Synthesis of 5′-NAD-Capped RNA

Höfer, Katharina; Abele, Florian; Schlotthauer, Jasmin; Jäschke, Andres

doi:10.1021/acs.bioconjchem.6b00072

Cited by 27 publications

(32 citation statements)

References 44 publications

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“…5’-NUD, 5’-NGD or 5’-NCD-RNA were prepared by reaction of nicotinamide riboside phosphoimidazolide with 5’- 32 P-phophorylated RNA 32 . Remaining 5’- 32 P-RNA was removed by XRN-1 digest.…”

Section: Methodsmentioning

confidence: 99%

Structure and function of the bacterial decapping enzyme NudC

et al. 2016

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RNA capping and decapping are thought to be distinctive features of eukaryotes. Recently, the redox cofactor NAD was discovered to be attached to small regulatory RNAs in bacteria in a cap-like manner, and Nudix hydrolase NudC was found to act as a NAD decapping enzyme in vitro and in vivo. Here, crystal structures of Escherichia coli NudC in complex with substrate NAD and with cleavage product NMN reveal the catalytic residues lining the binding pocket and principles underlying molecular recognition of substrate and product. Biochemical mutation analysis identifies the conserved Nudix motif as the catalytic center of the enzyme, which needs to be homodimeric as the catalytic pocket is composed of amino acids from both monomers. NudC is single-strand specific and has a purine preference for the 5’-terminal nucleotide. The enzyme strongly prefers NAD-RNA over NAD and binds to a diverse set of cellular RNAs in an unspecific manner.

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Section: Methodsmentioning

confidence: 99%

Structure and function of the bacterial decapping enzyme NudC

et al. 2016

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“…The 8-substituted adenosine monophosphates were synthesized based on published protocols [28,29] with slight modifications: Final phosphoanhydride formation was accomplished via phosphate activation of nicotinamide mononucleotide (NMN) with 1,1'-carbonyldiimidazole (CDI) resulting in the corresponding 5'-phosphorimidazolide, Im-NMN [30]. Phosphorimidazolides are commonly used and show coupling efficiencies similar to morpholidates [31].…”

Section: Synthesis Of 8-(furan-2-yl)-nadmentioning

confidence: 99%

“…5'-NAD-RNAs (RNA-1b -RNA-4b, see Table S1) were prepared using previously reported phosphorimidazolide coupling with Im-NMN with the free phosphate of 5'-P-RNA in aqueous solution in the presence of MgCl 2 (50-100 mM) at 50 • C [30]. 5'-P-RNA (10-100 nmol, 50-500 µM) prepared by solid-phase synthesis was incubated in the presence of a 100-500-fold excess of Im-NMN (50 mM, 1 mg per 50 µL reaction volume) and 50 Mm MgCl 2 for 2 h at 50 • C. The concentration of Im-NMN should be kept constant, using 1 mg/50 µL reaction volume, as higher concentrations did not lead to a higher conversion.…”

Section: Synthesis Of 5'-fur Nad-rnasmentioning

confidence: 99%

A Novel NAD-RNA Decapping Pathway Discovered by Synthetic Light-Up NAD-RNAs

et al. 2020

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The complexity of the transcriptome is governed by the intricate interplay of transcription, RNA processing, translocation, and decay. In eukaryotes, the removal of the 5’-RNA cap is essential for the initiation of RNA degradation. In addition to the canonical 5’-N7-methyl guanosine cap in eukaryotes, the ubiquitous redox cofactor nicotinamide adenine dinucleotide (NAD) was identified as a new 5’-RNA cap structure in prokaryotic and eukaryotic organisms. So far, two classes of NAD-RNA decapping enzymes have been identified, namely Nudix enzymes that liberate nicotinamide mononucleotide (NMN) and DXO-enzymes that remove the entire NAD cap. Herein, we introduce 8-(furan-2-yl)-substituted NAD-capped-RNA (FurNAD-RNA) as a new research tool for the identification and characterization of novel NAD-RNA decapping enzymes. These compounds are found to be suitable for various enzymatic reactions that result in the release of a fluorescence quencher, either nicotinamide (NAM) or nicotinamide mononucleotide (NMN), from the RNA which causes a fluorescence turn-on. FurNAD-RNAs allow for real-time quantification of decapping activity, parallelization, high-throughput screening and identification of novel decapping enzymes in vitro. Using FurNAD-RNAs, we discovered that the eukaryotic glycohydrolase CD38 processes NAD-capped RNA in vitro into ADP-ribose-modified-RNA and nicotinamide and therefore might act as a decapping enzyme in vivo. The existence of multiple pathways suggests that the decapping of NAD-RNA is an important and regulated process in eukaryotes.

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“…Somewhat surprisingly, exploring the 'middle ground'-which is the efficient synthesis of short capped oligonucleotides in high purity-still poses a challenge both for chemistry and biology. Short m 7 GpppRNAs, m 3 GpppRNAs, and NAD-RNAs have been synthesized by chemical reaction of RNA-5 0 phosphates with appropriate imidazole-activated nucleotides under aqueous conditions [109][110][111][112], but these reactions are only moderately efficient and require timeconsuming chromatographic and/or enzymatic work-ups to separate capped and uncapped RNAs. An improvement in solution synthesis of capped oligomers has been proposed by using a 4,4 0 -dimethoxytrityl (DMT) group as a lipophilic purification handle, which facilitates separation of capped RNAs from uncapped RNAs by RP HPLC [113].…”

Section: Phosphate Modifications For Chemical Capping Approachesmentioning

confidence: 99%

Applications of Phosphate Modification and Labeling to Study (m)RNA Caps

Warmiński

Sikorski

Kowalska

et al. 2017

Phosphate Labeling and Sensing in Chemical Biology

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The cap is a natural modification present at the 5 0 ends of eukaryotic messenger RNA (mRNA), which because of its unique structural features, mediates essential biological functions during the process of gene expression. The core structural feature of the mRNA cap is an N7-methylguanosine moiety linked by a 5 0 -5 0 triphosphate chain to the first transcribed nucleotide. Interestingly, other RNA 5 0 end modifications structurally and functionally resembling the m 7 G cap have been discovered in different RNA types and in different organisms. All these structures contain the 'inverted' 5 0 -5 0 oligophosphate bridge, which is necessary for interaction with specific proteins and also serves as a cleavage site for phosphohydrolases regulating RNA turnover. Therefore, cap analogs containing oligophosphate chain modifications or carrying spectroscopic labels attached to phosphate moieties serve as attractive molecular tools for studies on RNA metabolism and modification of natural RNA properties. Here, we review chemical, enzymatic, and chemoenzymatic approaches that enable preparation of modified cap structures and RNAs carrying such structures, with emphasis on phosphate-modified mRNA cap analogs and their potential applications.M. Warminski and P. J. Sikorski have contributed equally to this work.

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Synthesis of 5′-NAD-Capped RNA

Cited by 27 publications

References 44 publications

Structure and function of the bacterial decapping enzyme NudC

Structure and function of the bacterial decapping enzyme NudC

A Novel NAD-RNA Decapping Pathway Discovered by Synthetic Light-Up NAD-RNAs

Applications of Phosphate Modification and Labeling to Study (m)RNA Caps

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