Improved Method for the Purification of Oligonucleotides

描述（由申请人提供）：固相寡核苷酸合成产生一种复杂的混合物，除了目标序列外，还含有大量其他寡核苷酸。这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，以及分支物质。要从这些寡核苷酸中分离出所需的序列，纯化是必要的。对于较长的序列，相对于目标序列，副产物的量变得更大，纯化变得越来越困难。“三基-on”纯化是短寡核苷酸（20-40米）的标准技术。在这种方法中，核碱基脱保护和固体载体的裂解得到寡核苷酸混合物，其中一些仍然具有疏水的5'-二甲基氧三甲基（DMT）基团，这允许在反相吸附剂上进行亲和纯化，去除非DMT失效序列。不幸的是，这种方法不容易去除同样带有DMT组的删除和插入序列。此外，随着寡核苷酸变长，三基上方法的有效性降低，即使去除失效序列也是如此。先前制造更多疏水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，而且这些构建块都没有商业化。需要纯寡核苷酸，特别是长核苷酸的研究人员，仍然必须求助于费力的、低产量的纯化方法。本提案的主要目标是开发一种利用不同类型亲和相互作用的5'保护基团，这种亲和相互作用比DMT基团和反相介质之间的疏水相互作用强大得多。提出了一种具有新型5′保护基团的核苷类磷酰胺系。它们将使用标准的自动化方案并入越来越长的寡核苷酸中，并且由此产生的“标记”寡核苷酸将使用替代类型的市售吸附剂进行纯化。去除标签后，应该得到纯寡核苷酸，即使是长（bbb100 nt）的寡核苷酸。拟议的商业应用：拟议的亲和标记核苷磷酰胺将使研究人员能够使用技术上简单的程序制备前所未有的纯度越来越长的寡核苷酸。

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.