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Methods in Molecular Biology (Clifton,... 2021A lariat cap is a naturally occurring substitute of a conventional mRNA cap and is found in a particular genomic setting in a few eukaryotic microorganisms. It is...
A lariat cap is a naturally occurring substitute of a conventional mRNA cap and is found in a particular genomic setting in a few eukaryotic microorganisms. It is installed by the lariat capping ribozyme acting in cis. In principle, any RNA molecule in any organism can be equipped with a lariat cap in vivo when expressed downstream of a lariat capping ribozyme. Lariat capping is thus a versatile tool for studying the importance of the 5' end structure of RNA molecules. In this chapter, we present protocols to validate the presence of the lariat cap and measure the efficiency of in vivo cleavage by the lariat capping ribozyme.
Topics: Blotting, Northern; Dinucleoside Phosphates; Electrophoresis, Polyacrylamide Gel; Exonucleases; Flow Cytometry; Internal Ribosome Entry Sites; Models, Molecular; Nucleic Acid Conformation; RNA Caps; RNA, Catalytic; RNA, Messenger; Yeasts
PubMed: 32712925
DOI: 10.1007/978-1-0716-0716-9_15 -
Organic & Biomolecular Chemistry Feb 2011P(1),P(2)-Diimidazolyl derivatives of pyrophosphate and halomethylene-bis-phosphonates have been synthesized and characterized, and the mechanism of their formation was...
P(1),P(2)-Diimidazolyl derivatives of pyrophosphate and halomethylene-bis-phosphonates have been synthesized and characterized, and the mechanism of their formation was studied. These reagents enable synthesis of dinucleoside tetraphosphates and tetraphosphonates conveniently and in high yields.
Topics: Cyclization; Dinucleoside Phosphates; Diphosphates; Imidazoles; Molecular Structure; Organophosphonates
PubMed: 21082127
DOI: 10.1039/c0ob00542h -
Chembiochem : a European Journal of... Mar 2021Cyclic dinucleotide signaling systems, which are found ubiquitously throughout nature, allow organisms to rapidly and dynamically sense and respond to alterations in...
Cyclic dinucleotide signaling systems, which are found ubiquitously throughout nature, allow organisms to rapidly and dynamically sense and respond to alterations in their environments. In recent years, the second messenger, cyclic di-(3',5')-adenosine monophosphate (c-di-AMP), has been identified as an essential signaling molecule in a diverse array of bacterial genera. We and others have shown that defects in c-di-AMP homeostasis result in severe physiological defects and virulence attenuation in many bacterial species. Despite significant advancements in the field, there is still a major gap in the understanding of the environmental and cellular factors that influence c-di-AMP dynamics due to a lack of tools to sensitively and rapidly monitor changes in c-di-AMP levels. To address this limitation, we describe here the development of a luciferase-based coupled enzyme assay that leverages the cyclic nucleotide phosphodiesterase, CnpB, for the sensitive and high-throughput quantification of 3'3'-c-di-AMP. We also demonstrate the utility of this approach for the quantification of the cyclic oligonucleotide-based anti-phage signaling system (CBASS) effector, 3'3'-cGAMP. These findings establish CDA-Luc as a more affordable and sensitive alternative to conventional c-di-AMP detection tools with broad utility for the study of bacterial cyclic dinucleotide physiology.
Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Adenosine Monophosphate; Bacteria; Bacterial Proteins; Dinucleoside Phosphates; Enzyme Assays; High-Throughput Screening Assays; Hydrolysis; Luciferases; Luminescent Measurements; Mycobacterium tuberculosis
PubMed: 33142009
DOI: 10.1002/cbic.202000667 -
Analytical Biochemistry May 2003
Comparative Study
Topics: Chemistry Techniques, Analytical; Chromatography, Liquid; Dinucleoside Phosphates; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization; Time Factors
PubMed: 12694737
DOI: 10.1016/s0003-2697(03)00035-6 -
Journal of Chromatography. B,... May 2005In former studies, dinucleoside polyphosphates were quantified using ion-pair reversed-phase perfusion chromatography columns, which allows a detection limit in the...
