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的酶促合成 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·波梅兰茨 摘要 近年来，生物技术、研究和临床对合成RNA的需求急剧增加。 最近几年这阿利亚是由于新的CRISPR-Cas9基因组工程技术， 使用扩展的遗传字母表的具有皮摩尔亲和力的适体和适体酶， 哺乳动物生物学中的小RNA，都依赖于合成RNA。即使一些最好的公司 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，严重的 限制研究人员问“为什么不呢？“和“如果？“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。两项进展使得现在实现这一“重大挑战”是及时的。 化学. Benner实验室发明了用于NextGen测序的可移除的3 '-O氨氧基（ONH 2）基团。 现在，DNA Script被授权以“双重使用模式”进行酶辅助DNA合成，氨氧基产生200- 纯度和产率良好。在一个良性循环中，这促使我们开发了低成本的固相方法来制造 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（合作研究者）发现了酶，包括聚合酶及其变体， 将核糖核苷与RNA引物连接。这创造了一种基于以下的酶辅助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'-二磷酸连接的经典结构来学习 如何管理在酶辅助合成过程中发生在天然RNA中的折叠。甚至比与 在酶辅助的DNA合成中，这种折叠阻碍了全范围RNA序列的合成。小说 可转化、自脱保护和可软脱保护修饰应允许解决该问题。 作为目标2，我们将工程化Pol突变体，以找到接受图4.2中的4个标准核苷酸的那些。 利用3 '-ONH 2可逆终止子的结构。这些将通过（i）合并率， (ii)掺入的序列独立性，和（iii）这些的长度依赖性。的主要来源 错误（偶联失败导致单核苷酸缺失）将被严格计量 作为目标3，我们将实现一个半自动的RNA合成平台。我们还将使用连接酶和聚合酶， 变体以掺入在治疗性RNA、RNA和RNA合成中具有价值的“下一代”核苷酸类似物， 适体和适体酶以及RNA标记。这将吸引商业仪器制造商（例如DNA Script 和Nuclera都联系了这个平台），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，一个平行的RFA现在接受申请。

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