PubMed Health⌕ Search

Biomedical subjects

A D Ellington

Publications and source records attributed to A D Ellington.

At least 19 recordsLinked to original sources
PubMed1999

In vitro selection of an allosteric ribozyme that transduces analytes to amplicons.

We have selected an allosteric ribozyme ligase from a random sequence population that is activated up to 10,000-fold by oligonucleotide effectors. The ribozyme conforms to a classic two-state model for allostery in which the equilibrium between inactive and active conformers is dramatically altered by the presence of effector ligands. In the presence of the effector the allosteric ribozyme ligase generates templates that can subsequently be amplified using conventional amplification technologies, such as RT-PCR. Thus, the allosteric ribozyme can transduce (or convert) analytes into amplicons. We demonstrate two potential diagnostic applications of the selected allosteric ribozyme ligase: 'counting' short oligonucleotide effectors by RT-PCR, and counting a non-nucleic acid effector, ATP, by ligation.

Allosteric Site↗
PubMed1998

The fidelity of template-directed oligonucleotide ligation and its relevance to DNA computation.

Several different computational problems have been solved using DNA as a medium. However, the DNA computations that have so far been carried out have examined a relatively small number of possible sequence solutions in order to find correct sequence solutions. We have encoded a search algorithm in DNA that required the evaluation of >16 000 000 possible sequence solutions in order to find a single, correct sequence solution. Experimental evaluation of the search algorithm revealed bounds for the accuracies of answers to other large, computationally complex problems and suggested methods for the optimization of DNA computations in general. Short oligonucleotide substrates performed substantially better than longer substrates. Large, computationally complex problems whose evaluation requires hybridization and ligation can likely best be encoded and evaluated using short oligonucleotides at mesophilic temperatures.

Algorithms↗
PubMed1998

In vitro selection of phosphorothiolated aptamers.

A pool of RNA molecules that contained exclusively phosphorothioate internucleoside linkages was used as a starting point for the selection of aptamers that bind to basic fibroblast growth factor (bFGF), and appear to act as heparin mimics.

Base Sequence↗
PubMed1998

Adapting selected nucleic acid ligands (aptamers) to biosensors.

A flexible biosensor has been developed that utilizes immobilized nucleic acid aptamers to specifically detect free nonlabeled non-nucleic acid targets such as proteins. In a model system, an anti-thrombin DNA aptamer was fluorescently labeled and covalently attached to a glass support. Thrombin in solution was selectively detected by following changes in the evanescent-wave-induced fluorescence anisotropy of the immobilized aptamer. The new biosensor can detect as little as 0.7 amol of thrombin in a 140-pL interrogated volume, has a dynamic range of 3 orders of magnitude, has an inter-sensing-element measurement precision of better than 4% RSD over the range 0-200 nM, and requires less than 10 min for sample analysis. The aptamer-sensor format is generalizable and should allow sensitive, selective, and fast determination of a wide range of analytes.

Anisotropy↗
PubMed1998

RNA molecules that bind to and inhibit the active site of a tyrosine phosphatase.

Protein tyrosine phosphatases (PTPases) are essential proteins in many cellular processes. In vitro selection was used to evolve high affinity RNA aptamers to the Yersinia PTPase from two random pools varying in length. Selected aptamers from the two different pools share a 21-residue conserved sequence. They bind to their target with dissociation constants of 18 and 28 nM and inhibit the enzyme with IC50 values of 10 and 35 nM, but do not bind a related PTPase. Modification of the PTPase's active site cysteine with the alkylating agent iodoacetate results in a loss of binding affinity. These experiments suggest that the selected aptamers act by binding at or near the active site and might therefore be useful in defining the interactions between PTPases and their targets.

Base Sequence↗
PubMed1998

Crystal structures of a series of RNA aptamers complexed to the same protein target.

We have determined the crystal structures, at 2.8 A resolution, of two different RNA aptamers, each bound to MS2 coat protein. One of the aptamers contains a non-Watson-Crick base pair, while the other is missing one of the unpaired adenines that make sequence-specific contacts in the wild-type complex. Despite these differences, the RNA aptamers bind in the same location on the protein as the wild-type translational operator. Comparison of these new structures with other MS2-RNA complexes allows us to refine further the definition of the minimal recognition elements and suggests a possible application of the MS2 system for routine structure determination of small nucleic acid motifs.

Base Pairing↗
PubMed1998

Crystal structure of an RNA aptamer-protein complex at 2.8 A resolution.

The crystal structure, at 2.8 A resolution, of an RNA aptamer bound to bacteriophage MS2 coat protein has been determined. It provides an opportunity to compare the interactions of MS2 coat protein and wild type operator with those of an aptamer, whose secondary structure differs from the wild type RNA in having a three-base loop (compared to a tetraloop) and an additional base pair between this loop and the sequence-specific recognition element in the stem. The RNA binds in the same location on the coat protein as the wild type operator and maintains many of the same RNA-protein interactions. In order to achieve this, the RNA stem loop undergoes a concerted rearrangement of the 3' side while leaving the 5' side and the loop interactions largely unchanged, illustrating the ability of RNA to present similar molecular recognition surfaces from distinct primary and secondary structures.

Asparagine↗
PubMed1997

Ribozymes: red in tooth and claw.

A population of catalytic RNA molecules has been engineered to operate and evolve in vitro in a continuous manner. The novel continuity of the process allows the propagation of many generations in a very short time and without the manual manipulation necessary with traditional in vitro selection techniques.

Catalysis↗
PubMed1997

Surprising fidelity of template-directed chemical ligation of oligonucleotides.

