Purification of Oligonucleotides and Nucleoside Triphosphates

DESCRIPTION (provided by applicant): Solid-phase oligonucleotide (ON) synthesis produces a complex mixture that contains, in addition to the target sequence, a plethora of other ONs and by-products. Purification is necessary to isolate the desired sequence from these other oligonucleotides. "Trityl-on" purification is a standard technique for short ONs (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 S'-dimethoxytrityl (DMT) group, which allows affinity purification on reverse phase adsorbents. Unfortunately, as the oligonucleotide becomes longer or more complex, the effectiveness of the trityl-on method diminishes. Prior attempts to make more hydrophobic DMT analogs have met with some success, have never proceeded beyond analytical scale proof-of-concept studies. Researchers who require pure oligonucleotides, especially long ones or those bearing modifications, must still resort to laborious, low-yielding purification methods. Phase I funded the development of a new class of affinity tags involving highly fluorinated ("fluorous") ponytails as well as a highly fluorinated chromatography adsorbent (Fluoro-Pak), for which the fluorous-tagged ONs had an unprecedented affinity, allowing the purification of ONs up to 100 nucleotides in high yield. The main objective Phase II funding is to extend the fluorous affinity purification approach to include important modified ONs, such as those bearing biotins and fluorophores. These materials are more difficult to make and purify than standard ONs, and represent a considerable challenge for scientists interested in fluorescent diagnostic probes, modified RNAs, etc. A secondary objective of Phase II Is to apply this technology to the synthesis and purification of nucleoside S'-triphosphates, which are useful as building blocks for the enzymatic synthesis of nucleic acids that are used in diagnostic and sequencing applications. Relevance to public health: Therapeutic and diagnostic oligonucleotides (small fragments of DNA and RNA) are increasingly important in the detection of genetic mutations and the treatment of disease, but they are notoriously difficult to purify from the complex mixtures that result from their synthesis. The proposed work seeks to further develop an entirely new method for oligonucleotide purification, thus streamlining the development and production of these crucial substances.