Enzymatically stable 5′ mRNA cap analogs: Synthesis and binding studies with human DcpS decapping enzyme

Kalek, Marcin; Jemielity, Jacek; Darżynkiewicz, Zbigniew; Bojarska, Elzbieta; Stępiński, Janusz; Stolarski, Ryszard; Davis, Richard E.; Darżynkiewicz, Edward

doi:10.1016/j.bmc.2005.12.045

Cited by 49 publications

(76 citation statements)

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“…Hydrolysis of other cap analogs was monitored using a fluorimetric method, in which the increase of fluorescence intensity as a result of enzymatic cleavage of pyrophosphate bond in dinucleotides was recorded as previously described (Kalek et al 2006). The initial rate of hydrolysis was calculated by linear regression of substrate concentration versus time.…”

Section: Enzymatic Digestion Of Cap Analogs With Hit-45mentioning

confidence: 99%

Identification of the HIT-45 protein from Trypanosoma brucei as an FHIT protein/dinucleoside triphosphatase: Substrate specificity studies on the recombinant and endogenous proteins

Banerjee¹,

Palenchar²,

Łukaszewicz³

et al. 2009

RNA

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A new member of the FHIT protein family, designated HIT-45, has been identified in the African trypanosome Trypanosoma brucei. Recombinant HIT-45 proteins were purified from trypanosomal and bacterial protein expression systems and analyzed for substrate specificity using various dinucleoside polyphosphates, including those that contain the 59-mRNA cap, i.e., m 7 GMP. This enzyme exhibited typical dinucleoside triphosphatase activity (EC 3.6.1.29), having its highest specificity for diadenosine triphosphate (ApppA). However, the trypanosome enzyme contains a unique amino-terminal extension, and hydrolysis of cap dinucleotides with monomethylated guanosine or dimethylated guanosine always yielded m 7 GMP (or m 2,7 GMP) as one of the reaction products. Interestingly, m U, that occurs in these organisms.

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Section: Enzymatic Digestion Of Cap Analogs With Hit-45mentioning

confidence: 99%

Identification of the HIT-45 protein from Trypanosoma brucei as an FHIT protein/dinucleoside triphosphatase: Substrate specificity studies on the recombinant and endogenous proteins

Banerjee¹,

Palenchar²,

Łukaszewicz³

et al. 2009

RNA

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“…Analogs of this type were shown to bind human DcpS specifically and to be resistant to hydrolysis by this protein (Kalek et al 2006 GpCH 2 pp-Sepharose (2) (Fig. 1A) was performed using the strategy described in Knorre et al (1976) and Webb et al (1984).…”

Section: Introductionmentioning

confidence: 99%

“…1A) was performed using the strategy described in Knorre et al (1976) and Webb et al (1984). m 7 GpCH 2 pp (8) was obtained using a methodology previously devised by our group (Kalek et al 2005a(Kalek et al , 2006. The bisphosphonate moiety was introduced into the nucleotide by direct phosphonylation of guanosine with methylenebis(phosphonic dichloride) in trimethyl phosphate as the solvent (Kalek et al 2005b).…”

Section: Introductionmentioning

confidence: 99%

“…The resultant GpCH 2 p (5) was coupled to another phosphate moiety using a two-step procedure. It involved activation of the nucleoside 59-bisphosphonate with an imidazole exploiting the 2,29-dithiodipyridine/triphenylphosphine activation system (Mukaiyama and Hashimoto 1972), followed by actual coupling of the obtained imidazole derivative (6) to bis(triethylammonium) phosphate in the presence of ZnCl 2 (Kadokura et al 1997;Stepinski et al 2001;Jemielity et al 2003;Kalek et al 2006). Subsequent methylation of the intermediate 7 with CH 3 I produced the final cap analog (8).…”

