Biomedical subjects

T M Tarasow

Publications and source records attributed to T M Tarasow.

5 recordsLinked to original sources

PubMed1997

RNA-catalysed carbon-carbon bond formation.

The 'RNA world' hypothesis, which assumes that the chemical processes that led to the appearance of life were carried out by RNA molecules, has stimulated interest in catalytic reactions involving oligonucleotides such as catalytic RNA (ribozymes). Naturally occurring ribozymes have, for example, been shown to efficiently catalyse the formation and cleavage of nucleic-acid phosphodiester bonds, and this narrow range of RNA-catalysed reactions has been subsequently expanded by in vitro selection methods to include ester and amide bond formation S(N)2 reactions and porphyrin metallations. Carbon-carbon bond formation and the creation of asymmetric centres are both of great importance biochemically, but have not yet been accomplished by RNA catalysis. A widely used reaction that creates two new carbon-carbon bonds and up to four stereo-centres is the Diels-Alder cycloaddition, which occurs between a 1,3-butadiene and an alkene. Here we report the successful application of in vitro selection to isolate pyridine-modified RNA molecules that catalyse a Diels-Alder cycloaddition. We find that the RNA molecules accelerate the reaction rate by a factor of up to 800 relative to the uncatalysed reaction.

Alkenes↗

PubMed1997

Post-SELEX combinatorial optimization of aptamers.

In vitro selection techniques provide a means of isolating nucleic acid ligands for binding to particular protein targets. Although most aptamers have quite high affinities for their target proteins, it has been shown that post-SELEX modification can result in further enhancement of binding affinity, as well as other desired properties. This has led to the current development of a more systematic approach to aptamer optimization using a combinatorial screening methodology.

Base Sequence↗

PubMed1994

Enrichment for RNA molecules that bind a Diels-Alder transition state analog.

RNA molecules that bind a transition state analog for a Diels-Alder reaction (Kd = 0.35 +/- 0.05 mM) were isolated from a starting pool of approximately 10(14) sequences by affinity chromatography. After the initial rise and plateau of the amount of RNA that eluted with soluble analog, a step gradient elution was used to further enrich the pool for sequences with higher affinities for the target. To our knowledge, the isolation of RNA molecules that bind either a nonplanar or a hydrophobic ligand has not been reported previously. A conserved nucleotide sequence and secondary structure present in many of the RNA molecules are necessary but not sufficient for binding the analog. No catalysts of the targeted Diels-Alder reaction were found among the binders. The absence of catalysis contrasts with previous successful experiments with antibodies and suggests that other strategies may be needed to identify oligonucleotides with diverse catalytic activities.

Base Sequence↗

PubMed1993

Binding of a high-energy substrate conformer in antibody catalysis.

Enzymes can substantially increase the probability of a reaction by exploiting binding energy to preorganize their substrates into reactive conformations. Similar effects are likely to be important in a wide variety of designed catalysts, including catalytic antibodies. Transferred nuclear Overhauser effects have been used here to investigate how an antibody possessing chorismate mutase activity binds its flexible substrate molecule chorismate. The conversion of chorismate to prephenate by way of a Claisen rearrangement requires the substrate to adopt an energetically disfavored diaxial conformation in which the enolpyruvyl side chain is positioned over the six-membered ring. The antibody, which was elicited by a conformationally restricted transition state analog for this reaction, appears to bind this high-energy substrate conformer preferentially, as judged by diagnostic intramolecular transferred nuclear Overhauser effects. Inhibitor studies with the transition state analog confirm that preorganization takes place exclusively at the antibody active site. These results thus provide strong physical evidence for a direct relationship between the properties of a catalytic antibody and the structure of the transition state analog originally used to elicit the immune response.

Antibodies, Monoclonal↗

PubMedDate not supplied

Characterization of oligodeoxyribonucleotide-polyethylene glycol conjugates by electrospray mass spectrometry.

Electrospray mass spectrometry (ESMS) was used to characterize a number of differently functionalized polyethylene glycol-oligodeoxyribonucleotide conjugates. Sample preparation was found to be crucial to obtaining quality data. The resolution and precision of ESMS allowed for the identification of individual conjugates differing in MW by a single ethylene glycol unit with an accuracy of < or = 1 (0.02% of the molecular weight). In addition, ESMS was shown to be valuable in identifying chromatographically unresolvable components of a derivatized polyethylene glycol-oligonucleotide conjugate mixture.

Amides↗