Enzymatic Synthesis of RNA

RNA 的酶法合成

• 批准号: 10631998
• 负责人: STEVEN A BENNER
• 金额: $ 30万
• 依托单位: FOUNDATION FOR APPLIED MOLECULAR EVOLUTN
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10631998
• 关键词: Affinity; Alkalies; Architecture; Binding Proteins; Biology; Biomedical Research; Biotechnology; Budgets; CRISPR/Cas technology; Catalysis; Chemicals; Chemistry; Clinic; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Complement; Cost Measures; Coupling; Custom; DNA; DNA Folding; DNA biosynthesis; DNA chemical synthesis; DNA sequencing; Dependence; Engineering; Enzymatic Biochemistry; Enzymes; Error Sources; Failure; Foundations; France; Genetic; Genome engineering; Guanosine; Investigation; Laboratories; Learning; Length; Letters; Licensing; Ligase; Ligation; Medicine; Methods; Modification; Mole the mammal; Molecular Evolution; National Human Genome Research Institute; National Institute of General Medical Sciences; Nucleosides; Nucleotides; Oligonucleotides; Phase; Polymerase; Primer Extension; Procedures; RNA; RNA Folding; RNA Ligase (ATP); RNA Sequences; RNA chemical synthesis; RNA primers; Reagent; Research; Research Personnel; Ribonuclease H; Ribonucleosides; Role; Sales; Small RNA; Solid; Techniques; Technology; Technology Transfer; Transferase; Universities; Variant; Vertebral column; Work; aptamer; base; cost; deoxyribonucleoside triphosphate; improved; insertion/deletion mutation; insight; instrument; invention; mutant; next generation; next generation sequencing; novel; nucleobase; nucleoside triphosphate; nucleotide analog; phosphoramidite; programs; success; therapeutic RNA; tool; transcriptomics; tripolyphosphate

Enzymatic Synthesis of RNA Foundation for Applied Molecular Evolution Thomas Jefferson University Steven Benner Richard Pomerantz ABSTRACT The demand for synthetic RNA in biotechnology, research, and the clinic has increased dramatically in the last few years. This is due inter alia to novel CRISPR-Cas9 genome engineering techniques, the re-invention of aptamers and aptazymes with picomolar affinities using expanded genetic alphabets, and investigations of small RNAs in mammalian biology, all relying on synthetic RNA. Even with some of the best firms advancing classical phosphoramidite chemistry, 20 nmoles of an 120 nucleotide Ultramer® still costs $1080, a severe limit on researchers asking "Why not?" and "What if?" questions using synthetic RNA. The cost of RNA would be dramatically lowered if phosphoramidite chemistry were replaced by enzyme- assisted RNA synthesis. Two advances make it now timely to achieve this "Grand Challenge". Chemistry. The Benner lab invented a removable 3'-O aminoxy (ONH2) group for NextGen sequencing. Now licensed to DNA Script in a "dual use mode" for enzyme-assisted DNA synthesis, aminoxies generate 200- mers in good purity and yield. In a virtuous cycle, this led us to develop low cost solid-phase methods to make aminoxy triphosphates at < $1/micromole, and methods to make 3'-O-aminoxy ribonucleoside triphosphates. Enzymology. Marc Delarue (collaboration letter), DNA Script (collaboration letter), and Richard Pomerantz (co-Investigator) discovered enzymes, including polymerase  and its variants, that add ribonucleosides to an RNA primer. This creates an architecture for enzyme-assisted RNA synthesis based on aminoxy termination that complements a classical architecture that exploits RNA ligase. In Aim 1, we will use a classical architecture involving the ligation of nucleoside 3',5'-bisphosphates to learn how to manage folding that occurs in natural RNA during enzyme-assisted synthesis. Even more than with enzyme-assisted DNA synthesis, this folding obstructs the synthesis of a full range of RNA sequences. Novel transformable, self-deprotecting, and soft deprotectable modifications should allow this problem to be resolve. As Aim 2, we will engineer Pol  variants to find those that accept the 4 standard nucleotides in a Fig. 4.2 architecture that exploits 3'-ONH2 reversible terminators. These will be metricked by (i) rate of incorporation, (ii) sequence independence of incorporation, and (iii) length dependence of these. The principal sources of error (coupling failure leading to single nucleotide deletion) will be rigorously metricked As Aim 3, we will implement a semi-automatic platform for RNA synthesis. We will also use ligases and Pol  variants to incorporate "next generation" nucleotide analogs that have value in therapeutic RNA, RNA aptamers and aptazymes, and RNA tagging. This will attract commercial instrument makers (e.g. DNA Script and Nuclera were both contacted about this platform) to adapt their instrument to our chemistry/ enzymology. Even before this happens, our semi-automatic platform will allow this technology to be transferred to NHGRI centers that are chosen under NHGRI RFA-HG-20-019, a parallel RFA now accepting applications.

