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Havelund J.F.,University of Southern Denmark | Havelund J.F.,Danish Technological Institute DTI | Giessing A.M.B.,University of Southern Denmark | Hansen T.,University of Southern Denmark | And 4 more authors.
Journal of Molecular Biology | Year: 2011

Complete characterization of a biomolecule's chemical structure is crucial in the full understanding of the relations between their structure and function. The dominating components in ribosomes are ribosomal RNAs (rRNAs), and the entire rRNA-but a single modified nucleoside at position 2501 in 23S rRNA-has previously been characterized in the bacterium Escherichia coli. Despite a first report nearly 20 years ago, the chemical nature of the modification at position 2501 has remained elusive, and attempts to isolate it have so far been unsuccessful. We unambiguously identify this last unknown modification as 5-hydroxycytidine-a novel modification in RNA. Identification of 5-hydroxycytidine was completed by liquid chromatography under nonoxidizing conditions using a graphitized carbon stationary phase in combination with ion trap tandem mass spectrometry and by comparing the fragmentation behavior of the natural nucleoside with that of a chemically synthesized ditto. Furthermore, we show that 5-hydroxycytidine is also present in the equivalent position of 23S rRNA from the bacterium Deinococcus radiodurans. Given the unstable nature of 5-hydroxycytidine, this modification might be found in other RNAs when applying the proper analytical conditions as described here. © 2011 Elsevier Ltd. All rights reserved.


Schultheisz H.L.,Scripps Research Institute | Szymczyna B.R.,Scripps Research Institute | Scott L.G.,Scripps Research Institute | Williamson J.R.,Cassia, Llc
Journal of the American Chemical Society | Year: 2011

The use of stable isotope labeling has revolutionized NMR studies of nucleic acids, and there is a need for methods of incorporation of specific isotope labels to facilitate specific NMR experiments and applications. Enzymatic synthesis offers an efficient and flexible means to synthesize nucleoside triphosphates from a variety of commercially available specifically labeled precursors, permitting isotope labeling of RNAs prepared by in vitro transcription. Here, we recapitulate de novo pyrimidine biosynthesis in vitro, using recombinantly expressed enzymes to perform efficient single-pot syntheses of UTP and CTP that bear a variety of stable isotope labeling patterns. Filtered NMR experiments on 13C, 15N, 2H-labeled HIV-2 TAR RNA demonstrate the utility and value of this approach. This flexible enzymatic synthesis will make implementing detailed and informative RNA stable isotope labeling schemes substantially more cost-effective and efficient, providing advanced tools for the study of structure and dynamics of RNA molecules. © 2010 American Chemical Society.


Viladoms J.,Scripps Research Institute | Scott L.G.,Cassia, Llc | Fedor M.J.,Scripps Research Institute
Journal of the American Chemical Society | Year: 2011

Active-site guanines that occupy similar positions have been proposed to serve as general base catalysts in hammerhead, hairpin, and glmS ribozymes, but no specific roles for these guanines have been demonstrated conclusively. Structural studies place G33(N1) of the glmS ribozyme of Bacillus anthracis within hydrogen-bonding distance of the 2′-OH nucleophile. Apparent pK a values determined from the pH dependence of cleavage kinetics for wild-type and mutant glmS ribozymes do not support a role for G33, or any other active-site guanine, in general base catalysis. Furthermore, discrepancies between apparent pK a values obtained from functional assays and microscopic pK a values obtained from pH-fluorescence profiles with ribozymes containing a fluorescent guanosine analogue, 8-azaguanosine, at position 33 suggest that the pH-dependent step in catalysis does not involve G33 deprotonation. These results point to an alternative model in which G33(N1) in its neutral, protonated form donates a hydrogen bond to stabilize the transition state. © 2011 American Chemical Society.


Where chromatographic separation of sucrose from molasses produced is employed, the color of the molasses feed to the molasses separator operation has a relatively large effect on the color of the sucrose enriched extract produced from the separation process and, as a result, on the overall efficiency of sugar recovery from the molasses separation process. The subsequent sugar end processing efficiency of the separator sucrose enriched extract product to granulated sugar is quite dependent on the color loading of the extract being processed. This study evaluates the effect of manipulating and controlling the pH value of the A (high green) run-off syrup, B (intermediate) green run-off syrup and final molasses produced to achieve minimal color rise and a low final molasses color from the thick juice processed in the sugar end. The deliberate and precise control of green syrup pH value will produce molasses of the desired color relative to the quality of the beets being processed and the thick juice produced from such beet quality. It is further shown that the color of extract produced is highly dependent on the color of molasses feed to the molasses separation operation.


Cottrell J.W.,Scripps Research Institute | Scott L.G.,Cassia, Llc | Fedor M.J.,Scripps Research Institute
Journal of Biological Chemistry | Year: 2011

Understanding how self-cleaving ribozymes mediate catalysis is crucial in light of compelling evidence that human and bacterial gene expression can be regulated through RNA self-cleavage. The hairpin ribozyme catalyzes reversible phosphodiester bond cleavage through a mechanism that does not require divalent metal cations. Previous structural and biochemical evidence implicated the amidine group of an active site adenosine, A38, in a pH-dependent step in catalysis.Wedeveloped a way to determine microscopic pK a values in active ribozymes based on the pH-dependent fluorescence of 8-azaadenosine (8azaA). We compared the microscopic pK a for ionization of 8azaA at position 38 with the apparent pK a for the self-cleavage reaction in a fully functional hairpin ribozyme with a unique 8azaA at position 38. Microscopic and apparent pK a values were virtually the same, evidence that A38 protonation accounts for the decrease in catalytic activity with decreasing pH. These results implicate the neutral unprotonated form of A38 in a transition state that involves formation of the 5′-oxygen-phosphorus bond. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.


