Nicotinamide adenine dinucleotide (NAD) serves as an essential redox co-factor and mediator of multiple biological processes. Besides its well-established role in electron transfer reactions, NAD serves as a substrate for other biotransformations, which, at the molecular level, can be classified as protein post-translational modifications (protein deacylation, mono-, and polyADP-ribosylation) and formation of signaling molecules (e.g., cyclic ADP ribose). These biochemical reactions control many crucial biological processes, such as cellular signaling and recognition, DNA repair and epigenetic modifications, stress response, immune response, aging and senescence, and many others. However, the links between the biological effects and underlying molecular processes are often poorly understood. Moreover, NAD has recently been found to tag the 5′-ends of some cellular RNAs, but the function of these NAD-capped RNAs remains largely unrevealed. Synthetic NAD analogs are invaluable molecular tools to detect, monitor, structurally investigate, and modulate activity of NAD-related enzymes and biological processes in order to aid their deeper understanding. Here, we review the recent advances in the design and development of NAD analogs as probes for various cellular NAD-related enzymes, enzymatic inhibitors with anticancer or antimicrobial therapeutic potential, and other NAD-related chemical biology tools. We focus on research papers published within the last 10 years.

SUMMARY: Cells organize biochemical processes into biological condensates. P-bodies are cytoplasmic condensates enriched in factors important for mRNA degradation. P-bodies have been identified as sites of both mRNA storage and decay, but how these opposing outcomes may be achieved in condensates is unresolved. A critical step in mRNA degradation is removal of the 5’-7-methylguanosine cap by Dcp1/Dcp2, which is highly enriched in P-bodies. Dcp1/Dcp2 activity is repressed in condensates in vitro and requires the activator Edc3. Activation of decapping is amplified in condensates relative to the surrounding solution due to stabilization of an autoinhibited state in Dcp1/Dcp2. Edc3 couples a conformational change in the Dcp1/Dcp2 active site with alteration of the protein-protein interactions driving phase separation to activate decapping in condensates. The composition-dependent regulation of enzyme activity in condensates occurs over length scales ranging from microns to Ångstroms and may control the functional state of P-bodies and related phase-separated compartments. HIGHLIGHTS: mRNA decapping in droplets is repressed Catalytically inert droplets are activated by a change in condensate composition A switch in enzymatic activity requires a conformational change in condensates Condensates amplify enzyme activation compared to surrounding solution

IFITs (interferon-induced proteins with tetratricopeptide repeats) are components of the innate immune response that bind to viral and cellular RNA targets to inhibit translation and replication. The RNA target recognition is guided by molecular patterns, particularly at the RNA 5′ ends. IFIT1 preferably binds RNAs modified with the m⁷G cap-0 structure, while RNAs with cap-1 structure are recognized with lower affinity. Less is known about the propensity of IFIT1 to recognize noncanonical RNA 5′ ends, including hypermethylated and noncanonical RNA caps. Further insights into the structure-function relationship for IFIT1–RNA interactions are needed but require robust analytical methods. Here, we report a biophysical assay for quick, direct, in-solution affinity assessment of differently capped RNAs with IFIT1. The procedure, which relies on measuring microscale thermophoresis of fluorescently labeled protein as a function of increasing ligand concentration, is applicable to RNAs of various lengths and sequences without the need for their labeling or affinity tagging. Using the assay, we examined 13 canonically and noncanonically 5′-capped RNAs, revealing new binding preferences of IFIT1. The 5′ terminal m⁶A mark in the m⁷G cap had a protective function against IFIT1, which was additive with the effect observed for the 2′-Oposition (m⁶A_mcap-1). In contrast, an increased affinity for IFIT1 was observed for several noncanonical caps, including trimethylguanosine, unmethylated (G), and flavin-adenine dinucleotide caps. The results suggest new potential cellular targets of IFIT1 and may contribute to broadening the knowledge of the innate immune response mechanisms and the more effective design of chemically modified mRNAs.

We have focused our attention on alternative synthetic approaches for nucleotides and dinucleotides, that simplifies purification and/or improves sustainability. On one hand, we developed a liquid-phase synthesis using a benzene-centred tripodal support to prepare adenine nucleoside phosphoesters and methylenebisphosphonate derivatives. On the other hand, we used mechanochemistry, a technique that enables solid/solid reactions through mechanical grinding under solventless or solvent-free conditions, to get access to dinucleotides. Graphical Abstract