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.