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De Novo Sequence Determination of Modified Oligonucleotides

2009; American Chemical Society; Volume: 81; Issue: 10 Linguagem: Inglês

10.1021/ac802452p

1520-6882

Julie Farand, Francis Gosselin,

DNA and Nucleic Acid Chemistry

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.