Cofactors are pivotal compounds for the cell and many biotechnological processes. It is therefore interesting to ask how well cofactors can be bound by oligonucleotides designed not to convert but to store and release these biomolecules. Here we show that triplex-based DNA binding motifs can be used to bind nucleotides and cofactors, including NADH, FAD, SAM, acetyl CoA, and tetrahydrofolate (THF). Dissociation constants between 0.1 μM for SAM and 35 μM for THF were measured. A two-nucleotide gap still binds NADH. The selectivity for one ligand over the others can be changed by changing the sequence of the binding pocket. For example, a mismatch placed in one of the two triplets adjacent to the base-pairing site changes the selectivity, favoring the binding of FAD over that of ATP. Further, changing one of the two thymines of an A-binding motif to cytosine gives significant affinity for G, whereas changing the other does not. Immobilization of DNA motifs gives beads that store NADH. Exploratory experiments show that the beads release the cofactor upon warming to body temperature.

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Nicotinamide adenine dinucleotide (NAD) is a pivotal redox cofactor of primary metabolism. Its redox reactivity is based on the nicotinamide mononucleotide (NMN) moiety. We investigated whether NMN(+) can engage in pairing interactions, when incorporated into an oligonucleotide. Here we describe the incorporation of NMN(+) at the 3'-terminus of an oligodeoxynucleotide via a phosphoramidate coupling in solution. The stability of duplexes and triplexes with the NMN(+)-containing strand was measured in UV-melting curves. While the melting points of duplexes with different bases facing the nicotinamide were similar, triplex stabilities varied greatly between different base combinations, suggesting specific pairing. The most stable triplexes were found when a guanine and an adenine were facing the NMN(+) residue. Their triplex melting points were higher than those of the corresponding triplexes with a thymidine residue at the same position. These results show that NMN(+) residues can be recognized selectively in DNA helices and are thus compatible with the molecular recognition in nucleic acids.

… The surface of a sepharose bead with immobilized triplex motifs releases bound NADH when approaching 37 8C. The NADH then induces bioluminescence. For more details, see the Full Paper by C. Richert et al. on page 15879 ff.