BACKGROUND: Nucleic acid replication via oligonucleotide ligation has been shown to be extremely prone to errors. If this is the case, it is difficult to envision how the assembly and replication of short oligonucleotides could have contributed to the origin of life and to the evolution of a putative RNA world. In order to assess the fidelity of oligonucleotide replication more accurately, chemical ligation reactions were performed with constant-sequence DNA templates and random-sequence DNA pools as substrates. RESULTS: In keeping with earlier results, constant-sequence hairpin templates were not faithfully copied by random-sequence substrates. Linear templates, however, showed exceptional replication fidelity, particularly when random hexamers were ligated at 25 degrees C. Surprisingly, at low temperatures the formation of G.A base pairs was common and sometimes occurred even more readily than the formation of the corresponding Watson-Crick A-T and G-C base pairs. CONCLUSIONS: The fidelity of ligation reactions increases with temperature and decreases with the length of the random-sequence substrates. Oligonucleotides with a defined sequence can be copied faithfully in the absence of enzymes. Thus, to the extent that short oligonucleotides could readily have been generated by prebiotic mechanisms, it is possible that the earliest self-replicators arose via oligonucleotide ligation.

Base Composition↗
PubMed1997

Aptamers as therapeutic and diagnostic reagents: problems and prospects.

Aptamers are nucleic acid molecules that bind specific ligands. Barriers to the application of aptamers as therapeutic and diagnostic reagents have been overcome in the past several years. In particular, aptamers that bind biomedically relevant targets have proven to be efficacious at modifying cellular metabolism. Such aptamers can be stabilized by chemical modifications and potentially used in vivo. Researchers have begun to devise aptamer-based diagnostic assays that may rival more conventional immunoassays.

Biopolymers↗
PubMed1997

In vitro selection of aptamers that bind to ribosome-inactivating toxins.

Ribosome-inactivating proteins, such as ricin, pepocin and gypsophilin, catalyze the hydrolysis of a single N-glycosidic bond at a specific position in rRNAs. Aptamers targeting pepocin were selected from a random sequence RNA pool that spanned 30 positions. After 8 rounds, the anti-pepocin aptamers were sequenced and a conserved hairpin motif was identified. Interestingly, the selected motif is quite different from the toxin-binding domains of rRNAs.

Base Sequence↗
PubMed1996

Detecting immobilized protein kinase C isozymes with RNA aptamers.

Nucleic acids that can bind tightly and specifically to protein targets can be selected from random sequence pools. Just as antibodies have been used to detect proteins in a variety of formats, it is possible the nucleic acid binding species (aptamers) could be used to specifically detect proteins. Using a simple filter binding assay, aptamers have been used to detect protein kinase C (PKC) introduced into a cell extract. Aptamers that were relatively nonspecific could be used to simultaneously detect different PKC isozymes, while aptamers with higher specificities could discriminate between related isozymes. These results demonstrate the feasibility of using aptamers as diagnostic tools to detect proteins.

Animals↗
PubMed1996

Anti-peptide aptamers recognize amino acid sequence and bind a protein epitope.

In vitro selection of nucleic acid binding species (aptamers) is superficially similar to the immune response. Both processes produce biopolymers that can recognize targets with high affinity and specificity. While antibodies are known to recognize the sequence and conformation of protein surface features (epitopes), very little is known about the precise interactions between aptamers and their epitopes. Therefore, aptamers that could recognize a particular epitope, a peptide fragment of human immunodeficiency virus type I Rev, were selected from a random sequence RNA pool. Several of the selected RNAs could bind the free peptide more tightly than a natural RNA ligand, the Rev-binding element. In accord with the hypothesis that protein and nucleic acid binding cusps are functionally similar, interactions between aptamers and the peptide target could be disrupted by sequence substitutions. Moreover, the aptamers appeared to be able to bind peptides with different solution conformations, implying an induced fit mechanism for binding. Just as anti-peptide antibodies can sometimes recognize the corresponding epitope when presented in a protein, the anti-peptide aptamers were found to specifically bind to Rev.

Amino Acid Sequence↗
PubMed1996

Selection of RNAs that bind to duplex DNA at neutral pH.

RNA that are capable of binding duplex DNA in a site-specific manner have potential applications in gene therapy strategies. Such RNAs might be targeted to DNA sequences in a gene promoter and prevent initiation of transcription by occluding transcription factors and/or RNA polymerases. RNA oligonucleotides that bind homopurine/homopyrimidine DNA sequences by forming triple-helical complexes involving T.A.T and C+.G.C base-triplets can be rationally designed. However, the formation of such pyrimidine motif triple helices typically requires mildly acidic conditions. In addition, the proper oligonucleotide sequence must be optimally presented within a longer RNA transcript if it is to be synthesized in vivo. To address these issues, RNAs were selected from pools of random sequences for binding to a homopurine/homopyrimidine DNA sequence. RNAs selected for binding the duplex DNA target between pH 6.5 and pH 7.4 were characterized by sequence analysis and binding studies. All RNAs isolated by selection and amplification were found to contain a pyrimidine recognition sequence for binding the duplex DNA target via conventional triple helix formation. The selected approximately 85 nt RNAs have dissociation constants that approach, but do not surpass, the binding affinity of a 21 nt RNA oligonucleotide that binds the DNA target sequence by forming a canonical triple helix. The presence of a pyrimidine recognition sequence within a longer RNA transcript is not sufficient for high affinity. Experimental data and secondary structure predictions suggest that the context of the pyrimidine recognition sequence within selected RNAs is a very important determinant of DNA binding affinity. These studies provide insight into the development of RNA transcripts that may function as gene-specific repressors by forming triple helices with DNA in vivo.

Base Sequence↗