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Journal of the American Chemical Society May 2012We present a (13)C-based isotope labeling protocol for RNA. Using (6-(13)C)pyrimidine phosphoramidite building blocks, site-specific labels can be incorporated into a...
We present a (13)C-based isotope labeling protocol for RNA. Using (6-(13)C)pyrimidine phosphoramidite building blocks, site-specific labels can be incorporated into a target RNA via chemical oligonucleotide solid-phase synthesis. This labeling scheme is particularly useful for studying milli- to microsecond dynamics via NMR spectroscopy, as an isolated spin system is a crucial prerequisite to apply Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion type experiments. We demonstrate the applicability for the characterization and detection of functional dynamics on various time scales by incorporating the (6-(13)C)uridine and -cytidine labels into biologically relevant RNAs. The refolding kinetics of a bistable terminator antiterminator segment involved in the gene regulation process controlled by the preQ(1) riboswitch class I was investigated. Using (13)C CPMG relaxation dispersion NMR spectroscopy, the milli- to microsecond dynamics of the HIV-1 transactivation response element RNA and the Varkud satellite stem loop V motif was addressed.
Topics: Carbon Isotopes; Cytidine; HIV-1; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Nucleic Acid Conformation; Organophosphorus Compounds; Pyrimidine Nucleotides; RNA; RNA, Viral; Solid-Phase Synthesis Techniques; Spin Labels; Uridine
PubMed: 22489874
DOI: 10.1021/ja302148g -
British Journal of Haematology Nov 1979In pyrimidine 5'-nucleotidase deficiency, erythrocytes contain elevated levels of pyrimidine nucleotides. The composition of this nucleotide pool was examined by ion...
In pyrimidine 5'-nucleotidase deficiency, erythrocytes contain elevated levels of pyrimidine nucleotides. The composition of this nucleotide pool was examined by ion exchange chromatography on Dowex formate columns using a linear ammonium formate elution gradient. In contradistinction to normal erythrocytes, adenine nucleotides accounted for only 32% of the nucleotide pool. The remainder consisted of 50% cytidine and 16% uridine nucleotides. The remaining 2% was not identified. The most abundant compound appeared to be UDP glucose whilst high levels of CTP, CMP and an unidentified cytidine compound less polar than CMP accounted for most of the cytidine nucleotide pool. The possibility that the abnormal nucleotides were due to an elevated reticulocyte count was excluded and it was also shown that erythrocytes from subjects heterozygous for pyrimidine 5'-nucleotidase deficiency did not have detectable levels of the abnormal nucleotides.
Topics: Adenosine Triphosphate; Anemia, Hemolytic, Congenital Nonspherocytic; Anemia, Pernicious; Cytidine Diphosphate; Cytidine Monophosphate; Erythrocytes; Humans; Infant; Nucleotidases; Pyrimidine Nucleotides; Uridine Diphosphate; Uridine Monophosphate; Uridine Triphosphate
PubMed: 497119
DOI: 10.1111/j.1365-2141.1979.tb03769.x -
Advances in Pharmacology (San Diego,... 2011Given the widespread importance of purinergic receptor-evoked signaling, understanding how ATP and other nucleotides are released from cells in a regulated manner is an... (Review)
Review
Given the widespread importance of purinergic receptor-evoked signaling, understanding how ATP and other nucleotides are released from cells in a regulated manner is an essential physiological question. Nonlytic release of ATP, UTP, UDP-glucose, and other nucleotides occurs in all cell types and tissues via both constitutive mechanisms, that is, in the absence of external stimuli, and to a greater extent in response to biochemical or mechanical/physical stimuli. However, a molecular understanding of the processes regulating nucleotide release has only recently begun to emerge. It is generally accepted that nucleotide release occurs in two different scenarios, exocytotic release from the secretory pathway or via conductive/transport mechanisms, and a critical review of our current understanding of these mechanisms is presented in this chapter.
Topics: Animals; Cell Survival; Humans; Models, Biological; Purine Nucleotides; Pyrimidine Nucleotides
PubMed: 21586361
DOI: 10.1016/B978-0-12-385526-8.00008-4 -
Nanoscale Jun 2017Designed DNA nanostructures of impressive size have been described, but designed structures of the size of protein enzymes that bind organic ligands with high...
Designed DNA nanostructures of impressive size have been described, but designed structures of the size of protein enzymes that bind organic ligands with high specificity are rare. Here we report a four-helix motif consisting of three synthetic strands with 65 base pairs and 165 nucleotides in total that folds well. Furthermore, we show that in the interior of this small folded DNA nanostructure, cavities can be set up that bind pyrimidine nucleotides with micromolar affinity. Base-specific binding for both thymidine and cytidine derivatives is demonstrated. The binding affinity depends on the position in the structure, as expected for recognition beyond simple base pairing. The folding motif reported here can help to expand DNA nanotechnology into the realm of selective molecular recognition that is currently dominated by protein-based enzymes and receptors.
