Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.3 (RNase T1)
 1,228 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A new method using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for the direct analysis of the mass-silent post-transcriptionally modified nucleoside pseudouridine in nucleic acids has been developed. This method utilizes 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide to derivatize pseudouridine residues. After chemical derivatization all pseudouridine residues will contain a 252 Da 'mass tag' that allows the presence of pseudouridine to be identified using mass spectrometry. Pseudouridine residues can be identified in intact nucleic acids by obtaining a mass spectrum of the nucleic acid before and after derivatization. The mass difference (in units of 252 Da) will denote the number of pseudouridine residues present. To determine the sequence location of pseudouridine, a combination of enzymatic hydrolysis and mass spectrometric steps are used. Here, MALDI analysis of RNase T1 digestion products before and after modification are used to narrow the sequence location of pseudouridine to specific T1 fragments in the gene sequence. Further mass spectrometric monitoring of exonuclease digestion products from isolated T1 fragments is then used for exact sequence placement. This approach to pseudouridine identification is demonstrated using Escherichia coli tRNAS: This new method allows for the direct determination of pseudouridine in nucleic acids, can be used to identify modified pseudouridine residues and can be used with general modification mapping approaches to completely characterize the post-transcriptional modifications present in RNAs.
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PMID:Identification of the mass-silent post-transcriptionally modified nucleoside pseudouridine in RNA by matrix-assisted laser desorption/ionization mass spectrometry. 1135 94

Pseudouridine, an isomer of uridine, is probably the most common of many posttranscriptional RNA modifications found in nature. Although mass spectrometry has become widely used in the characterization of modified nucleic acids, its application to the recognition and sequence placement of pseudouridine has not been straightforward, particularly in the case of complex mixtures such as those resulting from selective enzymatic hydrolysis of RNA into oligonucleotides. We report results of a study of the characteristic dissociation reactions of pseudouridine-containing oligonucleotides following ionization by electrospray and use of those pathways in an LC/MS-based method applicable to direct analysis of RNase digests of RNA. As a consequence of the C-C (rather than C-N) glycosidic bond of pseudouridine, the otherwise common dissociation paths involving base loss do not occur, resulting in characteristic formation of a set of low-mass negative ions containing the intact glycosidic bond (m/z 225, 207, 189, 165, 164, 139), which permit recognition of pseudouridine-containing oligonucleotides. Those components can subsequently be subjected to sequence analysis by MS/MS, in which enhancement of selective sequence-determining ions (a-, w-, y-types), and absence of a - base ions, are observed at the site of pseudouridylation. Also, selected reaction pathways can be monitored in the LC/MS/MS analysis that are indicative of pseudouridine at the 5' terminus (m/z 225 --> 165), internal positions (m/z 207 --> 164), and in the RNase T1-derived product Psi pGp (m/z 668 --> 207) arising from the RNA sequence ...G Psi G... These procedures can be effectively integrated into an existing suite of LC/ESI-MS-based methods designed for the analysis of posttranscriptionally modified sites in RNA.
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PMID:Detection of the common RNA nucleoside pseudouridine in mixtures of oligonucleotides by mass spectrometry. 1605 77