RNA 的酶法合成 应用分子进化基金会 托马斯杰斐逊大学 史蒂文·本纳 理查德·波美兰兹 抽象的 生物技术、研究和临床对合成 RNA 的需求急剧增加 过去几年。这主要归功于新颖的 CRISPR-Cas9 基因组工程技术，即重新发明的 使用扩展遗传字母表具有皮摩尔亲和力的适体和适体酶，以及 哺乳动物生物学中的小RNA，全部依赖于合成RNA。即使一些最好的公司正在进步 经典的亚磷酰胺化学，20 纳摩尔的 120 核苷酸 Ultramer® 仍然花费 1080 美元，这是一个严重的问题 限制研究人员询问“为什么不呢？”和“如果呢？”使用合成 RNA 的问题。 如果亚磷酰胺化学被酶取代，RNA 的成本将大大降低 辅助RNA合成。两项进展使得现在能够及时实现这一“重大挑战”。 化学。 Benner 实验室发明了用于 NextGen 测序的可移除 3'-O 氨氧基 (ONH2) 基团。 现已授权 DNA Script 以“双重使用模式”进行酶辅助 DNA 合成，氨氧可产生 200- mer 具有良好的纯度和产率。在良性循环中，这促使我们开发低成本固相方法来制造 < 1 美元/微摩尔的氨氧基三磷酸，以及制备 3'-O-氨氧基核糖核苷三磷酸的方法。 酶学。 Marc Delarue（合作信）、DNA Script（合作信）和 Richard Pomerantz（共同研究员）发现了酶，包括聚合酶  及其变体，可以添加 将核糖核苷连接至RNA引物。这创建了一个基于酶辅助 RNA 合成的架构 氨氧基终止补充了利用 RNA 连接酶的经典架构。 在目标 1 中，我们将使用涉及核苷 3',5'-二磷酸连接的经典架构来学习 如何管理酶辅助合成过程中天然 RNA 中发生的折叠。甚至比与 在酶辅助 DNA 合成中，这种折叠阻碍了全范围 RNA 序列的合成。小说 可变形、自去保护和软去保护修改应该可以解决这个问题。 作为目标 2，我们将设计 Pol  变体，以找到接受图 4.2 中 4 个标准核苷酸的变体 利用 3'-ONH2 可逆终止子的架构。这些将通过 (i) 合并率来衡量， (ii) 掺入的序列独立性，以及 (iii) 这些的长度依赖性。主要来源 错误（导致单核苷酸缺失的偶联失败）将被严格衡量 作为目标 3，我们将实现一个用于 RNA 合成的半自动平台。我们还将使用连接酶和 Pol  掺入“下一代”核苷酸类似物的变体，这些核苷酸类似物在治疗性RNA、RNA中具有价值 适体和适体酶，以及 RNA 标签。这将吸引商业仪器制造商（例如 DNA Script 和 Nuclera 均就该平台进行了联系）以使他们的仪器适应我们的化学/酶学。 甚至在此之前，我们的半自动平台将允许这项技术转移到 NHGRI 根据 NHGRI RFA-HG-20-019 选择的中心，这是一个并行 RFA，现已接受申请。

项目成果

Promoter-independent synthesis of chemically modified RNA by human DNA polymerase θ variants.

由人类 DNA 聚合酶 α 变体进行化学修饰 RNA 的启动子独立合成。

• DOI: 10.1261/rna.079396.122
• 发表时间: 2023
• 期刊: RNA (New York, N.Y.)
• 作者: Tredinnick,Taylor;Kent,Tatiana;Minakhin,Leonid;Li,Ziyuan;Madzo,Jozef;Chen,XiaojiangS;Pomerantz,RichardT
• 通讯作者: Pomerantz,RichardT