Purification of Optically Labeled Oligonucleotides

描述（由申请人提供）：将光学标记物（例如荧光团、猝灭剂）引入生物分子中彻底改变了这些材料的研究。标记物质的纯化通常被忽视，但具有重要的实际意义。标签涉及化学反应，当然这从来都不是理想的，产生所需的材料加上副产品。在一种方法中，用反应性形式的标记物处理靶分子，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，自动化肽或寡核苷酸合成可用于掺入标记，再次导致必须从中分离所需材料的产物的分布。通常至关重要的是要有高纯度的材料，不含其他含有（或甚至缺乏）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进行分离。分离可能是困难的，并且由于疏水相互作用的弱点，采用小筒的更快方法的使用遭受低的材料回收。该提案的目标是为最终用户提供一种简单易行的纯化方法，该方法将提供前所未有纯度的光学标记材料。一种新型的亲和标签将被构建到光学标记中，在标记材料和改性的色谱支持物之间赋予强吸引力。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”盒的形式使用，允许用户仅“钓出”附着有光学标记的材料。最初的工作将集中在寡核苷酸探针纯化。

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.