De novo sequence determination of modified oligonucleotides.
We present the combined application of chemical and enzymatic digestions toward de novo sequence determination of a modified oligonucleotide. The unknown of interest, consisting of a random mixture of 2'-deoxy, 2'-fluoro, 2'-O-methyl, abasic and/or ribonucleotides, is a representative oligonucleotide used as a component of synthetic short interfering RNAs (siRNAs). The sequence is initially determined using chemical degradation, electrospray ionization (ESI), time-of-flight (TOF), and tandem (MS/MS) mass spectrometry. A nucleoside composition analysis is then performed via enzymatic digestion of the oligonucleotide to complement the chemical sequencing method. The identity and experimental count of each nucleoside within the oligonucleotide are determined by ultra performance liquid chromatography (UPLC) analysis. This additional analysis allows unambiguous sequence assignment of an unknown chemically modified oligonucleotide.
Department of Process Research, Merck Research Laboratories, 126 East Lincoln Avenue, Rahway, New Jersey 07065, USA. julie_farand@merck.com
Julie
J
Farand
Francis
F
Gosselin
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006868:Hydrolysis; D009705:Nucleosides / Q000032:analysis; D009711:Nucleotides / Q000737:chemistry; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry*; D034741:RNA, Small Interfering / Q000032:analysis / Q000737:chemistry; D017421:Sequence Analysis / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D009705:Nucleosides; D009711:Nucleotides; D009841:Oligonucleotides; D034741:RNA, Small Interfering