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 d evices, we have characterized the properties of aptamers selected from a li brary of approximately 10(14) variants through their interaction with S-ade nosylhomocysteine (SAH, AdoHcy), Competition studies with SAH and azaSAM an alogues revealed that the Hoogsteen face of adenine is the main contributor to binding, whereas specificity for SAW is conferred by a secondary contac t point at or near the sulfur/thioether of homocysteine (Hcy). Binding spec ificities were determined by both affinity chromatography and a novel metho d designed for the biosensor. The kinetic properties of individual aptamers , including the "classic" ATP aptamer that also emerged in our selection, w ere studied by biosensor analysis. Association rates were slow, but the com plexes were stable, suggesting micro- to submicromolar affinities. A soluti on affinity of similar to 0.1 mu M was found for the strongest binding vari ant under the conditions used for selection (5 mM Mg2+). Systematic studies of the effect of Mg2+ and Mn2+ on binding, however, revealed that the affi nity of the aptamers could be substantially improved, and at optimized cond itions of Mn2+ the affinity of one of the aptamers approached that of an an ti-SAM antibody with similar/identical binding specificity. Comparisons wit h the MAb suggest that the on rate is the limiting factor for high-affinity binding by these aptamers, and comparison with a truncated aptamer shows t hat shortening of RNA constructs may alter binding kinetics as well as sens itivity to ions.