Enzymatic Synthesis of RNA
RNA 的酶法合成
基本信息
- 批准号:10201263
- 负责人:
- 金额:$ 77.42万
- 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:AffinityAlkaliesArchitectureBindingBiologyBiomedical ResearchBiotechnologyBudgetsCRISPR/Cas technologyCatalysisChemicalsChemistryClinicClustered Regularly Interspaced Short Palindromic RepeatsCollaborationsComplementCost MeasuresCouplingCustomDNADNA FoldingDNA biosynthesisDNA chemical synthesisDNA sequencingDependenceEngineeringEnzymatic BiochemistryEnzymesError SourcesFailureFoundationsFranceGeneticGenome engineeringGuanosineInvestigationLaboratoriesLearningLengthLettersLicensingLigaseLigationMedicineMethodsModificationMolecular EvolutionNational Human Genome Research InstituteNational Institute of General Medical SciencesNucleosidesNucleotidesOligonucleotidesPhasePolymerasePrimer ExtensionProceduresRNARNA FoldingRNA Ligase (ATP)RNA SequencesRNA chemical synthesisRNA primersReagentResearchResearch PersonnelRibonuclease HRibonucleosidesRoleSalesSmall RNASolidTechniquesTechnologyTechnology TransferTimeTransferaseUniversitiesVariantVertebral columnWorkaptamerbasecostdeoxyribonucleoside triphosphateimprovedinsightinstrumentinventionmutantnext 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.
酶促RNA合成
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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STEVEN A BENNER其他文献
STEVEN A BENNER的其他文献
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{{ 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:
$ 77.42万 - 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:
$ 77.42万 - 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:
$ 77.42万 - 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:
$ 77.42万 - 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:
$ 77.42万 - 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:
$ 77.42万 - 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:
$ 77.42万 - 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:
$ 77.42万 - 项目类别:
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