Characterization of synthetic oligonucleotides containing biologically important modified bases by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
Characterization of synthetic oligonucleotides containing biologically important modified bases by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
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DOI：
10.1016/j.ab.2008.04.031
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发表时间：
2008-08-15
影响因子：
2.9
通讯作者：
Sowers, Lawrence C.
中科院分区：
文献类型：
--
作者：
Cui, Zhengfang;Theruvathu, Jacob A.;Sowers, Lawrence C.
含有修饰碱基的寡核苷酸通常用于生物化学和生物物理研究，以评估特定类型的化学损伤对 DNA 结构和功能的影响。与使用正常 DNA 碱基合成寡核苷酸相比，使用修饰碱基合成寡核苷酸通常需要修改合成或脱保护条件。此外，一些具有生物学意义的修饰碱基在合成和寡核苷酸分离过程中容易受到进一步损坏。在本文中，我们描述了基质辅助激光解吸/电离飞行时间质谱 (MALDI-TOF-MS) 在表征一系列修饰的合成寡核苷酸中的应用。讨论了获得高质量准确度以确认寡核苷酸组成的潜力和限制。还提出了同位素簇的检查作为确认寡核苷酸元素组成的方法。对未纯化的反应混合物进行 MALDI-TOF-MS 分析可用于确认合成顺序并揭示合成过程中的潜在问题。纯化过程中和纯化后的分析可以获得有关脱嘌呤和碱氧化的重要信息。它还可以揭示非标准合成、脱保护或纯化策略可能出现的意外问题。修饰寡核苷酸的正确表征对于正确解释使用这些底物进行的实验至关重要，MALDI-TOF-MS 分析提供了一种简单而广泛的表征方法，可用于寡核苷酸生产和使用的多个阶段。 (C) 2008 Elsevier Inc. 保留所有权利。
Oligonucleotides containing modified bases are commonly used for biochemical and biophysical studies to assess the impact of specific types of chemical damage on DNA structure and function. In contrast to the synthesis of oligonucleotides with normal DNA bases, oligonucleotide synthesis with modified bases often requires modified synthetic or deprotection conditions. Furthermore, several modified bases of biological interest are prone to further damage during synthesis and oligonucleotide isolation. In this article, we describe the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to the characterization of a series of modified synthetic oligonucleotides. The potential for and limits in obtaining high mass accuracy for confirming oligonucleotide composition are discussed. Examination of the isotope cluster is also proposed as a method for confirming oligonucleotide elemental composition. MALDI-TOF-MS analysis of the unpurified reaction mixture can be used to confirm synthetic sequence and to reveal potential problems during synthesis. Analysis during and after purification can yield important information on depurination and base oxidation. It can also reveal unexpected problems that can occur with nonstandard synthesis, deprotection, or purification strategies. Proper characterization of modified oligonucleotides is essential for the correct interpretation of experiments performed with these substrates, and MALDI-TOF-MS analysis provides a simple yet extensive method of characterization that can be used at multiple stages of oligonucleotide production and use. (C) 2008 Elsevier Inc. All rights reserved.