Purification of Oligonucleotides and Nucleoside Triphosphates

寡核苷酸和三磷酸核苷的纯化

• 批准号: 7228929
• 负责人: William H. Pearson
• 金额: $ 31.06万
• 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: 2004-09-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7228929
• 关键词: Affinity; Affinity Chromatography; Area; Base Sequence; Biotin; Characteristics; Chromatography; Class; Complex; Complex Mixtures; DNA; Detection; Development; Diagnostic; Disease; Effectiveness; Excision; Failure; Funding; Gene Mutation; Goals; Immobilization; Methods; Modification; Nucleic Acids; Nucleotides; Oligodeoxyribonucleotides; Oligonucleotides; Oligoribonucleotides; Personal Satisfaction; Phase; Production; Property; Public Health; RNA; Range; Reagent; Recovery; Research Personnel; Resort; Scientist; Solid; Standards of Weights and Measures; Surface; Techniques; Technology; Therapeutic; Time; Ursidae Family; Work; analog; base; concept; desire; fluorophore; functional group; interest; monomer; novel; nucleobase; nucleoside triphosphate; phosphoramidite; success

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.

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，除了目标序列外，还含有大量其他ON和副产物。要从这些寡核苷酸中分离出所需的序列，纯化是必要的。“三酰基-on”纯化是短离子（20-40米）的标准技术。在这种方法中，核碱基的脱保护和固体载体的裂解得到了寡核苷酸的混合物，其中一些仍然带有疏水的S'-二甲氧基三甲基（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，三基-on方法的有效性降低。先前尝试制造更多的疏水性DMT类似物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，特别是长核苷酸或带有修饰的寡核苷酸，仍然必须采用费力的、低产量的纯化方法。第一阶段资助了一种新型亲和标签的开发，涉及高氟化（“氟”）马尾以及高氟化色谱吸附剂（Fluoro-Pak），其中含氟标记的网络具有前所未有的亲和性，可以以高产量纯化多达100个核苷酸的网络。第二阶段资金的主要目标是扩展氟亲和纯化方法，以包括重要的改性氮化碳，例如那些含有生物素和荧光团的氮化碳。这些材料比标准的核糖核酸更难制造和纯化，对于对荧光诊断探针、修饰rna等感兴趣的科学家来说，这是一个相当大的挑战。第二阶段的第二个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'-三磷酸是用于诊断和测序应用的核酸酶合成的有用基石。与公共卫生的相关性：治疗性和诊断性寡核苷酸（DNA和RNA的小片段）在检测基因突变和治疗疾病方面日益重要，但众所周知，它们很难从合成后的复杂混合物中纯化出来。提出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。