Sequence confirmation of modified oligonucleotides using chemical degradation, electrospray ionization, time-of-flight, and tandem mass spectrometry.
We report the sequencing of highly modified oligonucleotides containing a mixture of 2'-deoxy, 2'-fluoro, 2'-O-methyl, abasic, and ribonucleotides. The passenger and guide strands each containing 48% and 86% of modified nucleotides, respectively, are representative sequences of synthetic short interfering RNAs (siRNAs). We describe herein the sequence confirmation of both strands using a series of robust chemical reactions, followed by analysis via ESI-TOF and ion trap mass spectrometry (ITMS). The following method enables the rapid sequence confirmation of highly modified oligonucleotides.
Farand
Julie
J
Department of Process Research, Merck Research Laboratories, 2950 Wilderness Place, Boulder, Colorado 80301, USA. julie_farand@merck.com
Beverly
Michael
M
D001483:Base Sequence; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D009841:Oligonucleotides / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D009841:Oligonucleotides