In former studies, dinucleoside polyphosphates were quantified using ion-pair reversed-phase perfusion chromatography columns, which allows a detection limit in the micromolar range. The aim of this study was both to describe a chromatographic assay with an increased efficiency of the dinucleoside separation, which enables the reduction of analytical run times, and to establish a chromatographic assay using conditions, which allow MALDI-mass spectrometric analysis of the resulting fractions. We compared the performance of conventional silica reversed phase chromatography columns, a perfusion chromatography column and a monolithic reversed-phase C18 chromatography column. The effects of different ion-pair reagents, flow-rates and gradients on the separation of synthetic diadenosine polyphosphates as well as of diadenosine polyphosphates isolated from human platelets were analysed. Sensitivity and resolution of the monolithic reversed-phase chromatography column were both higher than that of the perfusion chromatography and the conventional reversed phase chromatography columns. Using a monolithic reversed-phase C18 chromatography column, diadenosine polyphosphates were separable baseline not only in the presence of tetrabutylammonium hydrogensulfate (TBA) but also in the presence of triethylammonium acetate (TEAA) as ion-pair reagent. The later reagent is useful because, in contrast to TBA, it is compatible with MALDI mass-spectrometric methods. This makes TEAA particularly suitable for identification of unknown nucleoside polyphosphates. Furthermore, because of the lower backpressure of monolithic reversed-phase chromatography columns, we were able to significantly increase the flow rate, decreasing the amount of time for the analysis close to 50%, especially using TBA as ion-pair reagent. In summary, monolithic reversed phase C18 columns markedly increase the sensitivity and resolution of dinucleoside polyphosphate analysis in a time-efficient manner compared to reversed-phase perfusion chromatography columns or conventional reversed-phase columns. Therefore, further dinucleoside polyphosphate analytic assays should be based on monolithic silica C18 columns instead of perfusion chromatography or conventional silica reversed phase chromatography columns. In conclusion, the use of monolithic silica C18 columns will lead to isolation and quantification of up to now unknown dinucleoside polyphosphates. These chromatography columns may facilitate further research on the biological roles of dinucleoside polyphosphates.
Topics: Blood Platelets; Chromatography, Affinity; Chromatography, High Pressure Liquid; Dinucleoside Phosphates; Humans; Quaternary Ammonium Compounds; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tetraethylammonium
PubMed: 15797530
DOI: 10.1016/j.jchromb.2005.01.038 -
Protein Science : a Publication of the... Mar 2023Cyclic-di-nucleotide-based secondary messengers regulate various physiological functions, including stress responses in bacteria. Cyclic diadenosine monophosphate...
Cyclic-di-nucleotide-based secondary messengers regulate various physiological functions, including stress responses in bacteria. Cyclic diadenosine monophosphate (c-di-AMP) has recently emerged as a crucial second messenger with implications in processes including osmoregulation, antibiotic resistance, biofilm formation, virulence, DNA repair, ion homeostasis, and sporulation, and has potential therapeutic applications. The contrasting activities of the enzymes diadenylate cyclase (DAC) and phosphodiesterase (PDE) determine the equilibrium levels of c-di-AMP. Although c-di-AMP is suspected of playing an essential role in the pathophysiology of bacterial infections and in regulating host-pathogen interactions, the mechanisms of its regulation remain relatively unexplored in mycobacteria. In this report, we biochemically and structurally characterize the c-di-AMP synthase (MsDisA) from Mycobacterium smegmatis. The enzyme activity is regulated by pH and substrate concentration; conditions of significance in the homoeostasis of c-di-AMP levels. Substrate binding stimulates conformational changes in the protein, and pApA and ppApA are synthetic intermediates detectable when enzyme efficiency is low. Unlike the orthologous Bacillus subtilis enzyme, MsDisA does not bind to, and its activity is not influenced in the presence of DNA. Furthermore, we have determined the cryo-EM structure of MsDisA, revealing asymmetry in its structure in contrast to the symmetric crystal structure of Thermotoga maritima DisA. We also demonstrate that the N-terminal minimal region alone is sufficient and essential for oligomerization and catalytic activity. Our data shed light on the regulation of mycobacterial DisA and possible future directions to pursue.
Topics: Mycobacterium smegmatis; Bacterial Proteins; Dinucleoside Phosphates; Bacillus subtilis
PubMed: 36660887
DOI: 10.1002/pro.4568 -
Journal of the American Chemical Society Aug 2004The putative human tumor suppressor gene FHIT encodes Fhit, the fragile histidine triad protein. Fhit is thought to participate in a signal transduction pathway...
