and biomedical applications. A number of ligand-sensing, conformational-switching ribozymes (also known as allosteric ribozymes or aptazymes) have been generated by some combination of directed evolution or rational design. Such sensor elements typically fuse a molecular recognition domain (aptamer) with a catalytic signal generator (ribozyme). Although the rational design of aptazymes has begun to be explored, the relationships between the thermodynamics of aptazyme conformational changes and aptazyme performance in vitro and in vivo have not been examined in a quantitative framework. We have therefore developed a quantitative and predictive model for aptazymes as biosensors in vitro and as riboswitches in vivo. In the process, we have identified key relationships (or dimensionless parameters) that dictate aptazyme performance, and in consequence, established equations for precisely engineering aptazyme function. In particular, our analysis quantifies the intrinsic trade-off between ligand sensitivity and the dynamic range of activity. We were also able to determine how in vivo parameters, such as mRNA degradation rates, impact the design and function of aptazymes when used as riboswitches. Using this theoretical framework we were able to achieve quantitative agreement between our models and published data. In consequence, we are able to suggest experimental guidelines for quantitatively predicting the performance of aptazyme-based riboswitches. By identifying factors that limit the performance of previously published systems we were able to generate immediately testable hypotheses for their improvement. The robust theoretical framework and identified optimization parameters should now enable the precision design of aptazymes for biotechnological and clinical applications.
A combined in vitro / in vivo selection for polymerases with novel promoter specificities
Jijumon Chelliserrykattil, George Cai, Andrew D Ellington
BMC Biotechnology , 2001, DOI: 10.1186/1472-6750-1-13
Abstract: 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.The RNA polymerase from bacteriophage T7 has a relatively narrow specificity for a particular promoter sequence, making it an extremely useful tool for molecular biology and biotechnology applications. Most recently, the crystal structure of the polymerase in complex with a DNA promoter has revealed the structural basis for this specificity [1,2]. In addition, a number of researchers have examined the contribution of various nucleotides and functional groups in the promoter and various amino acids in the polymerase to specificity [3-5]. Based upon both structural and mutagenic analyses, it has proven possible to identify polymerase variants with altered specificities for promoters [6,7]. Polymerase variants with altered specificities have also been identified using genetic selections [8]. However, the variant polymerases and variant promoters that have so far been identified are close in sequence to the wild-type. For example, the only known polymerase variant that switches promoters contains a single amino acid change, recognizes a single nucleotide change in the promoter, and closely mimics interactions known to occur for T3 RNA polymerase [9]. The large sequence space that surrounds both polymerases and promoters has so far prevented more sweeping searches for more diverse variants. To address this problem, we have developed a com
Anticipatory evolution and DNA shuffling
Jamie M Bacher, Brian D Reiss, Andrew D Ellington
Genome Biology , 2002, DOI: 10.1186/gb-2002-3-8-reviews1021
Abstract: Proteins are machines created by evolution, but it is unclear just how finely evolution has guided their sequence, structure, and function. It is undoubtedly true that individual mutations in a protein affect both its structure and its function and that such mutations can be fixed during evolutionary history, but it is also true that there are other elements of protein sequence that have been acted upon by evolution. For example, the genetic code appears to be laid out so that mutations and errors in translation are minimally damaging to protein structure and function [1]. Could the probability that a beneficial mutation is found and fixed in the population also have been manipulated during the course of evolution, so that the proteins we see today are more capable of change than the proteins that may have been cobbled together following the 'invention' of translation? Have proteins, in fact, evolved to evolve? There is already some evidence that bacteria are equipped to evolve phenotypes that are more capable of further adaptation (reviewed in [2,3,4]). For example, mutator [5] and hyper-recombinogenic [6] strains arise as a result of selection experiments. The development of DNA shuffling (reviewed in [7,8]) and the appearance of several recent papers using this technique [9,10,11] provide us with a surprising new opportunity to ask and answer these fundamental questions at the level of individual genes, and perhaps even genomes.DNA shuffling, a method for in vitro recombination, was developed as a technique to generate mutant genes that would encode proteins with improved or unique functionality [12,13]. It consists of a three-step process that begins with the enzymatic digestion of genes, yielding smaller fragments of DNA. The small fragments are then allowed to randomly hybridize and are filled in to create longer fragments. Ultimately, any full-length, recombined genes that are recreated are amplified via the polymerase chain reaction. If a series of alleles o
Inhibition of Cell Proliferation by an Anti-EGFR Aptamer
Na Li,Hong Hanh Nguyen,Michelle Byrom,Andrew D. Ellington
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0020299
Abstract: Aptamers continue to receive interest as potential therapeutic agents for the treatment of diseases, including cancer. In order to determine whether aptamers might eventually prove to be as useful as other clinical biopolymers, such as antibodies, we selected aptamers against an important clinical target, human epidermal growth factor receptor (hEGFR). The initial selection yielded only a single clone that could bind to hEGFR, but further mutation and optimization yielded a family of tight-binding aptamers. One of the selected aptamers, E07, bound tightly to the wild-type receptor (Kd = 2.4 nM). This aptamer can compete with EGF for binding, binds to a novel epitope on EGFR, and also binds a deletion mutant, EGFRvIII, that is commonly found in breast and lung cancers, and especially in grade IV glioblastoma multiforme, a cancer which has for the most part proved unresponsive to current therapies. The aptamer binds to cells expressing EGFR, blocks receptor autophosphorylation, and prevents proliferation of tumor cells in three-dimensional matrices. In short, the aptamer is a promising candidate for further development as an anti-tumor therapeutic. In addition, Aptamer E07 is readily internalized into EGFR-expressing cells, raising the possibility that it might be used to escort other anti-tumor or contrast agents.
