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Direct High‐Throughput Screening Assay for mRNA Cap Guanine‐N7 Methyltransferase Activity
Author(s) -
Kasprzyk Renata,
Fido Mateusz,
Mamot Adam,
Wanat Przemyslaw,
Smietanski Miroslaw,
Kopcial Michal,
Cowling Victoria H.,
Kowalska Joanna,
Jemielity Jacek
Publication year - 2020
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.202001036
Subject(s) - methyltransferase , linker , guanine , rna , messenger rna , chemistry , substrate (aquarium) , high throughput screening , biochemistry , microbiology and biotechnology , biology , methylation , nucleotide , dna , gene , ecology , computer science , operating system
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.