The putative human tumor suppressor gene FHIT encodes Fhit, the fragile histidine triad protein. Fhit is thought to participate in a signal transduction pathway involving dinucleoside polyphosphates. Fhit catalyzes the Mg2+-dependent hydrolysis of P1-5'-O-adenosine-P3-5'-O-adenosine triphosphate (Ap3A) to AMP and MgADP. Mutation of His96 to glycine disables Fhit as a catalyst for the hydrolysis of phosphoanhydrides such as Ap3A. However, the mutated enzyme H96G-Fhit efficiently catalyzes the synthesis of phosphoanhydride bonds in reactions of nucleoside-5'-phosphimidazolides with nucleoside di- and triphosphates. H96G-Fhit can be employed in the synthesis of a wide range of dinucleoside tri- and tetraphosphates. We here describe the use of H96G-Fhit to catalyze the synthesis of Ap3A, Ap3C, Ap3G, Ap3T, Ap3U, Cp3U, Tp3U, dAp3U, Ap4A, Ap4U, and the fluorescent Ap4etheno-C.
Topics: Acid Anhydride Hydrolases; Catalysis; Dinucleoside Phosphates; Humans; Kinetics; Neoplasm Proteins; Phosphotransferases (Phosphate Group Acceptor); Protein Engineering
PubMed: 15291552
DOI: 10.1021/ja0400640 -
European Journal of Biochemistry May 1999T4 RNA ligase has been shown to synthesize nucleoside and dinucleoside 5'-polyphosphates by displacement of the AMP from the E-AMP complex with polyphosphates and...
T4 RNA ligase has been shown to synthesize nucleoside and dinucleoside 5'-polyphosphates by displacement of the AMP from the E-AMP complex with polyphosphates and nucleoside diphosphates and triphosphates. Displacement of the AMP by tripolyphosphate (P3) was concentration dependent, as measured by SDS/PAGE. When the enzyme was incubated in the presence of 0.02 mm [alpha-32P] ATP, synthesis of labeled Ap4A was observed: ATP was acting as both donor (Km, microm) and acceptor (Km, mm) of AMP from the enzyme. Whereas, as previously known, ATP or dATP (but not other nucleotides) were able to form the E-AMP complex, the specificity of a compound to be acceptor of AMP from the E-AMP complex was very broad, and with Km values between 1 and 2 mm. In the presence of a low concentration (0.02 mm) of [alpha-32P] ATP (enough to form the E-AMP complex, but only marginally enough to form Ap4A) and 4 mm of the indicated nucleotides or P3, the relative rate of synthesis of the following radioactive (di)nucleotides was observed: Ap4X (from XTP, 100); Ap4dG (from dGTP, 74); Ap4G (from GTP, 49); Ap4dC (from dCTP, 23); Ap4C (from CTP, 9); Ap3A (from ADP, 5); Ap4ddA, (from ddATP, 1); p4A (from P3, 200). The enzyme also synthesized efficiently Ap3A in the presence of 1 mm ATP and 2 mm ADP. The following T4 RNA ligase donors were inhibitors of the synthesis of Ap4G: pCp > pAp > pA2'p.
Topics: Adenosine Monophosphate; Bacteriophage T4; Catalysis; Dinucleoside Phosphates; Hydrogen-Ion Concentration; RNA Ligase (ATP); Substrate Specificity
PubMed: 10215898
DOI: 10.1046/j.1432-1327.1999.00338.x -
Justus Liebigs Annalen Der Chemie Mar 1965
Topics: Chemical Phenomena; Chemistry; Dinucleoside Phosphates; Nucleosides; Nucleotides; Oligonucleotides; Research; Ribose
PubMed: 14311888
DOI: 10.1002/jlac.19656830124 -
Chemistry & Biodiversity Sep 2017Protected dinucleoside-2',5'-monophosphate has been prepared to develop a prodrug strategy for 2-5A. The removal of enzymatically and thermally labile...
Protected dinucleoside-2',5'-monophosphate has been prepared to develop a prodrug strategy for 2-5A. The removal of enzymatically and thermally labile 4-(acetylthio)-2-(ethoxycarbonyl)-3-oxo-2-methylbutyl phosphate protecting group and enzymatically labile 3'-O-pivaloyloxymethyl group was followed at pH 7.5 and 37 °C by HPLC from the fully protected dimeric adenosine-2',5'-monophosphate 1 used as a model compound for 2-5A. The desired unprotected 2',3'-O-isopropylideneadenosine-2',5'-monophosphate (9) was observed to accumulate as a major product. Neither the competitive isomerization of 2',5'- to a 3',5'-linkage nor the P-O5' bond cleavage was detected. The phosphate protecting group was removed faster than the 3'-O-protection and, hence, the attack of the neighbouring 3'-OH on phosphotriester moiety did not take place.
Topics: Adenosine Monophosphate; Chromatography, High Pressure Liquid; Dimerization; Dinucleoside Phosphates; Prodrugs
PubMed: 28557349
DOI: 10.1002/cbdv.201700220