Section: Introductionmentioning

confidence: 99%

“…The introduction of a hydrophobic six-carbon atom chain with primary aliphatic amino group into nucleotide molecule changed its physicochemical properties, but did not significantly affect its reactivity in further synthetic steps, including conversion to the imidazole derivative, coupling to another nucleotide moiety, and immobilization on Sepharose. Dinucleotides 18 and 19 were obtained in a ZnCl 2 -catalyzed coupling reaction of 2-nt subunits, in which one subunit was activated as P-imidazolide (Mukaiyama and Hashimoto 1972;Kadokura et al 1997;Stepinski et al 2001;Jemielity et al 2003;Kalek et al 2006). The reaction of 13 with imidazole led to a moderately contaminated imidazolide derivative, which could not GTP-Sepharose are from Webb et al (1984).…”

Section: Introductionmentioning

confidence: 99%

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Affinity resins containing enzymatically resistant mRNA cap analogs—a new tool for the analysis of cap-binding proteins

Szczepaniak¹,

Zuberek²,

Darżynkiewicz³

et al. 2012

RNA

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Cap-binding proteins have been routinely isolated using m 7 GTP-Sepharose; however, this resin is inefficient for proteins such as DcpS (scavenger decapping enzyme), which interacts not only with the 7-methylguanosine, but also with the second cap base. In addition, DcpS purification may be hindered by the reduced resin capacity due to the ability of DcpS to hydrolyze m 7 GTP. Here, we report the synthesis of new affinity resins, m 7 GpCH 2 pp-and m 7 GpCH 2 ppA-Sepharoses, with attached cap analogs resistant to hydrolysis by DcpS. Biochemical tests showed that these matrices, as well as a hydrolyzable m 7 GpppA-Sepharose, bind recombinant mouse eIF4E specifically and at high capacity. In addition, purification of cap-binding proteins from yeast extracts confirmed the presence of all expected cap-binding proteins, including DcpS in the case of m 7 GpCH 2 pp-and m 7 GpCH 2 ppA-Sepharoses. In contrast, binding studies in vitro demonstrated that recombinant human DcpS efficiently bound only m 7 GpCH 2 ppA-Sepharose. Our data prove the applicability of these novel resins, especially m 7 GpCH 2 ppA-Sepharose, in biochemical studies such as the isolation and identification of cap-binding proteins from different organisms.

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Covalent Chemical 5′‐Functionalization of RNA with Diazo Reagents

Gampe¹,

Hollis-Symynkywicz²,

Zécri³

2016

Angewandte Chemie

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Functionalization of RNAa tt he 5'-terminus is important for analytical and therapeutic purposes.C urrently, these RNAs are synthesized de novo starting with achemically functionalized5 '-nucleotide,w hich is incorporated into RNA using chemical synthesis or biochemical techniques.M ethods for direct chemical modification of native RNAwould provide an attractive alternative but are currently underexplored. Herein, we report that diazoc ompounds can be used to selectively alkylate the 5'-phosphate of ribo(oligo)nucleotides to give RNAl abelled through an ative phosphate ester bond. We applied this method to functionalize oligonucleotides with biotin and an orthosteric inhibitor of the eukaryotic initiation factor 4E (eIF4E), an enzyme involved in mRNArecognition. The modified RNAbinds to eIF4E, demonstrating the utility of this labelling technique to modulate biological activity of RNA. This method complements existing techniques and may be used to chemically introduce abroad range of functional handles at the 5'-end of RNA.

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Enzymatically stable 5′ mRNA cap analogs: Synthesis and binding studies with human DcpS decapping enzyme

Cited by 49 publications

References 30 publications

Identification of the HIT-45 protein from Trypanosoma brucei as an FHIT protein/dinucleoside triphosphatase: Substrate specificity studies on the recombinant and endogenous proteins

Identification of the HIT-45 protein from Trypanosoma brucei as an FHIT protein/dinucleoside triphosphatase: Substrate specificity studies on the recombinant and endogenous proteins

Affinity resins containing enzymatically resistant mRNA cap analogs—a new tool for the analysis of cap-binding proteins

Covalent Chemical 5′‐Functionalization of RNA with Diazo Reagents

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