In Vitro Selection of RNA Aptamers Specific for Cyanocobalamin

RNA receptors (aptamers) capable of specifically binding cyanocobalamin (vitamin B₁₂) have been isolated by in vitro selection from a pool of 5 × 10¹⁴ RNAs of random sequence. After eight rounds of selection by affinity chromatography and enzymatic amplification, the pool was dominated by two sequences. The major sequence, comprising 60% of the pool, was studied further. It was found to bind vitamin B₁₂ in solution with a dissociation constant (K_d) of approximately 320 ± 90 nM and to bind cobinamide dicyanide with a K_d of 8.8 ± 0.5 μM. The aptamer does not detectably bind adenosylcobalamin (coenzyme B₁₂). The selection was conducted in 1 M LiCl, and binding is dependent on the presence of high concentrations of Li⁺ but independent of Mg²⁺. To define the binding site for cyanocobalamin, a second cyanocobalamin-binding selection was carried out using a pool of sequences derived from the major aptamer sequence randomized at a level of 30%. The sequence data from this selection revealed a 31-base highly conserved region, on the basis of which was synthesized a smaller aptamer of 35 nucleotides. This small aptamer binds cyanocobalamin in solution with a K_d of 88 ± 19 nM and cobinamide dicyanide with a K_d of 20 ± 9 μM. This aptamer has the highest affinity yet reported for a small molecule ligand. A number of covarying positions were found in the conserved region of the sequences from this second, mutagenized pool selection. On the basis of these data, an unusual pseudoknot secondary structure is proposed for the aptamer. Chemical modification protection experiments are consistent with this structure and have demonstrated that the RNA undergoes a conformational change upon binding its ligand. Possible contacts with the cyanocobalamin have also been mapped. A third selection was carried out in which the salt specificity of the aptamer was changed from LiCl to NaCl plus MgCl₂. Sequence analysis of the final round pool of RNAs from this selection revealed several conserved changes from the original vitamin B₁₂ aptamer sequence.