Improved Method for the Purification of Oligonucleotides

寡核苷酸纯化的改进方法

• 批准号: 6788544
• 负责人: William H. Pearson
• 金额: $ 9.94万
• 依托单位: BERRY AND ASSOCIATES, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6788544
• 关键词: adsorption; affinity chromatography; amidation /deamidation; chemical chain length; chemical structure function; fluorine; intermolecular interaction; method development; nucleic acid chemical synthesis; nucleic acid purification; nucleic acid quantitation /detection; nucleosides; oligonucleotides; silicon

DESCRIPTION (provided by applicant): Solid-phase oligonucleotide synthesis produces a complex mixture that contains, in addition to the target sequence, a plethora of other oligonucleotides. These include a distribution of shorter failure sequences resulting from failed couplings, a heterogeneous mixture of deletion and insertion sequences that are nearly the same length as the target sequence, and branched materials. Purification is necessary to isolate the desired sequence from these other oligonucleotides. For longer sequences, the amount of the by-products becomes larger relative to the target sequence, and purification becomes increasingly difficult. "Trityl-on" purification is a standard technique for short oligonucleotides (20-40 mers). In this approach, nucleobase deprotection and cleavage from the solid support gives a mixture of oligonucleotides, some of which still bear a hydrophobic 5'-dimethyoxytrityl (DMT) group, which allows affinity purification on reverse phase adsorbents, removing non-DMT-bearing failure sequences. Unfortunately, this approach does not easily remove deletion and insertion sequences, which also bear a DMT group. Further, as the oligonucleotide becomes longer, the effectiveness of the trityl-on method diminishes, even for removing failure sequences. Prior attempts to make more hydrophobic DMT analogs have met with some success, but generally involve difficult synthetic work, and none of these building blocks are commercially available. Researchers who require pure oligonucleotides, especially long ones, must still resort to laborious, low-yielding purification methods. The main objective of this proposal is to develop a 5'-protecting group that exploits a different type of affinity interaction, one that is much more powerful than the hydrophobic interaction between a DMT group and reverse phase media. A line of nucleoside phosphoramidites bearing novel 5'-protecting groups is proposed. They will be incorporated into increasingly long oligonucleotides using standard automated protocols, and the resultant "tagged" oligonucleotides will be purified using an alternate type of commercially available adsorbent. After removing the tag, pure oligonucleotides should result, even on long (>100 nt) oligonucleotides. Proposed commercial applications: The proposed affinity-labeled nucleoside phosphoramidites will enable researchers to prepare increasingly lengthy oligonucleotides of unprecedented purity, using technically simple procedures.

描述(申请人提供)：固相寡核苷酸合成产生一种复杂的混合物，除了靶序列外，还含有过多的其他寡核苷酸。这些包括由失败的偶联导致的较短失效序列的分布、与目标序列长度几乎相同的缺失和插入序列的异质混合物以及分支材料。要从这些其他寡核苷酸中分离出所需的序列，必须进行纯化。对于较长的序列，副产物的量相对于目标序列变得更大，并且纯化变得越来越困难。“Trityl-On”提纯是短链寡核苷酸(20-40 MERS)的标准技术。在这种方法中，通过去碱基保护和从固体载体上裂解得到寡核苷酸的混合物，其中一些仍然带有疏水的5‘-二甲氧基三甲基(DMT)基团，这允许在反相吸附剂上进行亲和纯化，去除不含DMT的失败序列。不幸的是，这种方法不容易去除也带有DMT基团的缺失和插入序列。此外，随着寡核苷酸变得更长，即使对于去除失败序列，三苯基-ON方法的有效性也会降低。以前制造更疏水性的DMT类似物的尝试已经取得了一些成功，但通常涉及困难的合成工作，并且这些构建块都不是商业上可用的。需要纯寡核苷酸的研究人员，特别是长寡核苷酸，仍然必须求助于费力、低产的纯化方法。该建议的主要目的是开发利用不同类型的亲和作用的5‘-保护基团，这种亲和作用比DMT基团和反相介质之间的疏水作用强得多。提出了一种新型5‘-保护基核苷亚磷酰胺的合成方法。它们将使用标准的自动化程序被加入到越来越长的寡核苷酸中，得到的“标记”寡核苷酸将使用另一种可商业获得的吸附剂进行提纯。去掉标签后，即使是长的(&gt；100个核苷酸)寡核苷酸，也应该产生纯的寡核苷酸。拟议的商业应用：拟议的亲和标记核苷亚磷酰胺将使研究人员能够使用技术简单的程序制备越来越长的、具有前所未有纯度的寡核苷酸。