RNA Aptamers to S-Adenosylhomocysteine: Kinetic Properties, Divalent Cation Dependency, and Comparison with Anti-S-adenosylhomocysteine Antibody†

To explore the potential of RNA aptamers as small-molecule discriminating devices, we have characterized the properties of aptamers selected from a library of approximately 1014 variants through their interaction with S-adenosylhomocysteine (SAH, AdoHcy). Competition studies with SAH and azaSAM analogues revealed that the Hoogsteen face of adenine is the main contributor to binding, whereas specificity for SAH is conferred by a secondary contact point at or near the sulfur/thioether of homocysteine (Hcy). Binding specificities were determined by both affinity chromatography and a novel method designed for the biosensor. The kinetic properties of individual aptamers, including the “classic” ATP aptamer that also emerged in our selection, were studied by biosensor analysis. Association rates were slow, but the complexes were stable, suggesting micro- to submicromolar affinities. A solution affinity of ∼0.1 μM was found for the strongest binding variant under the conditions used for selection (5 mM Mg2+). Sy...