Scott L.G.,Cassia, Llc | Hennig M.,University of North Carolina at Chapel Hill
Methods in Enzymology | Year: 2016

Naturally occurring RNA lacks fluorine-19 (19F), thus, their specifically fluorinated counterparts are particularly well suited to noninvasively monitoring the dynamic conformational properties and ligand-binding interactions of the RNA. For nuclear magnetic resonance (NMR) spectroscopy, 19F-NMR of fluorine-substituted RNA provides an attractive, site-specific probe for structure determination in solution. Advantages of 19F include high NMR sensitivity (83% of 1H), high natural abundance (100%), and the extreme sensitivity of 19F to the chemical environment leading to a large range of chemical shifts. The preparation of base-substituted 2-fluoropurine and 5-fluoropyrimidine 5′-triphosphates (2F-ATP/5F-CTP/5F-UTP) can be carried out using efficient enzymatic synthesis methods. Both pyrimidine analogs, 5-fluorouridine and 5-fluorocytidine, as well as, 2-fluoroadenosine are readily incorporated into RNA transcribed in vitro using T7 RNA polymerase. © 2016 Elsevier Inc. All rights reserved.


Giessing A.M.B.,University of Southern Denmark | Scott L.G.,Cassia, Llc | Kirpekar F.,University of Southern Denmark
Journal of the American Society for Mass Spectrometry | Year: 2011

LC/MS analysis of ribonucleosides is traditionally performed by reverse phase chromatography on silica based C18 type stationary phases using MS compatible buffers and methanol or acetonitrile gradients. Due to the hydrophilic and polar nature of nucleosides, down-scaling C18 analytical methods to a two-column nano-flow setup is inherently difficult. We present a nanochip LC/MS ion-trap strategy for routine characterization of RNA nucleosides in the fmol range. Nucleosides were analyzed in positive ion mode by reverse phase chromatography using a 75 μ×150 mm, 5 μ particle porous graphitic carbon (PGC) chip with an integrated 9 mm, 160 nL trapping column. Nucleosides were separated using a formic acid/acetonitrile gradient. The method was able to separate isobaric nucleosides as well as nucleosides with isotopic overlap to allow unambiguous MS n identification on a low resolution ion-trap. Synthesis of 5-hydroxycytidine (oh 5C) was achieved from 5-hydroxyuracil in a novel three-step enzymatic process. When operated in its native state using formic acid/acetonitrile, PGC oxidized oh 5C to its corresponding glycols and formic acid conjugates. Reduction of the PGC stationary phase was achieved by flushing the chip with 2.5 mM oxalic acid and adding 1 mM oxalic acid to the online solvents. Analyzed under reduced chromatographic conditions oh 5C was readily identified by its MH + m/z 260 and MS n fragmentation pattern. This investigation is, to our knowledge, the first instance where oxalic acid has been used as an online reducing agent for LC/MS. The method was subsequently used for complete characterization of nucleosides found in tRNAs using both PGC and C18 chips. © American Society for Mass Spectrometry, 2011.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 300.00K | Year: 2013

This proposal outlines a plan to synthesize standard samples of a range of glycans as reference compounds in glycomics research. Cassia, LLC shall work in collaboration with the Paulson laboratory at The Scripps Research Institute to make these compoundscommercially available to the research community. The Specific Aims include enzymatic synthesis of 20-32 glycans as either terminal fragments, or N- or O-linked glycans. In addition, these compounds shall be synthesized as heavy versions including 13Cisotope labels to facilitate their use as internal standards for identification and quantitation of glycans using mass spectrometry. The glycans shall be characterized using NMR and mass spectrometry, and the compounds shall be supplied as standards forthe National Institute of General Medical Sciences (NIGMS) microarray facility. The specific aims in Phase I of this Fast Track SBIR proposal are designed to supply the first wave of glycan products, and to begin to transfer the technology of enzymaticglycan synthesis from the Paulson laboratory to Cassia. These aims set the foundation for synthesis of expanded glycan libraries in Phase II, and successful commercialization of a broad range of glycan reference compounds in Phase III Commercialization.PUBLIC HEALTH RELEVANCE


PubMed | Cassia, Llc and University of North Carolina at Chapel Hill
Type: | Journal: Methods in enzymology | Year: 2016

Naturally occurring RNA lacks fluorine-19 ((19)F), thus, their specifically fluorinated counterparts are particularly well suited to noninvasively monitoring the dynamic conformational properties and ligand-binding interactions of the RNA. For nuclear magnetic resonance (NMR) spectroscopy, (19)F-NMR of fluorine-substituted RNA provides an attractive, site-specific probe for structure determination in solution. Advantages of (19)F include high NMR sensitivity (83% of (1)H), high natural abundance (100%), and the extreme sensitivity of (19)F to the chemical environment leading to a large range of chemical shifts. The preparation of base-substituted 2-fluoropurine and 5-fluoropyrimidine 5-triphosphates (2F-ATP/5F-CTP/5F-UTP) can be carried out using efficient enzymatic synthesis methods. Both pyrimidine analogs, 5-fluorouridine and 5-fluorocytidine, as well as, 2-fluoroadenosine are readily incorporated into RNA transcribed in vitro using T7 RNA polymerase.

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