Molecular Recognition of cAMP by an RNA Aptamer

• Published in: Biochemistry (Easton) Vol. 39; no. 30; pp. 8983 - 8992
• Main Authors: Koizumi, Makoto, Breaker, Ronald R
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 01.08.2000
• Subjects: 8,5'-O-cycloadenosine; Adenosine Triphosphate - metabolism; Base Sequence; Binding Sites; Cyclic AMP - chemistry; Cyclic AMP - genetics; Cyclic AMP - metabolism; Glycosides - chemistry; Glycosides - metabolism; Ligands; Molecular Sequence Data; N3,5-cycloadenosine; Nucleic Acid Conformation; RNA - genetics; RNA - isolation & purification; RNA - metabolism; Sequence Homology, Nucleic Acid; Substrate Specificity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi000149n

Two classes of RNA aptamers that bind the second messenger adenosine 3‘,5‘-cyclic monophosphate (cAMP; 1) were isolated from a random-sequence pool using in vitro selection. Class I and class II aptamers are formed by 33- and 31-nucleotide RNAs, respectively, and each is comprised of similar stem−loop and single-stranded structural elements. Class II aptamers, which dominate the final selected RNA population, require divalent cations for complex formation and display a dissociation constant (K D) for cAMP of ∼10 μM. A representative class II aptamer exhibits substantial discrimination against 5‘- and 3‘-phosphorylated nucleosides such as ATP, 5‘-AMP, and 3‘-AMP. However, components of cAMP such as adenine and adenosine also are bound, indicating that the adenine moiety is the primary positive determinant of ligand binding. Specificity of cAMP binding appears to be established by hydrogen bonding interactions with the adenine base as well as by steric interactions with groups on the ribose moiety. In addition, the aptamer recognizes 8,5‘-O-cycloadenosine (2) but not N 3,5‘-cycloadenosine (3), indicating that this RNA might selectively recognize the anti conformation of the N-glycosidic bond of cAMP.