Topics: DNA; Nanostructures; Nucleic Acid Conformation; Pyrimidine Nucleotides
PubMed: 28327725
DOI: 10.1039/c7nr00094d -
Cardiovascular Drugs and Therapy Sep 1998The metabolism of pyrimidine nucleotides in the myocardium is poorly understood. The turnover of these nucleotides is high, whereas their concentration is rather low.... (Review)
Review
The metabolism of pyrimidine nucleotides in the myocardium is poorly understood. The turnover of these nucleotides is high, whereas their concentration is rather low. The de novo pathway of synthesis does not seem very efficient, although the utilization of nucleosides could represent the major pathway for pyrimidine nucleotide synthesis. In rat blood, cytidine could be the major precursor for pyrimidine nucleotide synthesis. The precursor, whatever its exact nature (uridine or cytidine), could be species dependent, and the liver could a major role in providing blood nucleosides. Owing to the essential role of pyrimidine nucleotides in the synthesis of macromolecules, acute or chronic alteration of the metabolism of these nucleotides could have crucial consequences on heart viability and function. Providing pyrimidine precursors to the heart, isolated or in situ, induces functional and metabolic effects on the heart. The experimental results suggest that such interventions could be beneficial in clinical situations such as cardioplegia, heart preservation, or recovery from ischemia.
Topics: Animals; Humans; Myocardial Ischemia; Myocardium; Pyrimidine Nucleotides; Rats
PubMed: 9794091
DOI: 10.1023/a:1007752700936 -
Expert Opinion on Therapeutic Patents 2023Pyrimidine nucleotides are essential for the parasite's growth and replication. Parasites have only a pathway for the biosynthesis of pyrimidine nucleotides.... (Review)
Review
INTRODUCTION
Pyrimidine nucleotides are essential for the parasite's growth and replication. Parasites have only a pathway for the biosynthesis of pyrimidine nucleotides. Dihydroorotate dehydrogenase (DHODH) enzyme is involved in the rate-limiting step of the pyrimidine biosynthesis pathway. DHODH is a biochemical target for the discovery of new antimalarial agents.
AREA COVERED
This review discussed the development of patented DHODH inhibitors published between 2007 and 2023 along with their chemical structures and activities.
EXPERT OPINION
DHODH enzyme is involved in the rate-limiting fourth step of the pyrimidine biosynthesis pathway. Thus, inhibition of DHODH using species-selective inhibitors has drawn much attention for treating malaria because they inhibit parasite growth without affecting normal human functions. Looking at the current scenario of antimalarial drug resistance with most of the available antimalarial drugs, there is a huge need for targeted newer agents. Newer agents with unique mechanisms of action may be devoid of drug toxicity, adverse effects, and the ability of parasites to quickly gain resistance, and DHODH inhibitors can be those newer agents. Many DHODH inhibitors were patented in the past, and the dependency of on pyrimidine provided a new approach for the development of novel antimalarial agents.
Topics: Humans; Dihydroorotate Dehydrogenase; Antimalarials; Plasmodium falciparum; Oxidoreductases Acting on CH-CH Group Donors; Patents as Topic; Pyrimidines; Enzyme Inhibitors; Pyrimidine Nucleotides
PubMed: 37942637
DOI: 10.1080/13543776.2023.2280596 -
European Journal of Biochemistry Feb 1985Extensive 1H and 15H NMR investigations of the nucleotide moieties capable of hydrogen bonding to ribonuclease A were carried out in order to gain more detailed...
Extensive 1H and 15H NMR investigations of the nucleotide moieties capable of hydrogen bonding to ribonuclease A were carried out in order to gain more detailed information on the specificity of nucleotide-enzyme interaction. The 1H investigations focussed on those protons presumed to be involved in hydrogen bonding between the various nucleotides and the enzyme. In particular these were the imino protons of the uridine nucleotides and the amino protons of the cytidine nucleotides. The technique of 15N-1H double quantum filtering was applied for observation of the resonances of the latter in the nucleotide-enzyme complex. The downfield shift observed for the imino proton resonance of the uridine nucleotides was indicative of hydrogen bond formation to the enzyme. 15N NMR spectra of the free nucleotides and the nucleotide-enzyme complexes were also acquired to examine the possibility of hydrogen bond formation at the N3 site of both pyrimidine bases and the amino group of the cytidine nucleotides. The downfield shift observed for the 15N3 resonance of the uridine nucleotides and the upfield shift observed for the corresponding resonance of the cytidine nucleotides was evidence that the N3 moiety acts as hydrogen donor or hydrogen acceptor in the nucleotide-enzyme complex. The effect of complex formation on the 15N1 resonance of the respective bases was also studied. Both 1H and 15N NMR results indicated subtle differences between the complexes of the 2' and 3' nucleotides. The extent of hydrogen bonding as well as the arrangement of the nucleotide base at the active site of the enzyme varies in dependence on the position of the phosphate group. It is established that hydrogen bonding, though not the main binding force between the nucleotides and the enzyme, is certainly a major factor of RNase A specificity for pyrimidine nucleotides.
