Purification of Oligonucleotides and Nucleoside Triphosphates

描述（由申请人提供）：固相寡核苷酸（ON）合成产生一种复杂的混合物，除了靶序列外，该混合物还含有大量其他ON和副产物。纯化对于从这些其他寡核苷酸中分离所需序列是必要的。“Trityl-on”纯化是用于短ON（20-40聚体）的标准技术。在这种方法中，核碱基脱保护和从固体支持物上切割得到寡核苷酸的混合物，其中一些仍然带有疏水性S '-二甲氧基三苯甲基（DMT）基团，这允许在反相吸附剂上进行亲和纯化。不幸的是，随着寡核苷酸变得更长或更复杂，三苯甲基-on方法的有效性降低。先前制备更疏水的DMT类似物的尝试已经取得了一些成功，但从未超出分析规模的概念验证研究。需要纯寡核苷酸的研究人员，特别是长寡核苷酸或那些带有修饰的寡核苷酸，仍然必须求助于费力的、低产量的纯化方法。第一阶段资助了一类新的亲和标签的开发，涉及高度氟化的（“氟”）马尾辫以及高度氟化的色谱吸附剂（Fluoro-Pak），其中氟标记的ON具有前所未有的亲和力，允许以高产率纯化高达100个核苷酸的ON。第二阶段资金的主要目标是扩展荧光亲和纯化方法，以包括重要的修饰ON，如那些轴承生物素和荧光团。这些材料比标准ON更难制备和纯化，并且对于对荧光诊断探针、修饰的RNA等感兴趣的科学家来说代表了相当大的挑战。第II阶段的第二目标是将该技术应用于核苷S '-三磷酸的合成和纯化，核苷S'-三磷酸可用作用于诊断和测序应用中的核酸的酶促合成的构件。与公共卫生的相关性：治疗和诊断寡核苷酸（DNA和RNA的小片段）在检测基因突变和治疗疾病方面越来越重要，但众所周知，它们很难从合成的复杂混合物中纯化出来。拟议的工作旨在进一步开发一种全新的寡核苷酸纯化方法，从而简化这些关键物质的开发和生产。

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.