ExciTides: NTP-derived probes for monitoring pyrophosphatase activity based on excimer-to-monomer transitions

Wanat, Przemysław; Kasprzyk, Renata; Kopcial, Michal; Sikorski, Pawel J.; Strzelecka, Dominika; Jemielity, Jacek; Kowalska, Joanna

doi:10.1039/c8cc04968h

Cited by 6 publications

(7 citation statements)

References 30 publications

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“…For the Py-labeled probe 9 , the fluorescence intensity at the emission maximum increased by 50% after complete DcpS cleavage (Figure 4c and Figure S3). Nevertheless, certain previously developed probes provided higher sensitivity than the one formulated here [15,16]. Modest changes in fluorescence emission upon the enzymatic cleavage of compounds 6 – 9 were independently confirmed with the nonspecific pyrophosphatase PDE-I (Figure S4).…”

Section: Resultssupporting

confidence: 58%

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N1-Propargylguanosine Modified mRNA Cap Analogs: Synthesis, Reactivity, and Applications to the Study of Cap-Binding Proteins

et al. 2019

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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.

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Section: Resultssupporting

confidence: 58%

“…Chemically modified cap analogs including fluorescent probes are useful tools for the study of cap-binding proteins. To this end, nucleotides labeled with antibodies [12], fluorescent tags [13,14,15,16], EPR tags [17], and radioisotopes [18] have been developed.…”

Section: Introductionmentioning

confidence: 99%

N1-Propargylguanosine Modified mRNA Cap Analogs: Synthesis, Reactivity, and Applications to the Study of Cap-Binding Proteins

et al. 2019

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“…However this method did not enable monitoring of reaction progress in real‐time and had a lower z factor value (0.63). Recently, we also reported doubly labeled mononucleotide excimer probes for enzymatic studies—including DcpS protein and cell imaging . These probes enabled reaction monitoring using visible light; however, they are structurally more complex and had lower affinities for DcpS than probe 4 b .…”

Section: Discussionmentioning

confidence: 99%

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

Kasprzyk

Starek

Ciechanowicz

et al. 2019

Chemistry A European J

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The m 7 Gc ap is au nique nucleotide structure at the 5'-end of all eukaryotic mRNAs. The cap specificallyinteracts with numerous cellular proteins and participates in biological processes that are essential for cell growth and function. To provide small molecularp robest os tudy important cap-recognizing proteins, we synthesized m 7 Gn ucleotides labeled with fluorescent tags via the terminal phosph(on)ate group and studied how their emission properties changed upon protein binding or enzymaticc leavage. Only the pyrene-labeled compounds behaved as sensitive turn-on probes. Ap yrene-labeled m 7 GTP analogue showed up to eightfold enhanced fluorescencee mission upon binding to eukaryotic translation initiation factor 4E (eIF4E) and over 30-folde nhancementu pon cleavageb yd ecapping scavenger (DcpS) enzyme. These observations serveda st he basis for developing binding-and hydrolytic-activity assays. The assay utility was validated with previously characterizedl ibraries of eIF4E ligands and DcpS inhibitors. The DcpS assay was also appliedtostudy hydrolytic activity and inhibition of endogenous enzyme in cytoplasmic extracts from HeLa and HEK cells.

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“…To this end, a GpppA analogue equipped with an alkyne handle (1)w as synthesized in af ew steps, starting from an appropriate ADP precursor functionalized with propargylamine at the 3'-O position ( Figure S2). P-imidazolideo f( N-propargyl)-3'-carbamoyloadenosine diphosphate [36] was coupled with the triethylammonium Figure 1. The principle of monitoring N7 methylation by using af luorescence-based approach.…”

Section: Synthesismentioning

confidence: 99%

“…The other alkyne-functionalized GpppA and GpppG analogues (2-4)a nd corresponding probes (7-9)w ere synthesized by using as imilara pproach and previously reported functionalization strategies ( Figure S2A, B). [36,37] Additionally,t he product analogue 5 wass ynthesized by N7methylation of compound 1 and functionalized to yield probes 10 a,b ( Figure S2C). The structures of the fluorescent nucleotides were confirmedb yH RMS.…”

Section: Synthesismentioning

confidence: 99%

Direct High‐Throughput Screening Assay for mRNA Cap Guanine‐N7 Methyltransferase Activity

Kasprzyk

Fido

Mamot

et al. 2020

Chemistry A European J

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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.

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ExciTides: NTP-derived probes for monitoring pyrophosphatase activity based on excimer-to-monomer transitions

Cited by 6 publications

References 30 publications

N1-Propargylguanosine Modified mRNA Cap Analogs: Synthesis, Reactivity, and Applications to the Study of Cap-Binding Proteins

N1-Propargylguanosine Modified mRNA Cap Analogs: Synthesis, Reactivity, and Applications to the Study of Cap-Binding Proteins

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

Direct High‐Throughput Screening Assay for mRNA Cap Guanine‐N7 Methyltransferase Activity

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