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.

酶促RNA合成

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