Sequencing of single and double stranded RNA oligonucleotides by acid hydrolysis and MALDI mass spectrometry.
Treatment of RNA oligonucleotides with strong acids at pH 1-2 rapidly leads to hydrolysis of the phosphodiester bonds at the 5'-position of ribose. Analysis of the resulting degradation products by MALDI coupled to an Orbitrap high resolution mass spectrometer shows almost complete mass ladders from both sides of the nucleotides without interfering fragments from base losses or internal fragments. From the mass differences between adjacent peaks of a mass ladder, the sequence can be determined. Low cleavage efficiency at the termini leads to 2mers and 3mers which can be identified by MS/MS. In this way the complete sequences of different siRNA 21mer single and double strands could be verified. This simple and fast method can be applied for controlling sequences of synthetic oligomers, as well as for de-novo sequencing. Moreover, the method is applicable for localization and identification of RNA modifications as demonstrated using the examples of an oligonucleotide with phosphorothioate backbone and of one containing 2'-methoxy-ribose modifications.
Bahr
Ute
U
Cluster of Excellence Macromolecular Complexes, Institute of Pharmaceutical Chemistry, University of Frankfurt, 60438 Frankfurt, Germany. bahr@pharmchem.uni-frankfurt.de
Aygün
Hüseyin
H
Karas
Michael
M
D000143:Acids / Q000737:chemistry*; D001483:Base Sequence; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D009843:Oligoribonucleotides / Q000032:analysis / Q000737:chemistry* / Q000235:genetics*; D012313:RNA / Q000032:analysis / Q000737:chemistry* / Q000235:genetics*; D017423:Sequence Analysis, RNA / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000143:Acids; D009843:Oligoribonucleotides; D012313:RNA