Sequencing RNA by a combination of exonuclease digestion and uridine specific chemical cleavage using MALDI-TOF.
The determination of DNA sequences by partial exonuclease digestion followed by Matrix-Assisted Laser Desorption Time of Flight Mass Spectrometry (MALDI-TOF) is a well established method. When the same procedure is applied to RNA, difficulties arise due to the small (1 Da) mass difference between the nucleotides U and C, which makes unambiguous assignment difficult using a MALDI-TOF instrument. Here we report our experiences with sequence specific endonucleases and chemical methods followed by MALDI-TOF to resolve these sequence ambiguities. We have found chemical methods superior to endonucleases both in terms of correct specificity and extent of sequence coverage. This methodology can be used in combination with exonuclease digestion to rapidly assign RNA sequences.
Tolson
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SmithKline Beecham Pharmaceuticals, New Frontiers Science Park (North), Coldharbour Road, The Pinnacles, Harlow, Essex CM19 5AD, UK. David_A_Tolson.@sbphrd.com
Nicholson
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D000814:Aniline Compounds; D001483:Base Sequence; D004722:Endoribonucleases / Q000378:metabolism; D005092:Exonucleases / Q000378:metabolism*; D006834:Hydrazines; D007834:Lasers; D013058:Mass Spectrometry / Q000379:methods*; D012313:RNA / Q000737:chemistry*; D017423:Sequence Analysis, RNA / Q000379:methods*; D014529:Uridine / Q000737:chemistry*
D000814:Aniline Compounds; D006834:Hydrazines; C029424:hydrazine; D012313:RNA; D004722:Endoribonucleases; D005092:Exonucleases; C110454:ribonuclease CL3; C027909:endoribonuclease Phy M; C023650:aniline; D014529:Uridine