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Nucleic Acids Symposium Series 2004 48(1):263-264; doi:10.1093/nass/48.1.263
© 2004 by Oxford University Press
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A novel method for sequence placement of modified nucleotides in mixtures of transfer RNA

Tracy M. Wagner, Vinod Nair, Rebecca Guymon, Steven C. Pomerantz, Pamela F. Crain, Darrell R. Davis and James A. McCloskey

Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112, USA

Sequence placement of post-transcriptionally modified nucleosides in tRNA can be experimentally difficult, particularly in cases involving new or unexpected modifications or sequence sites. We describe a mass spectrometry-based approach to this problem, involving the following steps: crude isolations of one or several tRNAs by HPLC from an unfractionated tRNA mixture; digestion to oligonucleotide mixtures by RNase T1; analysis by combined HPLC/electrospray ionization-MS for recognition of modifications; and direct gas-phase sequencing of selected targets in the mixture by LC/MS/MS. Isoacceptor identity can be established in favorable cases when tRNA gene sequences are available.


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