A completely randomized RNA pool as well as a degenerate pool comprised of an RNA sequence which binds citrulline with a dissociation constant of 0 muM were used to select for tight binding arginine specific RNA aptamers. A modified in vitro selection scheme, based on affinity chromatography was applied to allow the enrichment of high affinity solution binders. The selection scheme included a negative selection with the non-cognate ligand citrulline, and a heat denaturation step prior to affinity elution with an excess of the cognate ligand arginine. After 20 cycles the majority of the pools bound specifically to the arginine matrix even after denaturation/renaturation in the presence of 20 mM of a non-cognate amino acid. When denatured and eluted in the presence of 20 mM arginine, the selected RNAs quantitatively washed off the column. These RNA aptamers were cloned and sequenced. Equilibrium dialysis performed with the most abundant clone among the selected sequence revealed Kd values of 330 nM for the RNA/arginine affinity, which is nearly a 200-fold improvement over the tightest binding arginine binding RNAs known to date. Arginine recognition by this RNA is highly enantioselectice: L- arginine is bound 12 000-fold better than D-arginine. Chemical modification analysis revealed that the secondary structure of the aptamer might contain a pseudoknot motif. Our tight binding arginine aptamers join a number of natural and in vitro selected RNAs which recognize arginine. The RNAs described here compare in their binding affinity with the tightest binding RNA aptamers for low molecular weight molecules isolated in other in vitro selection experiments.
In a previous study, an RNA aptamer for the specific recognition of arginine was evolved from a parent sequence that bound citrulline specifically. The two RNAs differ at only 3 positions out of 44. The solution structures of the two aptamers complexed to their cognate amino acids have now been determined by two-dimensional nuclear magnetic resonance spectroscopy. Both aptamers contain two asymmetrical internal loops that are not well ordered in the free RNA but that fold into a compact structure upon ligand binding. Those nucleotides common to both RNAs include a conserved cluster of purine residues, three of which form an uneven plane containing a G:G pair, and two other residues nearly perpendicular to that surface. Two of the three variant nucleotides are stacked on the cluster of purines and form a triple contact to the amino acid side chain, whereas the edge of the third variant nucleotide is capping the binding pocket.
5-(3"-Aminopropynyl)-2'-deoxyuridine (dJ), a modified nucleoside with a side chain carrying a cationic functional group, was incorporated into an oligonucleotide library, which was amplified using the Vent DNA polymerase in a polymerase chain reaction (PCR). When coupled to an in vitro selection procedure, PCR amplification generated receptors that bind ATP. This is the first example of an in vitro selection generating oligonucleotide receptors where the oligonucleotide library has incorporated a cationic nucleotide functionality. The selection yielded functionalized receptors having sequences differing from a motif known to arise in a standard selection experiment using only natural nucleotides. Surprisingly, both the natural and the functionalized motifs convergently evolved to bind not one, but two ATP molecules cooperatively. Likewise, the affinity of the receptors for ATP had converged; in both cases, the receptors are half saturated at the 3 mM concentrations of ATP presented during the selection. The convergence of phenotype suggests that the outcome of this selection experiment was determined by features of the environment during which selection occurs, in particular, a highly loaded affinity resin used in the selection step. Further, the convergence of phenotype suggests that the optimal molecular phenotype has been achieved by both selections for the selection conditions. This interplay between environmental conditions demanding a function of a biopolymer and the ability of the biopolymer to deliver that function is strictly analogous to that observed during natural selection, illustrating the nature of life as a self-sustaining chemical system capable of Darwinian evolution.
Mirror-image oligonucleotide ligands (Spiegelmers) that bind to the pharmacologically relevant target gonadotropin-releasing hormone I (GnRH) with high affinity and high specificity have been identified using the Spiegelmer technology. GnRH is a decapeptide that plays an important role in mammalian reproduction and sexual maturation and is associated with several benign and malignant diseases. First, aptamers that bind to D-GnRH with dissociation constants of 50-100 nM were isolated out of RNA and DNA libraries. The respective enantiomers of the DNA and RNA aptamers were synthesized, and their binding to L-GnRH was shown. These Spiegelmers bind to L-GnRH with similar affinity to that of the corresponding aptamers that bind to D-GnRH. We further demonstrated dose-dependent inhibition of GnRH-induced Ca(2+) release in Chinese hamster ovary cells that were stably transfected with the human GnRH receptor.
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