Topics: Chemical Phenomena; Chemistry; Hydrogen; Hydrogen Bonding; Magnetic Resonance Spectroscopy; Nitrogen Isotopes; Protein Binding; Pyrimidine Nucleotides; Ribonuclease, Pancreatic
PubMed: 3971970
DOI: 10.1111/j.1432-1033.1985.tb08708.x -
Chembiochem : a European Journal of... Jul 2014Isotope labeling has revolutionized NMR studies of small nucleic acids, but to extend this technology to larger RNAs, site-specific labeling tools to expedite NMR...
Isotope labeling has revolutionized NMR studies of small nucleic acids, but to extend this technology to larger RNAs, site-specific labeling tools to expedite NMR structural and dynamics studies are required. Using enzymes from the pentose phosphate pathway, we coupled chemically synthesized uracil nucleobase with specifically (13) C-labeled ribose to synthesize both UTP and CTP in nearly quantitative yields. This chemoenzymatic method affords a cost-effective preparation of labels that are unattainable by current methods. The methodology generates versatile (13) C and (15) N labeling patterns which, when employed with relaxation-optimized NMR spectroscopy, effectively mitigate problems of rapid relaxation that result in low resolution and sensitivity. The methodology is demonstrated with RNAs of various sizes, complexity, and function: the exon splicing silencer 3 (27 nt), iron responsive element (29 nt), Pro-tRNA (76 nt), and HIV-1 core encapsidation signal (155 nt).
Topics: Molecular Dynamics Simulation; Nuclear Magnetic Resonance, Biomolecular; Pyrimidine Nucleotides; RNA; Stereoisomerism
PubMed: 24954297
DOI: 10.1002/cbic.201402130 -
Acta Microbiologica Et Immunologica... Dec 2014Upon HIV infection, cells become activated and cell surface thiols are present in increased number. Earlier we demonstrated in vitro anti-HIV effect of thiolated...
Upon HIV infection, cells become activated and cell surface thiols are present in increased number. Earlier we demonstrated in vitro anti-HIV effect of thiolated pyrimidine nucleotide UD29, which interferes thiol function. To further analyse the redox processes required for HIV-1 entry and infection, toxicity assays were performed using HIV-1 infected monolayer HeLaCD4-LTR/ β-gal cells and suspension H9 T cells treated with several thiolated nucleotide derivatives of UD29. Selective cytotoxicity of thiolated pyrimidines on HIV-1 infected cells were observed. Results indicate that thiolated pyrimidine derivates may interfere with -SH (thiol) groups concentrated in lipid rafts of cell membrane and interacts HIV-1 infected (activated) cells resulting in a selective cytotoxicity of HIV-1 infected cells, and reducing HIV-1 entry.
Topics: Cell Line; Cell Survival; HIV-1; Humans; Membrane Microdomains; Pyrimidine Nucleotides; Sulfhydryl Compounds
PubMed: 25496973
DOI: 10.1556/AMicr.61.2014.4.6 -
Journal of Plant Physiology Nov 2003Purine and pyrimidine nucleotides participate in many biochemical processes in plants. They are building blocks for nucleic acid synthesis, an energy source, precursors... (Review)
Review
Purine and pyrimidine nucleotides participate in many biochemical processes in plants. They are building blocks for nucleic acid synthesis, an energy source, precursors for the synthesis of primary products, such as sucrose, polysaccharides, phospholipids, as well as secondary products. Therefore, biosynthesis and metabolism of nucleotides are of fundamental importance in the growth and development of plants. Nucleotides are synthesized both from amino acids and other small molecules via de novo pathways, and from preformed nucleobases and nucleosides by salvage pathways. In this article the biosynthesis, interconversion and degradation of purine and pyrimidine nucleotides in higher plants are reviewed. This description is followed by an examination of physiological aspects of nucleotide metabolism in various areas of growth and organized development in plants, including embryo maturation and germination, in vitro organogenesis, storage organ development and sprouting, leaf senescence, and cultured plant cells. The effects of environmental factors on nucleotide metabolism are also described. This review ends with a brief discussion of molecular studies on nucleotide synthesis and metabolism.
Topics: Plant Development; Plants; Purine Nucleotides; Pyrimidine Nucleotides
PubMed: 14658380
DOI: 10.1078/0176-1617-01169