In eukaryotes, mature mRNA is formed through modifications of precursor mRNA, one of which is 5’ cap biosynthesis, involving RNA cap guanine‐N7 methyltransferase (N7‐MTase). N7‐MTases are also encoded by some eukaryotic viruses and facilitate their replication. N7‐MTase inhibitors have therapeutic potential, but their discovery is difficult because long RNA substrates are usually required for activity. Herein, we report a universal N7‐MTase activity assay based on small‐molecule fluorescent probes. We synthesized 12 fluorescent substrate analogues (GpppA and GpppG derivatives) varying in the dye type, dye attachment site, and linker length. GpppA labeled with pyrene at the 3’‐O position of adenosine acted as an artificial substrate with the properties of a turn‐off probe for all three tested N7‐MTases (human, parasite, and viral). Using this compound, a N7‐MTase inhibitor assay adaptable to high‐throughput screening was developed and used to screen synthetic substrate analogues and a commercial library. Several inhibitors with nanomolar activities were identified.

The mRNA 5′ cap consists of N7-methylguanosine bound by a 5′,5′-triphosphate bridge to the first nucleotide of the transcript. The cap interacts with various specific proteins and participates in all key mRNA-related processes, which may be of therapeutic relevance. There is a growing demand for new biophysical and biochemical methods to study cap–protein interactions and identify the factors which inhibit them. The development of such methods can be aided by the use of properly designed fluorescent molecular probes. Herein, we synthesized a new class of m7Gp3G cap derivatives modified with an alkyne handle at the N1-position of guanosine and, using alkyne-azide cycloaddition, we functionalized them with fluorescent tags to obtain potential probes. The cap derivatives and probes were evaluated in the context of two cap-binding proteins, eukaryotic translation initiation factor (eIF4E) and decapping scavenger (DcpS). Biochemical and biophysical studies revealed that N1-propargyl moiety did not significantly disturb cap–protein interaction. The fluorescent properties of the probes turned out to be in line with microscale thermophoresis (MST)-based binding assays.

We describe a new type of nucleotide-derived fluorescent probe designed for monitoring pyrophosphatase activity based on excimer-to-monomer transitions, called ExciTide. The nucleotides were designed with two self-interacting dye moieties and synthesised using copper-catalysed azide-alkyne cycloaddition click chemistry. We applied these probes for enzyme activity monitoring and inhibitor evaluation. Some of the probes permeated into living cells, yielding interesting prospects for future applications.