Nicotinamide adenine dinucleotide (NAD) is a widely studied, important coenzyme, which is involved in many critical biological redox reactions. Oxidized NAD (NAD+) accepts two electrons while its reduced form (NADH) donates two electrons to a variety of substrates. Recently, NAD has been identified as a non-canonical initiating nucleotide (NCIN) in both prokaryotic and eukaryotic RNA. Since a pyrophosphate links the adenine and nicotinamide nucleotides in NAD, such an NCIN resembles the 7- methylguanylate cap found on the 5’ end of most eukaryotic messenger RNAs. The reason that RNA starts with NAD is not clear, but the current paradigm in the field assumes the NCINs primarily exist to modulate RNA function (i.e. they are “epitranscriptomic modifications”). This proposal examines another hypothesis that contends the opposite is true, i.e., that the RNA component is needed to modulate coenzyme activity. If the RNA influences coenzyme activity then reactions catalyzed by the NAD-utilizing enzymes should proceed at different rates or extents, and the sequence of the RNA should influence the reaction. The first prediction will be tested by comparing the ability of various enzymes to use NAD+-, NADP+-, NADH-, NADPH- capped RNA to their ability to use free dinucleotides. The enzymes chosen for analysis are glyceraldehyde 3-phosphate dehydrogenase, lactate dehydrogenase (LDH), pyruvate dehydrogenase, isocitrate dehydrogenase, a-ketoglutarate dehydrogenase; malate dehydrogenase, glutamate dehydrogenase, glucose-6-phosphate dehydrogenase, dihydrofolate reductase, DNA ligase, mono-ADP ribosyltransferase, poly (ADP-ribose) polymerase, and NAD-dependent deacetylase. Each enzyme that reacts with capped-RNA will be examined in more detail to estimate the RNA affinity and RNA specificity. Affinity will be monitored using enzyme kinetics and direct binding assays. Specificity will be investigated using a library of previously reported cellular NAD-capped RNAs, and by sequencing RNAs that co-immuno-precipitate with each enzyme. Preliminary results show LDH uses NAD-capped RNA as a coenzyme, suggesting that this could be the first study to show NCINs can participate in cellular chemical reactions, and possibly form a new class of key ribonucleoproteins or ribozymes.