Spatial Control of DNA Reaction Networks by DNA Sequence
Peter B. Allen,Xi Chen,Andrew D. Ellington
Molecules , 2012, DOI: 10.3390/molecules171113390
Abstract: We have developed a set of DNA circuits that execute during gel electrophoresis to yield immobile, fluorescent features in the gel. The parallel execution of orthogonal circuits led to the simultaneous production of different fluorescent lines at different positions in the gel. The positions of the lines could be rationally manipulated by changing the mobilities of the reactants. The ability to program at the nanoscale so as to produce patterns at the macroscale is a step towards programmable, synthetic chemical systems for generating defined spatiotemporal patterns.
Coupling Two Different Nucleic Acid Circuits in an Enzyme-Free Amplifier
Yu Jiang,Bingling Li,Xi Chen,Andrew D. Ellington
Molecules , 2012, DOI: 10.3390/molecules171113211
Abstract: DNA circuits have proven to be useful amplifiers for diagnostic applications, in part because of their modularity and programmability. In order to determine whether different circuits could be modularly stacked, we used a catalytic hairpin assembly (CHA) circuit to initiate a hybridization chain reaction (HCR) circuit. In response to an input nucleic acid sequence, the CHA reaction accumulates immobilized duplexes and HCR elongates these duplexes. With fluorescein as a reporter each of these processes yielded 10-fold signal amplification in a convenient 96-well format. The modular circuit connections also allowed the output reporter to be readily modified to a G-quadruplex-DNAzyme that yielded a fluorescent signal.
Evolution of phage with chemically ambiguous proteomes
Jamie M Bacher, James J Bull, Andrew D Ellington
BMC Evolutionary Biology , 2003, DOI: 10.1186/1471-2148-3-24
Abstract: The bacteriophage Qβ initially grows poorly in the presence of the amino acid analogue 6-fluorotryptophan. After 25 serial passages, the fitness of the phage on the analogue was substantially increased; there was no loss of fitness when the evolved phage were passaged in the presence of tryptophan. Seven mutations were fixed throughout the phage in two independent lines of descent. None of the mutations changed a tryptophan residue.A relatively small number of mutations allowed an unnatural amino acid to be functionally incorporated into a highly interdependent set of proteins. These results support the 'ambiguous intermediate' hypothesis for the emergence of divergent genetic codes, in which the adoption of a new genetic code is preceded by the evolution of proteins that can simultaneously accommodate more than one amino acid at a given codon. It may now be possible to direct the evolution of organisms with novel genetic codes using methods that promote ambiguous intermediates.Organismal proteomes are generally thought of as being chemically distinct, in the sense that a genetic code is maintained by codon:anticodon interactions and the specificities of aminoacyl-tRNA synthetases will almost always lead to the translation of mRNAs into proteins of defined sequence and chemical composition. While alternative codes are known [1], these also yield chemically distinct proteomes. The evolution of an organism with novel codon:anticodon interactions and aminoacyl-tRNA synthetase specificities may produce proteins whose sequences and compositions differ from those generated by an organism with the 'Universal' code, but still will not produce proteins that have multiple, different amino acids at a given sequence position.This chemical distinctness of organismal proteomes is maintained by the relatively low rate of amino acid misincorporation that occurs during protein biosynthesis. Many aminoacyl-tRNA synthetases have been found to have at least a thousand-fold preference f
Beyond allostery: Catalytic regulation of a deoxyribozyme through an entropy-driven DNA amplifier
Grace Eckhoff, Vlad Codrea, Andrew D Ellington, Xi Chen
Journal of Systems Chemistry , 2010, DOI: 10.1186/1759-2208-1-13
Abstract: A variety of functional nucleic acids have been engineered over the past two decades, including not only simple binding elements (aptamers [1,2]) and catalysts (ribozymes [3] and deoxyribozymes [4]), but also more 'intelligent' molecular parts, such as aptamer beacons and allosteric ribozymes that can sense biomolecules [5,6], process molecular information [7,8], and regulate biochemical systems [9]. However, most regulatory nucleic acid elements are based on allosteric control, which has a fundamental limitation: one input molecule generally yields only one output molecule. Such stoichiometric or sub-stoichiometric regulation is often insufficient for effective metabolic regulation or diagnostic signal transduction, especially when the concentrations of input molecules are low.In contrast, natural catalytic cascades, such as the phosphorylation of proteins by kinases, readily amplify low input signals. Although in principle ribozymes and deoxyribozymes could participate in similar cascades as catalysts [10-12], no generalizable method for implementing such cascades has yet been established. On the other hand, DNA and RNA can catalyze chemical reactions not only by forming intricate tertiary structures, but also by simply forming Watson-Crick base pairs. In fact, by serving as a hybridization template, DNA can control and catalyze a wide range of chemical reactions [13], some of which can yield products capable of regulating downstream reactions. More recently, Zhang and coworkers have designed a scheme for highly efficient, enzyme-free, entropy-driven catalytic reactions that relies only on the dynamic hybridization of DNA strands [14-17]. Because of its chemical simplicity, this scheme is expected to allow the development of enzyme-free DNA circuits substantially more complex and robust [18] than previous enzyme-dependent examples [19-22]. Similar strand displacement-based schemes using DNA hairpins as substrates have also been devised [23,24]. Moreover, it has be
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