RNA aptamers that bind flavin and nicotinamide redox cofactors

Abstract

RNA molecules that specifically bind riboflavin (Rb) and beta-nicotinamide mononucleotide (NMN) have been isolated by in vitro selection. A simple structural motif containing intramolecular G-quartets was found to bind tightly to oxidized riboflavin (Kd = 1-5 micromolar). DNA versions of the consensus sequence also bind, but with weaker affinity. DMS protection experiments show that the quartet structure of these aptamers is stabilized by interaction with the flavin. As a measure of their redox specificity, the aptamers do not show significant differential binding between oxidized and reduced forms of a 5-deazariboflavin derivative that is a close structural analog of riboflavin. In contrast to the lack of redox specificity of the riboflavin aptamers, RNAs selected for binding to the nicotinamide portion of NAD discriminate between NAD and NADH in solution by over an order of magnitude. A mutagenized pool based on one of the NMN aptamer sequences was used to reselect for NMN binding. Comparison of the reselected sequences led to the identification of the binding region of the aptamer. A complex secondary structure containing two interacting stem-loops is proposed for the minimal NMN-binding RNA. The same mutagenized pool was used to select for increased discrimination between NMN and NMNH. From these reselected sequences, a mutation within the binding region was identified that increases specificity for NMN. These experiments show that RNA can bind these cofactors with low micromolar affinity and, in the case of nicotinamide cofactors, can discriminate between the two redox states. These cofactor binding motifs may provide a framework for generating new ribozymes that catalyze redox reactions similar to those found in basic metabolic pathways.