Nucleic acid separations using superficially porous silica particles.
Nucleic acid separations using superficially porous silica particles.
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DOI：
10.1016/j.chroma.2016.02.057
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发表时间：
2016-04-01
期刊：
影响因子：
--
通讯作者：
Dickman MJ
中科院分区：
文献类型：
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作者：
Close ED;Nwokeoji AO;Milton D;Cook K;Hindocha DM;Hook EC;Wood H;Dickman MJ
表面多孔的二氧化硅颗粒能够实现核酸的高分辨率分离。C18表面多孔二氧化硅颗粒的孔径显著影响分辨率。最佳分离的小寡核苷酸获得80 μ m孔径。在孔径为150 μ m时观察到寡核苷酸的最佳分辨率（> 19聚体）。在孔径为400 μ m的情况下，较大的dsDNA/RNA分子的分辨率得到了提高。离子对反相液相色谱已广泛用于核酸分离。广泛的替代固定相已与离子对反相色谱结合使用，包括全多孔颗粒、无孔颗粒、大孔颗粒和整体固定相。在这项研究中，我们利用表面多孔二氧化硅颗粒结合离子对反相液相色谱法分析核酸。我们已经研究了一系列不同的孔径和相，用于分析各种核酸，包括寡核苷酸、寡核糖核苷酸、硫代磷酸寡核苷酸和高分子量dsDNA和RNA。表面多孔二氧化硅颗粒的孔径显示出显著影响核酸的分辨率。小寡核苷酸的最佳分离，如在RNase映射实验中产生的那些，获得80 μ m孔径，可以很容易地与质谱分析接口。在孔径为150 μ m时观察到较大寡核苷酸（> 19聚体）的分辨率提高。使用孔径为400 μ m的表面多孔二氧化硅颗粒实现了较大dsDNA/RNA分子的最佳分辨率。此外，我们已经利用150 μ m孔径的实心颗粒来分离完全硫代磷酸化寡核苷酸的典型杂质，这些杂质通常在这类重要的治疗性寡核苷酸的合成中产生。
Superficially porous silica particles enable high resolution separation of nucleic acids. The pore size of the C18 superficially porous silica particles significantly effects resolution. Optimum separations of small oligonucleotides obtained with 80 Å pore sizes. Optimum resolution of oligonucleotides (>19 mers) was observed with pore sizes of 150 Å. Improved resolution of larger dsDNA/RNA molecules was achieved with pore sizes of 400 Å Ion pair reverse-phase liquid chromatography has been widely employed for nucleic acid separations. A wide range of alternative stationary phases have been utilised in conjunction with ion pair reverse-phase chromatography, including totally porous particles, non-porous particles, macroporous particles and monolithic stationary phases. In this study we have utilised superficially porous silica particles in conjunction with ion pair reverse-phase liquid chromatography for the analysis of nucleic acids. We have investigated a range of different pore-sizes and phases for the analysis of a diverse range of nucleic acids including oligonucleotides, oligoribonucleotides, phosphorothioate oligonucleotides and high molecular weight dsDNA and RNA. The pore size of the superficially porous silica particles was shown to significantly affect the resolution of the nucleic acids. Optimum separations of small oligonucleotides such as those generated in RNase mapping experiments were obtained with 80 Å pore sizes and can readily be interfaced with mass spectrometry analysis. Improved resolution of larger oligonucleotides (>19 mers) was observed with pore sizes of 150 Å. The optimum resolution for larger dsDNA/RNA molecules was achieved using superficially porous silica particles with pore sizes of 400 Å. Furthermore, we have utilised 150 Å pore size solid-core particles to separate typical impurities of a fully phosphorothioated oligonucleotide, which are often generated in the synthesis of this important class of therapeutic oligonucleotide.