Research Project
6833405
[unreadable] 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. [unreadable] [unreadable]
