Enzymatic Synthesis of RNA

RNA 的酶法合成

基本信息

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
项目类别：
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

项目摘要

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础托马斯·杰斐逊大学史蒂文·本纳理查德·波美兰兹（Richard Pomerantz）抽象的生物技术，研究和诊所中合成RNA的需求在最近几年。这是由于新颖的CRISPR-CAS9基因组工程技术的外套，重新发明使用扩展的遗传字母的具有皮摩尔亲和力的适体和忠实剂，并研究哺乳动物生物学中的小RNA，均依赖于合成RNA。即使有一些最好的公司前进经典的磷光化学化学，120个核苷酸Ultramer®的20个nmoles仍然成本1080美元，这是严重的限制研究人员询问“为什么不呢？”和“如果？”使用合成RNA的问题。如果磷酸化学被酶取代，RNA的成本将大大降低辅助RNA合成。现在有两个进步使得实现这一“大挑战”的及时。化学。本纳实验室（Benner Lab）发明了一个可移动的3'-o aminooxy（ONH2）组进行NextGen测序。现在以“双重使用模式”为酶辅助DNA合成的DNA脚本许可，Aminoxies会产生200- 纯度和产量。在虚拟周期中，这使我们开发了低成本的固相方法氨基三磷酸盐<$ 1/micromole，以及制造3'-o-aminooxy-ariminooxy-ribbonauleoside三磷酸盐的方法。酶学。 Marc Delarue（合作信），DNA脚本（合作信）和理查德 Pomerantz（共同投资者）发现了包括聚合酶及其变体的酶 RNA底漆的丝封剂。这创建了基于酶辅助的RNA合成的结构 Aminoximal终止完成了利用RNA连接酶的经典体系结构。在AIM 1中，我们将使用涉及3'，5'双磷酸盐的核能连接的古典体系结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。甚至比酶辅助的DNA合成，这种折叠阻碍了整个RNA序列的合成。小说可转换，自我抑制和软脱抛弃的修改应允许解决此问题。作为目标2，我们将设计pol的变体，以找到那些接受图4.2中4个标准核苷酸的变体。利用3'-ONH2可逆终结器的体系结构。这些将由（i）就业率，（ii）公司的序列独立性和（iii）这些的长度依赖性。主要来源误差（导致单核苷酸缺失的耦合故障）将被严格衡量作为目标3，我们将实施一个半自动平台以进行RNA合成。我们还将使用连接酶和pol 在治疗RNA中具有价值的“下一代”核苷酸类似物的变体RNA 适中剂和适量齐，以及RNA标记。这将吸引商业仪器制造商（例如DNA脚本在此平台上都联系了Nuclera和Nuclera，以使其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该技术转移到NHGRI 根据NHGRI RFA-HG-20-019选择的中心，现在接受应用程序。

项目成果

期刊论文数量（1）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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.)
影响因子：
0
作者：
Tredinnick,Taylor;Kent,Tatiana;Minakhin,Leonid;Li,Ziyuan;Madzo,Jozef;Chen,XiaojiangS;Pomerantz,RichardT
通讯作者：
Pomerantz,RichardT