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Enzymatic Synthesis of RNA

RNA 的酶法合成

基本信息

批准号：
10201263
负责人：
STEVEN A BENNER
金额：
$ 77.42万
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/10201263
关键词：
Affinity Alkalies Architecture Binding 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 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 Time Transferase Universities Variant Vertebral column Work aptamer base cost deoxyribonucleoside triphosphate improved 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.

酶促RNA合成