- A nano-chip-LC/MSn based strategy for characterization of modified nucleosides using reduced porous graphitic carbon as a stationary phase
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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 MSn identification on a low resolution ion-trap. Synthesis of 5-hydroxycytidine (oh5C) was achieved from 5-hydroxyuracil in a novel three-step enzymatic process. When operated in its native state using formic acid/acetonitrile, PGC oxidized oh5C 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 oh5C was readily identified by its MH + m/z 260 and MSn 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.
- Giessing, Anders Michael Bernth,Scott, Lincoln Greyson,Kirpekar, Finn
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- SYNTHESIS AND STRUCTURE OF HIGH POTENCY RNA THERAPEUTICS
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This invention provides expressible polynucleotides, which can express a target protein or polypeptide. Synthetic mRNA constructs for producing a protein or polypeptide can contain one or more 5′ UTRs, where a 5′ UTR may be expressed by a gene of a plant. In some embodiments, a 5′ UTR may be expressed by a gene of a member of Arabidopsis genus. The synthetic mRNA constructs can be used as pharmaceutical agents for expressing a target protein or polypeptide in vivo.
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- Synthesis, base pairing properties and trans-lesion synthesis by reverse transcriptases of oligoribonucleotides containing the oxidatively damaged base 5-hydroxycytidine
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The synthesis of a caged RNA phosphoramidite building block containing the oxidatively damaged base 5-hydroxycytidine (5-HOrC) has been accomplished. To determine the effect of this highly mutagenic lesion on complementary base recognition and coding properties, this building block was incorporated into a 12-mer oligoribonucleotide for Tm and CD measurements and a 31-mer template strand for primer extension experiments with HIV-, AMV-and MMLV-reverse transcriptase (RT). In UV-melting experiments, we find an unusual biphasic transition with two distinct Tm's when 5-HOrC is paired against a DNA or RNA complement with the base guanine in opposing position. The higher T m closely matches that of a C-G base pair while the lower is close to that of a C-A mismatch. In single nucleotide extension reactions, we find substantial misincorporation of dAMP and to a lesser extent dTMP, with dAMP almost equaling that of the parent dGMP in the case of HIV-RT. A working hypothesis for the biphasic melting transition does not invoke tautomeric variability of 5-HOrC but rather local structural perturbations of the base pair at low temperature induced by interactions of the 5-HO group with the phosphate backbone. The properties of this RNA damage is discussed in the context of its putative biological function.
- Kuepfer, Pascal A.,Leumann, Christian J.
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scheme or table
p. 9422 - 9432
(2012/04/04)
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- Reactions of 5-bromo substituted pyrimidine nucleosides with aqueous alkalies: kinetics and mechanisms.
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Kinetics for the parallel and consecutive steps of the reactions of 5-bromocytidine, 5-bromouridine and its 5'-O-methyl and 2',3'-O-isopropylidene derivatives with aqueous alkalies were studied by LC. The mechanisms of the partial reactions involved are discussed.
- K?ppi,L?nnberg
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p. 768 - 775
(2007/10/02)
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