Identification of RNA linkage isomers by anion exchange purification with electrospray ionization mass spectrometry of automatically desalted phosphodiesterase-II digests.
Among the possible contaminants unique to RNA are linkage isomers that are difficult to identify by standard oligonucleotide analysis techniques. In a prior study, we used nonporous and monolithic polymer anion exchangers for purification and demonstrated a method to identify the presence of the linkage isomers. We also suggested a confirming technique employing phosphodiesterase-II (PDase-II), an enzyme incapable of cleaving 2'-5' linkages. We now present a method identifying the location of the linkage in the RNA isomer by anion exchange purification and electrospray ionization mass spectrometry (ESI-MS) of the digestion products. Because the ion-pair reversed-phase liquid chromatography (IP-RPLC) desalting methods we previously employed do not effectively separate oligonucleotides less than 6 bases from salt, we employed a direct reversed-phase method to automatically desalt the digestion products and then assessed the desalted digests by ESI-MS. The length and base composition of the fragments identified indicate that PDase-II cleaves up to and skips over the aberrant linkage and then resumes cleavage 1 or 2 bases to the 3' side of the 2'-5' linkage.
Thayer
J R
JR
Dionex Corporation, Sunnyvale, CA 94085, USA. jim.thayer@dionex.com
Puri
Nitin
N
Burnett
Chris
C
Hail
Mark
M
Rao
Srinivasa
S
D002852:Chromatography, Ion Exchange / Q000379:methods*; D005092:Exonucleases / Q000378:metabolism*; D007536:Isomerism; D012313:RNA / Q000737:chemistry* / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D012313:RNA; D005092:Exonucleases; C022650:spleen exonuclease