De Novo Sequence Determination of Modified Oligonucleotides

被引：25

作者：

Farand, Julie ^{[1
]}

Gosselin, Francis ^{[1
]}

机构：

[1] Merck Res Labs, Dept Proc Res, Rahway, NJ 07065 USA

来源：

ANALYTICAL CHEMISTRY | 2009年 / 81卷 / 10期

关键词：

TANDEM MASS-SPECTROMETRY; QUADRUPOLE-ION-TRAP; ELECTROSPRAY-IONIZATION; LIQUID-CHROMATOGRAPHY; NUCLEIC-ACIDS; PERFORMANCE; DNA; THERAPEUTICS; PEPTIDE; RNA;

D O I：

10.1021/ac802452p

中图分类号：

O65 [分析化学];

学科分类号：

070302 ; 081704 ;

摘要：

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.

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收藏

页码：3723 / 3730

页数：8

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