MOLECULAR RECOGNITION OF AMINO-ACIDS BY RNA-APTAMERS - AN L-CITRULLINE BINDING RNA MOTIF AND ITS EVOLUTION INTO AN L-ARGININE BINDER

A pool of similar to 10(15) different RNA molecules was enriched by in vitro selection for specific binding to L-citrulline [L-(+)-2-amino-5-ureidovaleric acid] in solution. Cloning and sequencing of the binding pool revealed two highly conserved regions present in all of the selected RNAs. These consensus sequences are 10 and 6 bases in length and are part of a defined secondary structure. All of the sequenced RNAs had the potential to fold into the same binding motif. On the basis of this motif, a 44 nucleotide long RNA was constructed. The dissociation constant of the 44-mer RNA and L-citrulline was determined by equilibrium gel filtration and by analytical column chromatography and was found to be 62-68 mu M. Specificity of the L-citrulline-aptamer was tested by affinity elution with D-citrulline, L-arginine [L-(+)-2-amino-5-guanidinovaleric acid], L-albizziin [L-(+)-2-amino-3-ureidopropionic acid], L-glutamine, and urea. The binding to L-citrulline was weakly enantioselective: D-citrulline had a K-d of 180 mu M which corresponds to a Delta Delta G of 2.5 kJ mol(-1) (0.6 kcal mol(-1)). The other citrulline analogues had no or very weak affinity to the RNA-aptamer. One of the L-citrulline-binding sequences (clone 16) was resynthesized with a 30% mutation rate per base position. This doped pool was subjected to further cycles of selection and amplification on agaroses that were derivatized with L-citrulline, L-arginine, L-lysine, L-albizziin, and L-glutamine. After three cycles, the pool bound to L-citrulline and after four cycles, aptamers for L-arginine showed up. No aptamers to L-albizziin, L-glutamine, or L-lysine could be enriched. Of 22 sequences derived from the arginine pool, 11 had the potential to fold into a motif that was only slightly different from the L-citrulline-binding motif and was highly specific for its cognate ligand. However, the arginine-binding pool also contained a large number of different and much less abundant motifs with no similarity to the ''parent'' motif. The most abundant RNA had values of K-d of 56-76 mu M for L-arginine and a K-d Of 410 mu M for D-arginine, which corresponds to a Delta Delta G of 4.6 kJ mol(-1) (1.1 kcal mol(-1)). This study provides the first example of the reselection of an RNA-aptamer to change its binding specificity.