Fluorescent Turn-On Probes for the Development of Binding and Hydrolytic Activity Assays for mRNA Cap-Recognizing Proteins

Chemistry : a European Journal
Renata KasprzykJacek Jemielity

Abstract

The m7 G cap is a unique nucleotide structure at the 5'-end of all eukaryotic mRNAs. The cap specifically interacts with numerous cellular proteins and participates in biological processes that are essential for cell growth and function. To provide small molecular probes to study important cap-recognizing proteins, we synthesized m7 G nucleotides labeled with fluorescent tags via the terminal phosph(on)ate group and studied how their emission properties changed upon protein binding or enzymatic cleavage. Only the pyrene-labeled compounds behaved as sensitive turn-on probes. A pyrene-labeled m7 GTP analogue showed up to eightfold enhanced fluorescence emission upon binding to eukaryotic translation initiation factor 4E (eIF4E) and over 30-fold enhancement upon cleavage by decapping scavenger (DcpS) enzyme. These observations served as the basis for developing binding- and hydrolytic-activity assays. The assay utility was validated with previously characterized libraries of eIF4E ligands and DcpS inhibitors. The DcpS assay was also applied to study hydrolytic activity and inhibition of endogenous enzyme in cytoplasmic extracts from HeLa and HEK cells.

References

Mar 15, 1996·International Journal of Cancer. Journal International Du Cancer·C G KevilJ S Alexander
Apr 30, 1998·Current Opinion in Cell Biology·N Sonenberg, A C Gingras
Feb 26, 1999·Archives of Otolaryngology--head & Neck Surgery·S FranklinC A Nathan
Aug 29, 2002·The EMBO Journal·Hudan LiuMegerditch Kiledjian
Apr 8, 2003·Combinatorial Chemistry & High Throughput Screening·David M Jameson, John C Croney
Apr 28, 2004·Bioorganic & Medicinal Chemistry Letters·Amarnath NatarajanJose A Halperin
Aug 25, 2004·Analytical Biochemistry·Zaneta Nikolovska-ColeskaShaomeng Wang
Oct 20, 2004·Biochemistry·Anna NiedzwieckaRyszard Stolarski
Apr 27, 2005·Chembiochem : a European Journal of Chemical Biology·Chikara Dohno, Isao Saito
Oct 27, 2005·Nucleosides, Nucleotides & Nucleic Acids·Marcin KalekEdward Darzynkiewicz
Jan 25, 2006·Bioorganic & Medicinal Chemistry·Marcin KalekEdward Darzynkiewicz
Dec 8, 2006·Journal of Medicinal Chemistry·Brian K Shoichet
Apr 24, 2007·Journal of Medicinal Chemistry·Brian Y FengChristopher P Austin
Aug 21, 2007·Analytical Biochemistry·Mark O Palmier, Steven R Van Doren
Oct 5, 2007·Methods in Enzymology·Anna NiedzwieckaRyszard Stolarski
Dec 7, 2007·Genes & Development·Hans-Guido WendelScott W Lowe
Feb 5, 2008·Cancer Research·Jeremy R GraffEric G Marcusson
Oct 9, 2008·ACS Chemical Biology·Jasbir SinghMark E Gurney
Nov 26, 2009·Nucleic Acids Research·Yi-Jiun ChenSheng-Chung Lee
Aug 4, 2010·Proceedings of the National Academy of Sciences of the United States of America·Luc FuricNahum Sonenberg
Mar 5, 2011·Nature Protocols·Ana M Rossi, Colin W Taylor
Aug 5, 2011·Assay and Drug Development Technologies·Moran Jerabek-WillemsenStefan Duhr
Oct 1, 2011·Wiley Interdisciplinary Reviews. RNA·Ivan TopisirovicAaron J Shatkin
Jan 14, 2012·Angewandte Chemie·Toshikazu OnoEric T Kool
Mar 7, 2012·Journal of Biomolecular Screening·Carlo ViscoPaola Magnaghi
Jun 4, 2013·Human Molecular Genetics·James P Van MeerbekeCharlotte J Sumner
Jun 6, 2013·Human Molecular Genetics·Rocky G GogliottiChristine J DiDonato
Jun 26, 2013·Future Medicinal Chemistry·Marcin ZiemniakJacek Jemielity
Jul 23, 2013·Nature Protocols·Christian WürthUte Resch-Genger
Aug 26, 2014·Assay and Drug Development Technologies·Jonathan J CherryJill Jarecki
Sep 23, 2014·Nature Reviews. Drug Discovery·Ugur SahinÖzlem Türeci

❮ Previous
Next ❯

Citations

Sep 24, 2020·Proceedings. Biological Sciences·C Marondedze
Apr 8, 2020·Chemistry : a European Journal·Renata KasprzykJacek Jemielity
May 22, 2019·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Michal KopcialJacek Jemielity

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.