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IMPROVED IDENTIFICATION OF THE POST-TRANSCRIPTIONALLY MODIFIED NUCLEOSIDE, PSEUDOURIDINE, IN RNAs

by DURAIRAJ, ANITA

Abstract (Summary)
Pseudouridine, the most abundant post-transcriptional modification in RNA, was derivatized with the water-soluble carbodiimide, N-cyclohexyl-N’-â-(4-methylmorpholinium) ethylcarbodiimide (CMC). Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was utilized as the mode of detection for CMC-derivatized pseudouridine. Initial studies involving method optimization were conducted on a simple homopolymer and heteropolymer DNA system. These studies were directed toward optimizing the CMC derivatization reaction conditions and ensuring its compatibility with MALDI-MS. After optimization, the method was applied to the determination of pseudouridine in a small RNA, Escherichia coli tRNATyrII. In addition to pseudouridine, this work revealed that the post-transcriptionally modified nucleoside, 2-methylthio-N6-isopentenyladenosine (ms2i6A) was also susceptible to derivatization by CMC. Method application studies were further expanded to include the detection of pseudouridines in a complicated mixture of E.coli tRNAs. For the complex mixture of tRNAs, endonuclease mapping and a signature digestion product approach was implemented with the CMC derivatization / MALDI-MS method for accurate identification and sequence placement of pseudouridine to individual tRNAs in the mixture. Further studies included the application of the CMC derivatization / MALDI-MS method for pseudouridine detection in a large RNA. Preliminary data was also generated from an affinity purification based approach for the detection of CMC derivatized oligonucleotides. These studies reveal the potential utility of the CMC derivatization / MALDI-MS approach as a fast and facile method for screening for pseudouridine in RNA.
Bibliographical Information:

Advisor:

School:University of Cincinnati

School Location:USA - Ohio

Source Type:Master's Thesis

Keywords:mass spectrometry pseudouridine rna cmc

ISBN:

Date of Publication:01/01/2007

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