PubMed Health⌕ Search

Biomedical subjects

Andrew D Ellington

Publications and source records attributed to Andrew D Ellington.

At least 19 recordsLinked to original sources
PubMed2003

Exponential growth by cross-catalytic cleavage of deoxyribozymogens.

We have designed an autocatalytic cycle based on the highly efficient 10-23 RNA-cleaving deoxyribozyme that is capable of exponential amplification of catalysis. In this system, complementary 10-23 variants were inactivated by circularization, creating deoxyribozymogens. Upon linearization, the enzymes can act on their complements, creating a cascade in which linearized species accumulate exponentially. Seeding the system with a pool of linear catalysts resulted not only in amplification of function but in sequence selection and represents an in vitro selection experiment conducted in the absence of any protein enzymes.

Base Sequence↗
PubMed2003

Simultaneous detection of diverse analytes with an aptazyme ligase array.

Allosteric ribozymes (aptazymes) can transduce the noncovalent recognition of analytes into the catalytic generation of readily observable signals. Aptazymes are easily engineered, can detect diverse classes of biologically relevant molecules, and have high signal-to-noise ratios. These features make aptazymes useful candidates for incorporation into biosensor arrays. Allosteric ribozyme ligases that can recognize a variety of analytes ranging from small organics to proteins have been generated. Upon incorporation into an array format, multiple different aptazyme ligases were able to simultaneously detect their cognate analytes with high specificity. Analyte concentrations could be accurately measured into the nanomolar range. The fact that analytes induced the formation of new covalent bonds in aptazyme ligases (as opposed to noncovalent bonds in antibodies) potentiated stringent washing of the array, leading to improved signal-to-noise ratios and limits of detection.

Biosensing Techniques↗
PubMed2003

Peptide-templated nucleic acid ligation.

Short oligonucleotide and peptide replicators have been described. To determine whether cross-replication could have occurred between such systems, we have attempted to show that peptides can specifically template the ligation of nucleic acids. A complex between a 35-mer anti-Rev RNA aptamer and a 17-mer arginine-rich motif (ARM) peptide from the HIV-1 Rev protein served as a model system. Aptamer half-molecules were activated for ligation via two activation chemistries, representing two distinct kinetic possibilities for early replicators. Cyanogen bromide activation was transient relative to oligonucleotides that terminated with a 5'-iodine and a 3'phosphorothioate, respectively. The Rev ARM specifically enhanced the degree or rate of ligation by both methods: there was a 10-fold increase in the production of full-length aptamer in the presence of cyanogen bromide and a 5.9- to 7.6-fold enhancement in the rate of ligation for stably activated aptamer half-molecules. These results support the possibility that life could have originated with peptide replicators and transitioned to nucleic acid replicators or that peptide and nucleic acid replicators could have been interdependent.

Amino Acid Sequence↗
PubMed2002

Group I aptazymes as genetic regulatory switches.

BACKGROUND: Allosteric ribozymes (aptazymes) that have extraordinary activation parameters have been generated in vitro by design and selection. For example, hammerhead and ligase ribozymes that are activated by small organic effectors and protein effectors have been selected from random sequence pools appended to extant ribozymes. Many ribozymes, especially self-splicing introns, are known control gene regulation or viral replication in vivo. We attempted to generate Group I self-splicing introns that were activated by a small organic effector, theophylline, and to show that such Group I aptazymes could mediate theophylline-dependent splicing in vivo. RESULTS: By appending aptamers to the Group I self-splicing intron, we have generated a Group I aptazyme whose in vivo splicing is controlled by exogenously added small molecules. Substantial differences in gene regulation could be observed with compounds that differed by as little as a single methyl group. The effector-specificity of the Group I aptazyme could be rationally engineered for new effector molecules. CONCLUSION: Group I aptazymes may find applications as genetic regulatory switches for generating conditional knockouts at the level of mRNA or for developing economically viable gene therapies.

Allosteric Regulation↗
PubMed2002

Construction and selection of bead-bound combinatorial oligonucleoside phosphorothioate and phosphorodithioate aptamer libraries designed for rapid PCR-based sequencing.

Chemically synthesized combinatorial libraries of unmodified or modified nucleic acids have not previously been used in methods to rapidly select oligonucleotides binding to target biomolecules such as proteins. Phosphorothioate oligonucleotides (S-ODNs) or phosphorodithioate oligonucleotides (S2-ODNs) with sulfurs replacing one or both of the non-bridging phosphate oxygens bind to proteins more tightly than unmodified oligonucleotides and have the potential to be used as diagnostic reagents and therapeutics. We have applied a split synthesis methodology to create one-bead one-S-ODN and one-bead one-S2-ODN libraries. Binding and selection of specific beads to the transcription factor NF-kappaB p50/p50 protein were demonstrated. Sequencing both the nucleic acid bases and the positions of any 3'-O-thioate/dithioate linkages was carried out by using a novel PCR-based identification tag of the selected beads. This approach allows us to rapidly and conveniently identify S-ODNs or S2-ODNs that bind to proteins.

Base Sequence↗
PubMed2002

Automated selection of aptamers against protein targets translated in vitro: from gene to aptamer.

