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: 10552062
• 负责人: Maksim Royzen
• 金额: $ 19.37万
• 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/10552062
• 关键词: Address; Adenosine; Anions; Biochemical; Biological; Biological Assay; Biomedical Research; Biotinylation; Cells; Cellular biology; Chemicals; Chemistry; Communities; Complex; Coumarins; Coupling; Cyclooctenes; DNA; Diels Alder reaction; Electrons; Elements; Exposure to; Failure; Gene Expression Regulation; Genetic Diseases; 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; Predisposition; 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; chemical synthesis; cross reactivity; design; endonuclease; flexibility; functional group; genomic tools; improved; instrument; instrumentation; mRNA Translation; phosphodiester; posttranscriptional; 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.

标题：含长RNA寡核苷酸的化学合成和非色谱纯化 自然发生的变化。 摘要： RNA的固相合成是一种重要的基因组工具，其提供了对RNA的精确控制。 寡核苷酸序列，并提供了RNA修饰的位点特异性掺入的机会， 荧光标记和生化标签。21世纪的发现创造了一个强大的需求， RNA链的合成，长度为100-200个核苷酸（-nt）。一个主要的限制，否则高度 优化的方法是纯化，这是众所周知的困难，劳动密集型，并需要昂贵的HPLC 仪器仪表因此，含有RNA修饰的长寡核苷酸的固相合成是可行的。 很少尝试。这项技术开发提案旨在通过开发一种非 - 层析RNA纯化方法，其将快10倍且便宜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。 提出的净化策略是基于生物正交逆电子需求狄尔斯-阿尔德（IEDDA） 反式环辛烯（TCO）和四嗪（Tz）之间的化学反应， 结构复杂，并从固相过程中产生的失败链增加长RNA链 合成. TCO和Tz对彼此具有高度选择性，并且与其它官能化的化合物具有最小的交叉反应性。 在RNA中发现。生物正交点击化学是高度有效的，即使在非常低的浓度下。 TCO和Tz。在初步研究中，我们已经表明，我们的策略允许有效的合成， 纯化76-nt长的tRNA和101-nt长的sgRNA，产率比常规方法高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， 使整个过程在7小时以下，进一步提高分离RNA的总产率和纯度。在Aim中 1，我们建议通过开发一种新的光不稳定连接体来加快这一过程，该连接体将允许快速光裂解 使用可见光。为了提高RNA的纯度和产率，我们建议优化固相合成 确保在每个合成循环期间完全覆盖所有失效序列的程序。优化 这一过程将适用于越来越长的RNA链，从76-nt tRNA到188-nt长的U2 snRNA。在目标2中， 我们计划通过合成一个144个核苷酸长的人工mRNA来说明我们的技术的力量， 和起始密码子附近的m6 A修饰。这将是报道的最长的含有RNA的寡核苷酸 修改.人工mRNA将被用于研究RNA修饰对转录速率的影响。 体外翻译我们计划与NHGRI技术开发团队合作， 广泛提供给研究人员。