UV Melting and Footprinting Studies of Three Structures of an RNA Aptamer with the Same Sequence

Abstract

Using systematic evolution of ligands by exponential enrichment (SELEX), we have previously selected a class of competitive RNA aptamers against the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors. One aptamer in this class was found to have a minimal, functional sequence of 58 nucleotides. In vitro transcription generates two RNA transcripts with the same sequence (M1 and M2), both of which are required to act together to inhibit the AMPA receptors. Although the M1 and M2 species have the same sequence, they cannot be inter-converted through the unfolding/refolding processes. To probe the secondary structures and the stabilities of these species, we carried out UV melting and RNA footprinting experiments. In these experiments, we used chemically synthesized AN58 (sAN58) as our control. sAN58 is a potent inhibitor by itself and possesses a different structure from either M1 or M2 (which are likely caused by co-transcriptional folding). Specifically, we have examined the effects of salt and urea on the melting profiles of the aptamers. All three RNA species had clearly distinct melting profiles with multiple melting transitions. M1, for instance, is found to have the highest melting temperature and the largest number of transitions. On the other hand, the melting profile of M2 is more similar to the sAN58 profile. These results suggest that all three RNA species have different structures, with M1 appearing to be the most stable of the three. Footprinting was also used to probe the secondary structure of the RNAs and to identify the nucleotides that interact with the S1S2 AMPA receptor ligand core.