Reagents for proteome research must of necessity be generated by high throughput methods. Aptamers are potentially useful as reagents to identify and quantitate individual proteins, yet are currently produced for the most part by manual selection procedures. We have developed automated selection methods, but must still individually purify protein targets. Therefore, we have attempted to select aptamers against protein targets generated by in vitro transcription and translation of individual genes. In order to specifically immobilize the protein targets for selection, they are also biotinylated in vitro. As a proof of this method, we have selected aptamers against translated human U1A, a component of the nuclear spliceosome. Selected sequences demonstrated exquisite mimicry of natural binding sequences and structures. These results not only reveal a potential path to the high throughput generation of aptamers, but also yield insights into the incredible specificity of the U1A protein for its natural RNA ligands.

Automation↗
PubMed2002

Anticipatory evolution and DNA shuffling.

DNA shuffling has proven to be a powerful technique for the directed evolution of proteins. A mix of theoretical and applied research has now provided insights into how recombination can be guided to more efficiently generate proteins and even organisms with altered functions.

DNA↗
PubMed2002

Increasing the thermal stability of an oligomeric protein, beta-glucuronidase.

The reporter enzyme beta-glucuronidase was mutagenized and evolved for thermostability. After four cycles of screening the best variant was more active than the wild-type enzyme, and retained function at 70 degrees C, whereas the wild-type enzyme lost function at 65 degrees C. Variants derived from sequential mutagenesis were shuffled together, and re-screened for thermostability. The best variants retained activities at even higher temperatures (80 degrees C), but had specific activities that were now less than that of the wild-type enzyme. The mutations clustered near the tetramer interface of the enzyme, and many of the evolved variants showed much greater resistance to quaternary structure disruption at high temperatures, which is also a characteristic of naturally thermostable enzymes. Together, these results suggest a pathway for the evolution of thermostability in which enzymes initially become stable at high temperatures without loss of activity at low temperatures, while further evolution leads to enzymes that have kinetic parameters that are optimized for high temperatures.

Directed Molecular Evolution↗
PubMed2002

In vitro selection of a deoxyribozyme that can utilize multiple substrates.

Deoxyribozymes that could catalyze the formation of an internucleotide phosphorothioester linkage were selected from a random sequence pool. During the course of the selection, the pool was successively challenged with five oligonucleotide substrates, each of which terminated in the same hexanucleotide sequence. Selected deoxyribozyme ligases could use all five substrates, albeit to different degrees, and appeared to form secondary structures that allow differential pairing between the deoxyribozyme and each substrate. These results suggest that early replicases may have been able to bind a variety of oligonucleotide substrates while catalyzing ligation via a common junction.

Animals↗
PubMed2002

ATP-dependent allosteric DNA enzymes.

Effector-activated ribozymes that respond to small organic molecules have previously been generated by appending binding species (aptamers) to ribozymes. In order to determine if deoxyribozymes can similarly be activated by effector molecules, we have appended an anti-adenosine aptamer to a selected deoxyribozyme ligase. The resultant constructs are specifically activated by ATP. Optimization of the joining region resulted in ligases that are activated up to 460-fold by ATP. The selected deoxyribozyme catalyzes ligation largely via a templating mechanism. Effector activation is surprisingly achieved by suppression of the rate of the background, templated ligation reaction in the absence of the effector molecule, probably by misalignment of the oligonucleotide substrates. This novel allosteric mechanism has not previously been observed for nucleic-acid catalysts and is rare even in protein catalysts.

Adenosine Triphosphate↗
PubMed2002

Reflections on a novel therapeutic candidate.

Aptamers composed of L-nucleic acids, Spiegelmers, were selected to specifically bind GnRH. Spiegelmer inhibition of GnRH activity was demonstrated in both cellular and animal models. Rabbit studies showed minimal immunogenic response to the agents.

Animals↗
PubMed2002

Protein-dependent ribozymes report molecular interactions in real time.

Most approaches to monitoring interactions between biological macromolecules require large amounts of material, rely upon the covalent modification of an interaction partner, or are not amenable to real-time detection. We have developed a generalizable assay system based on interactions between proteins and reporter ribozymes. The assay can be configured in a modular fashion to monitor the presence and concentration of a protein or of molecules that modulate protein function. We report two applications of the assay: screening for a small molecule that disrupts protein binding to its nucleic acid target and screening for protein protein interactions. We screened a structurally diverse library of antibiotics for small molecules that modulate the activity of HIV-1 Rev-responsive ribozymes by binding to Rev. We identified an inhibitor that subsequently inhibited HIV-1 replication in cells. A simple format switch allowed reliable monitoring of domain-specific interactions between the blood-clotting factor thrombin and its protein partners. The rapid identification of interactions between proteins or of compounds that disrupt such interactions should have substantial utility for the drug-discovery process.

Amino Acid Sequence↗
PubMed2002

Selecting nucleic acids for biosensor applications.

In vitro selection can be used to generate nucleic acid binding species (aptamers) and catalysts (ribozymes) that can recognize a variety of molecules. Because nucleic acid function is largely derived from readily tabulated secondary structures, it has proven possible to engineer aptamers and ribozymes to function as biosensors. Labeling nucleic acids with reporter molecules has yielded simple antibody substitutes, but by relying on ligand-dependent conformational changes it has also proven possible to generate biosensors that can recognize and specifically report the presence of ligands in homogenous solution. It may prove possible to generate signaling aptamers and allosteric ribozymes (aptazymes) that are responsive to a large fraction of an organismal proteome or metabolome using automated methods. Nucleic acid biosensor arrays for non-nucleic acid targets could likely be generated with the same facility as DNA chips.

Base Sequence↗