Published online 23 December 2005
Article |
Identification of a 14mer RNA that recognizes and binds flavin mononucleotide with high affinity
1School of Pharmacy, University of Wisconsin 777 Highland Avenue, Madison, WI 53705, USA 2Department of Chemistry, University of Wisconsin 777 Highland Avenue, Madison, WI 53705, USA
*To whom correspondence should be addressed. Tel: +1 608 262 7810; Fax: +1 608 262 5345; Email: smecozzi{at}wisc.edu
Received September 23, 2005. Revised November 3, 2005. Accepted November 16, 2005.
Aptamers are nucleic acids developed by in vitro evolution techniques that bind to specific ligands with high affinity and selectivity. Despite such high affinity and selectivity, however, in vitro evolution does not necessarily reveal the minimum structure of the nucleic acid required for selective ligand binding. Here, we show that a 35mer RNA aptamer for the cofactor flavin mononucleotide (FMN) identified by in vitro evolution can be computationally evolved to a mere 14mer structure containing the original binding pocket and eight scaffolding nucleotides while maintaining its ability to bind in vitro selectively to FMN. Using experimental and computational methodologies, we found that the 14mer binds with higher affinity to FMN (KD 
4 µM) than to flavin adenine dinucleotide (KD 12 µM) or to riboflavin (KD 13 µM),despite the negative charge of FMN. Different hydrogen-bond strengths resulting from differing ring-system electron densities associated with the aliphatic-chain charges appear to contribute to the selectivity observed for the binding of the 14mer to FMN and riboflavin. Our results suggest that high affinity and selectivity in ligand binding is not restricted to large RNAs, but can also be a property of extraordinarily short RNAs.