Purification of Optically Labeled Oligonucleotides

光学标记寡核苷酸的纯化

• 批准号: 6833405
• 负责人: William H. Pearson
• 金额: $ 9.98万
• 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: 2004-08-15 至 2005-02-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6833405
• 关键词: affinity chromatography; affinity labeling; amines; bioengineering /biomedical engineering; fluorescent dye /probe; high performance liquid chromatography; nucleic acid purification; oligonucleotides; technology /technique development

DESCRIPTION (provided by applicant): The introduction of optical labels (e.g. fluorophores, quenchers) into biomolecules has revolutionized the study of these materials. Generally overlooked but of great practical importance is the purification of labeled materials. Labeling involves chemical reactions, which of course are never ideal, producing the desired material plus byproducts. In one method, the target molecule is treated with a reactive form of the label, resulting in linkage to a pre-existing functional group. The desired labeled material may be contaminated with over-labeled and under-labeled materials, as well as starting materials and other byproducts. Alternatively, automated peptide or oligonucleotide synthesis can be used to incorporate labels, again leading to a distribution of products from which the desired material must be isolated. It is often crucial to have highly pure materials, free from other compounds that contain (or even lack) a fluorescent label or quencher. Researchers currently use tedious reversed-phase (RP) HPLC (sometimes repetitively) to purify labeled materials, a method that relies on the interaction of the hydrophobic label with the nonpolar chromatography matrix for separation. Separations can be difficult, and the use of quicker methods employing small cartridges suffers from low material recovery because of the weakness of hydrophobic interactions. The goal of this proposal is to provide the end user with a simple and easy purification method that will provide optically labeled materials of unprecedented purity. A new type of affinity tag will be built into the optical labels, imparting a strong attraction between labeled materials and a modified chromatography support. This affinity interaction is much stronger than the hydrophobic interaction encountered in RP-HPLC, and may be employed in the form of traditional HPLC columns or simple "catch and release" cartridges, allowing the user to "fish out" only materials that have the optical label attached. Initial work will focus on oligonucleotide probe purification.

描述（由申请人提供）：将光学标记（例如荧光团、猝灭剂）引入生物分子彻底改变了这些材料的研究。通常被忽视但具有重要实际意义的是标记材料的纯化。标签涉及化学反应，这当然永远不会理想，会产生所需的材料和副产品。在一种方法中，用反应形式的标记处理靶分子，导致与预先存在的官能团连接。所需的标记材料可能被过度标记和标记不足的材料以及起始材料和其他副产物污染。或者，可以使用自动肽或寡核苷酸合成来掺入标签，再次导致必须从中分离出所需材料的产品分布。拥有高纯度的材料通常至关重要，不含其他含有（甚至缺乏）荧光标记或猝灭剂的化合物。研究人员目前使用繁琐的反相 (RP) HPLC（有时重复）来纯化标记材料，这种方法依赖于疏水标记与非极性色谱基质的相互作用来进行分离。分离可能很困难，并且由于疏水相互作用的弱点，使用小柱的更快方法会受到材料回收率低的影响。该提案的目标是为最终用户提供一种简单易用的纯化方法，该方法将提供前所未有的纯度的光学标记材料。光学标签中将内置一种新型亲和标签，在标记材料和改进的色谱支持物之间产生强大的吸引力。这种亲和相互作用比 RP-HPLC 中遇到的疏水相互作用强得多，并且可以以传统 HPLC 柱或简单的“捕获和释放”柱的形式使用，允许用户仅“捞出”附有光学标签的材料。初步工作将集中于寡核苷酸探针的纯化。