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.

Tailoring RNA modular units on a common scaffold: A modular ribozyme with a catalytic unit for β-nicotinamide mononucleotide-activated RNA ligation

“…Therefore, in the RNA World, b-nicotinamide riboside or its monophosphate (b-NMN) may have played multiple roles as a cofactor of redox reactions and an activator of nucleotides. This model was partly supported by the isolation of RNA aptamers recognizing NAD(H) (Burgstaller and Famulok 1994;Lauhon and Szostak 1995) and NAD(H)-dependent ribozymes catalyzing redox conversion of alcohol and aldehyde (Tsukiji et al 2003). The results of the present study provided additional evidence to support the multifunctionality of b-nicotinamide riboside in the RNA World.…”

Arginine-modified DNA Aptamers That Show Enantioselective Recognition of the Dicarboxylic Acid Moiety of Glutamic Acid

“…We used the latter template, expecting that the incidence rates of aptamers might increase because G-rich sequences were found in some aptamers obtained by SELEX. [45][46][47] The diversity of the random pool based on the central random region was estimated to be approximately 10 12 unique sequences. The following primers that annealed to the primer regions were used for amplification of the selected oligonucleotides during the aptamer selection process.…”

Conserved Sequence Motif of RNA Aptamers Binding to the G-rich Sequence RNA

“…Target molecules include proteins, amino acids, nucleotides, antibiotics and RNA. [14][15][16][17][18][19][20][21][22][23][24][25][26] We used SELEX technique to get the information for the RNA-RNA interaction and got the similar results with affinity chromatography and gel mobility shift assay. According to this result, we knew that similar results could be got irrespective of SELEX techniques employed.…”