Isotopically Labeled Nucleotides for Biomolecular NMR

用于生物分子 NMR 的同位素标记核苷酸

• 批准号: 6935072
• 负责人: James R Williamson
• 金额: $ 12.5万
• 依托单位: CASSIA, LLC
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-15 至 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6935072
• 关键词: Bacillus stearothermophilus; Bacillus subtilis; Escherichia coli; Saccharomyces cerevisiae; adenine phosphoribosyltransferase; affinity chromatography; bacterial proteins; bioreactors; biotechnology; deuterium; fermentation; fungal proteins; microorganism metabolism; nuclear magnetic resonance spectroscopy; pentosyltransferase; phosphates; plasmids; radiotracer; ribonucleosides; ribonucleotides; synthetic enzyme; technology /technique development; transfection /expression vector

DESCRIPTION (provided by applicant): The goal of the proposed research is to scale up the production and manufacture of high-value ribonucleoside triphosphates containing stable isotope labels. The use for these products is in biomolecular studies of RNA structure using Nuclear Magnetic Resonance (NMR) spectroscopy. The market for these products is structural biology groups in academic and industrial laboratories engaged in structure determination or NMR screening of RNA. Stable isotope labeling with 13C, 15N, and 2H is a requirement for application of multidimensional NMR studies for structural biology of macromolecules. Existing methods for labeling of RNA and DNA suffer from three main drawbacks. First, the production of isotopically labeled nucleotides is complex and time consuming and difficult to carry out in individual laboratories. Second, it is extremely expensive, since the materials for a single sample can cost many thousands of dollars. Third, the existing methods only permit uniform isotope labeling, while there is considerable advantage to selective isotope labeling. Recently, we have developed technology that will ameliorate or eliminate these three drawbacks. The technology involves the efficient single-pot synthesis of labeled ribonucleotides from labeled precursors using a series of enzymatic transformations. In order to make these reagents widely available to the structural biology community, we propose to optimize and scale up production of these reagents for commercial distribution.

描述（由申请人提供）：拟议研究的目标是扩大含有稳定同位素标记的高价值核糖核苷三磷酸的生产和制造。这些产品用于使用核磁共振 (NMR) 光谱对 RNA 结构进行生物分子研究。这些产品的市场是学术和工业实验室中从事 RNA 结构测定或 NMR 筛选的结构生物学小组。 13C、15N 和 2H 稳定同位素标记是大分子结构生物学多维 NMR 研究应用的要求。现有的 RNA 和 DNA 标记方法存在三个主要缺点。首先，同位素标记的核苷酸的生产复杂且耗时，并且难以在单个实验室中进行。其次，它非常昂贵，因为单个样品的材料可能花费数千美元。第三，现有方法只能实现均匀同位素标记，而选择性同位素标记具有相当大的优势。 最近，我们开发了可以改善或消除这三个缺点的技术。 该技术涉及使用一系列酶促转化从标记前体中高效单锅合成标记核糖核苷酸。为了使这些试剂广泛应用于结构生物学界，我们建议优化和扩大这些试剂的生产以进行商业分销。