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 10(14) 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 approximately 0.1 microM was found for the strongest binding variant under the conditions used for selection (5 mM Mg(2+)). Systematic studies of the effect of Mg(2+) and Mn(2+) on binding, however, revealed that the affinity of the aptamers could be substantially improved, and at optimized conditions of Mn(2+) the affinity of one of the aptamers approached that of an anti-SAH antibody with similar/identical binding specificity. Comparisons with 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 that shortening of RNA constructs may alter binding kinetics as well as sensitivity to ions.
Gebhardt
K
K
Biotechnology Centre of Oslo, University of Oslo, Blindern, Norway. kgebhard@embnet.uio.no
Shokraei
A
A
Babaie
E
E
Lindqvist
B H
BH
D000225:Adenine / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D015374:Biosensing Techniques; D002413:Cations, Divalent / Q000494:pharmacology; D002846:Chromatography, Affinity; D003540:Cystathionine / Q000737:chemistry; D015723:Gene Library; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry*; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D012435:S-Adenosylhomocysteine / Q000276:immunology / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000031:analogs & derivatives / Q000737:chemistry
D000911:Antibodies, Monoclonal; D002413:Cations, Divalent; D009841:Oligonucleotides; C092076:aza-S-adenosylmethionine; D003540:Cystathionine; D012313:RNA; D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; D000225:Adenine