Enzymatic Synthesis of RNA
RNA 的酶法合成
基本信息
- 批准号:10631998
- 负责人:
- 金额:$ 30万
- 依托单位:
- 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:
- 关键词:AffinityAlkaliesArchitectureBinding ProteinsBiologyBiomedical ResearchBiotechnologyBudgetsCRISPR/Cas technologyCatalysisChemicalsChemistryClinicClustered Regularly Interspaced Short Palindromic RepeatsCollaborationsComplementCost MeasuresCouplingCustomDNADNA FoldingDNA biosynthesisDNA chemical synthesisDNA sequencingDependenceEngineeringEnzymatic BiochemistryEnzymesError SourcesFailureFoundationsFranceGeneticGenome engineeringGuanosineInvestigationLaboratoriesLearningLengthLettersLicensingLigaseLigationMedicineMethodsModificationMole the mammalMolecular EvolutionNational Human Genome Research InstituteNational Institute of General Medical SciencesNucleosidesNucleotidesOligonucleotidesPhasePolymerasePrimer ExtensionProceduresRNARNA FoldingRNA Ligase (ATP)RNA SequencesRNA chemical synthesisRNA primersReagentResearchResearch PersonnelRibonuclease HRibonucleosidesRoleSalesSmall RNASolidTechniquesTechnologyTechnology TransferTransferaseUniversitiesVariantVertebral columnWorkaptamerbasecostdeoxyribonucleoside triphosphateimprovedinsertion/deletion mutationinsightinstrumentinventionmutantnext generationnext generation sequencingnovelnucleobasenucleoside triphosphatenucleotide analogphosphoramiditeprogramssuccesstherapeutic RNAtooltranscriptomicstripolyphosphate
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
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:
- 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
{{
item.title }}
{{ item.translation_title }}
- DOI:
{{ item.doi }} - 发表时间:
{{ item.publish_year }} - 期刊:
- 影响因子:{{ item.factor }}
- 作者:
{{ item.authors }} - 通讯作者:
{{ item.author }}
数据更新时间:{{ journalArticles.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ monograph.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ sciAawards.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ conferencePapers.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ patent.updateTime }}
STEVEN A BENNER其他文献
STEVEN A BENNER的其他文献
{{
item.title }}
{{ item.translation_title }}
- DOI:
{{ item.doi }} - 发表时间:
{{ item.publish_year }} - 期刊:
- 影响因子:{{ item.factor }}
- 作者:
{{ item.authors }} - 通讯作者:
{{ item.author }}
{{ truncateString('STEVEN A BENNER', 18)}}的其他基金
Basic Research for Diagnostics and Surveillance in Lower Resource Environments
低资源环境诊断和监测基础研究
- 批准号:
10669039 - 财政年份:2021
- 资助金额:
$ 30万 - 项目类别:
Easily Used Kits to Evolve Reagents that Covalently Tag and Inactivate Proteins
易于使用的试剂盒可进化出共价标记和灭活蛋白质的试剂
- 批准号:
10626917 - 财政年份:2021
- 资助金额:
$ 30万 - 项目类别:
Easily Used Kits to Evolve Reagents that Covalently Tag and Inactivate Proteins
易于使用的试剂盒可进化出共价标记和灭活蛋白质的试剂
- 批准号:
10478279 - 财政年份:2021
- 资助金额:
$ 30万 - 项目类别:
Basic Research for Diagnostics and Surveillance in Lower Resource Environments
低资源环境诊断和监测基础研究
- 批准号:
10468606 - 财政年份:2021
- 资助金额:
$ 30万 - 项目类别:
Easily Used Kits to Evolve Reagents that Covalently Tag and Inactivate Proteins
易于使用的试剂盒可进化出共价标记和灭活蛋白质的试剂
- 批准号:
10298982 - 财政年份:2021
- 资助金额:
$ 30万 - 项目类别:
Equipment Supplement to 1R01GM141391-01A1 (Easily Used Kits to Evolve Reagents that Covalently Tag and Inactivate Proteins)
1R01GM141391-01A1 的设备补充(易于使用的试剂盒,用于进化共价标记和灭活蛋白质的试剂)
- 批准号:
10580301 - 财政年份:2021
- 资助金额:
$ 30万 - 项目类别:
PHS2019-02 Omnibus Solic of the NIH, CDC, and FDA for SBIR Apps No Clinical Trial (Parent SBIR R43/4
PHS2019-02 NIH、CDC 和 FDA 的 SBIR 应用程序综合 Solic 尚未进行临床试验(母公司 SBIR R43/4
- 批准号:
10476977 - 财政年份:2021
- 资助金额:
$ 30万 - 项目类别:
Reagents to Chemically Tag Specific Coronavirus Spike Proteins
化学标记特定冠状病毒刺突蛋白的试剂
- 批准号:
10259048 - 财政年份:2021
- 资助金额:
$ 30万 - 项目类别:
相似海外基金
Understanding the Volatile Evolution of Arc Magmas: Alkalies and Mixed (H2O + CO2) Volatile Solubility in Basalts and Basaltic Andesites
了解弧岩浆的挥发性演化:玄武岩和玄武安山岩中的碱金属和混合 (H2O CO2) 挥发性溶解度
- 批准号:
0838563 - 财政年份:2009
- 资助金额:
$ 30万 - 项目类别:
Standard Grant
Clinker's coating:role of alkalies and refractories texture
熟料的涂层:碱和耐火材料质地的作用
- 批准号:
200906-1996 - 财政年份:1999
- 资助金额:
$ 30万 - 项目类别:
Collaborative Research and Development Grants
Clinkers coating:role of alkalies and refractories texture
熟料涂层:碱和耐火材料质地的作用
- 批准号:
200906-1996 - 财政年份:1998
- 资助金额:
$ 30万 - 项目类别:
Collaborative Research and Development Grants
Clinker's coating:role of alkalies and refractories texture
熟料的涂层:碱和耐火材料质地的作用
- 批准号:
200906-1996 - 财政年份:1997
- 资助金额:
$ 30万 - 项目类别:
Collaborative Research and Development Grants