February 1, 1995Journal of the American Chemical Society
RNA aptamers that bind flavin and nicotinamide redox cofactors
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Discussion
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Implication
In vitro selection demonstrates high-affinity RNA aptamer binding to flavin and nicotinamide cofactors, indicating RNA can recognize redox states to potentially drive catalytic ribozyme functions.
Key Points
- To isolate and characterize RNA aptamers capable of binding essential metabolic redox cofactors, specifically riboflavin and nicotinamide mononucleotide.
- Conducted in vitro selection to isolate RNA aptamers binding oxidized riboflavin and beta-nicotinamide mononucleotide (NMN).
- Performed dimethyl sulfate (DMS) protection assays and affinity measurements to determine structural motifs and binding constants (Kd).
- Reselected from a mutagenized RNA pool to identify the minimal binding domain and enhance discrimination between oxidized and reduced nicotinamide cofactors.
- Riboflavin-binding aptamers utilized intramolecular G-quartets to bind oxidized riboflavin with high affinity (Kd = 1 to 5 µM), with structure stabilized by flavin interaction but lacking redox specificity.
- Nicotinamide-binding aptamers discriminated between oxidized NAD and reduced NADH in solution by over an order of magnitude.
- Reselection from mutagenized pools identified a minimal NMN-binding motif with two interacting stem-loops and pinpointed specific mutations that increased discrimination between NMN and NMNH.
Cite This Study
Lauhon et al. (1995) studied this question.