A novel method for sequence placement of modified nucleotides in mixtures of transfer RNA.
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.
Wagner
Tracy M
TM
Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
Nair
Vinod
V
Guymon
Rebecca
R
Pomerantz
Steven C
SC
Crain
Pamela F
PF
Davis
Darrell R
DR
McCloskey
James A
JA
D002851:Chromatography, High Pressure Liquid; D013058:Mass Spectrometry; D009711:Nucleotides / Q000032:analysis*; D012343:RNA, Transfer / Q000737:chemistry*; D006163:Ribonuclease T1 / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D017423:Sequence Analysis, RNA / Q000379:methods*; D016944:Sulfolobus
D009711:Nucleotides; D012343:RNA, Transfer; D006163:Ribonuclease T1