Synthesis and non-chromatographic purification of long RNA oligonucleotides containing naturally occurring modification

含有天然修饰的长 RNA 寡核苷酸的合成和非色谱纯化

• 批准号: 10364172
• 负责人: Maksim Royzen
• 金额: $ 23.24万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT ALBANY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-18 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10364172
• 关键词: Address; Adenosine; Alder plant; Anions; Biochemical; Biological; Biological Assay; Biomedical Research; Cells; Cellular biology; Chemicals; Chemistry; Communities; Complex; Coumarins; Coupling; Cyclooctenes; DNA; Electrons; Elements; Ensure; Exposure to; Failure; Gene Expression Regulation; Genetic Diseases; Genetic Translation; Genomics; Goals; High Pressure Liquid Chromatography; In Vitro; Initiator Codon; Isotope Labeling; Label; Length; Light; Messenger RNA; Methodology; Methods; Modification; Molecular; National Human Genome Research Institute; Nucleotides; Oligonucleotides; Oryctolagus cuniculus; Perception; Phase; Procedures; Process; Production; Proteins; Public Health; RNA; RNA chemical synthesis; RNA purification; RNA-Protein Interaction; Reporting; Research; Research Personnel; Reticulocytes; Role; S phase; Site; Solid; Speed; System; Technology; Time; Transfer RNA; Translations; U2 small nuclear RNA; Ultraviolet Rays; Vertebral column; Visible Radiation; Work; analog; base; chemical synthesis; cross reactivity; design; endonuclease; flexibility; functional group; genomic tools; improved; instrument; instrumentation; phosphodiester; process optimization; programs; technology development; therapeutic gene; tool

Title: Chemical synthesis and non-chromatographic purification of long RNA oligonucleotides containing naturally occurring modifications. ABSTRACT: Solid phase synthesis of RNA is an important genomic tool, which offers precise control over the oligonucleotide sequence and offers an opportunity for site-specific incorporation of RNA modifications, fluorescent labels and biochemical tags. Discoveries of the twenty-first century created a strong need for a robust synthesis of RNA strands, that are 100-200 nucleotides (-nt) in length. A major limitation of otherwise highly optimized process is purification, which is notoriously difficult, labor intensive and requires expensive HPLC instrumentation. As the result, solid phase synthesis of long oligonucleotides containing RNA modifications is rarely attempted. This technology-development proposal aims to address this limitation by developing a non- chromatographic RNA purification method which will be 10-times faster and 2-orders of magnitude cheaper and will allow isolation of strands that are 100-200-nt long in good yield and 98% purity. The proposed purification strategy is based on bio-orthogonal inverse electron demand Diels-Alder (IEDDA) chemistry between trans-cyclooctene (TCO) and tetrazine (Tz) that allows to selectively tag and purify structurally complex and increasing long RNA strands from the failure strands that accrue during solid phase synthesis. TCO and Tz are highly selective for each other and have minimal cross-reactivity with other functional groups found in RNA. The bio-orthogonal click chemistry is highly efficient, even at very low concentrations of TCO and Tz. During preliminary studies we have shown that our strategy allows efficient synthesis and purification of 76-nt long tRNA and 101-nt long sgRNA with yields that were 10-times higher than conventional purification methods. During the proposed research program, we aim to improve a number of important elements of our design to bring the overall process to under 7 hrs, further improve the overall yield and purity of the isolated RNA. In Aim 1, we propose to expedite the process by developing a new photolabile linker that will allow fast photocleavage using visible light. To improve purity and yield of isolated RNA, we propose to optimize the solid phase synthesis procedure to ensure that all failure sequences are fully capped during each synthetic cycle. The optimized process will be applied to increasingly longer RNA strands, from 76-nt tRNA to 188-nt long U2 snRNA. In Aim 2, we plan to illustrate the power of our technology by synthesizing a 144-nt long artificial mRNA, containing m1A and m6A modifications near the start codon. This will be the longest reported oligonucleotide containing RNA modifications. The artificial mRNA will be utilized to investigate the impact of RNA modifications on the rate of in vitro translation. We plan to work with NHGRI technology development team to make the proposed technology widely available to researchers.

长RNA寡核苷酸的化学合成及非层析纯化 自然发生的修改。 摘要： 固相合成RNA是一种重要的基因组学工具，它可以精确地控制 寡核苷酸序列并提供了RNA修饰的位点特异性掺入的机会， 荧光标签和生化标签。21世纪的发现产生了对强健的 合成核糖核酸链，长度为100-200个核苷酸(-nT)。在其他方面高度集中的主要限制 优化的过程是纯化，这是出了名的困难，劳动密集型，需要昂贵的高效液相 仪器仪表。其结果是，固相合成含有RNA修饰的长寡核苷酸 很少有人尝试过。这项技术开发提案旨在通过开发一种非 层析纯化RNA的方法，速度将快10倍，成本低2个数量级， 将允许以良好的产率和98%的纯度分离100-200-nT长的链。 提出的提纯策略是基于生物正交逆电子需求Diels-Alder(IEDDA) 反式环辛烯(TCO)和四嗪(TZ)之间的化学，允许选择性地标记和提纯 结构复杂且不断增加的长RNA链来自在固相过程中积累的故障链 综合。TCO和TZ对彼此具有很高的选择性，与其他官能团的交叉反应最小 在RNA中发现的基团。生物正交点击化学是高效的，即使在非常低的浓度 TCO和TZ。在初步研究中，我们的策略允许有效的合成和 纯化76个核苷酸的长tRNA和101个核苷酸的长sgRNA，产量比常规提纯提高10倍 纯化方法。 在拟议的研究计划中，我们的目标是改进我们设计的一些重要元素，以 将整个过程控制在7小时以内，进一步提高了总RNA的得率和纯度。在AIM 1，我们建议通过开发一种新的光敏连接物来加快这一过程，这种连接物将允许快速光切割 利用可见光。为了提高分离RNA的纯度和得率，我们建议优化固相合成 确保在每个合成周期内完全封顶所有故障序列的程序。经过优化的 这一过程将应用于越来越长的RNA链，从76个核苷酸的tRNA到188个核苷酸的U2 SnRNA。在目标2中， 我们计划通过合成包含M1a的144个核苷酸长的人造mRNA来说明我们技术的力量 和起始密码子附近的m6A修饰。这将是已报道的最长的含有RNA的寡核苷酸 修改。人工合成的信使核糖核酸将被用来研究RNA修饰对核糖核酸酶活性的影响。 体外翻译。我们计划与NHGRI技术开发团队合作，使拟议的技术 研究人员可以广泛使用。