Advancing the enzymatic toolkit for 2′-fluoro arabino nucleic acid (FANA) manipulation: phosphorylation, ligation, replication, and templating RNA transcription
文献类型：期刊论文
| 作者 | Liu, Yingyu1,2; Wang, Jun1,2; Wu, Yashu2,3; Wang, Yajun2,3 |
| 刊名 | CHEMICAL SCIENCE
 |
| 出版日期 | 2024-06-24 |
| ISSN号 | 2041-6520 |
| DOI | 10.1039/d4sc02904f |
| 通讯作者 | Wang, Yajun(wangyj@ibmc.ac.cn) |
| 英文摘要 | 2 '-Fluoro arabino nucleic acid (FANA), classified as a xeno nucleic acid (XNA), stands as a prominent subject of investigation in synthetic genetic polymers. Demonstrating efficacy as antisense oligonucleotides (ASOs) and exhibiting the ability to fold into functional structures akin to enzymes and aptamers, FANA holds substantial promise across diverse biological and therapeutic domains. Owing to structural similarities to DNA, the utilization of naturally occurring DNA polymerases for DNA-mediated FANA replication is well-documented. In this study, we explore alternative nucleic acid processing enzymes typically employed for DNA oligonucleotide (ON) phosphorylation, ligation, and amplification, and assess their compatibility with FANA substrates. Notably, T4 polynucleotide kinase (T4 PNK) efficiently phosphorylated the 5 '-hydroxyl group of FANA using ATP as a phosphate donor. Subsequent ligation of the phosphorylated FANA with an upstream FANA ON was achieved with T4 DNA ligase, facilitated by a DNA splint ON that brings the two FANA ONs into proximity. This methodology enabled the reconstruction of RNA-cleaving FANA 12-7 by ligating two FANA fragments amenable to solid-phase synthesis. Furthermore, Tgo DNA polymerase, devoid of 3 ' to 5 ' exonuclease activity [Tgo (exo-)], demonstrated proficiency in performing polymerase chain reaction (PCR) with a mixture of dNTPs and FANA NTPs (fNTPs), yielding DNA-FANA chimeras with efficiency and fidelity comparable to traditional DNA PCR. Notably, T7 RNA polymerase (T7 RNAP) exhibited recognition of double-stranded fA-DNA chimeras containing T7 promoter sequences, enabling in vitro transcription of RNA molecules up to 649 nt in length, even in the presence of highly structured F30 motifs at the 3 ' end. Our findings significantly expand the enzymatic toolkit for FANA manipulation, encompassing phosphorylation, ligation, chimeric amplification, and templating T7 RNAP-catalyzed RNA transcription. These advancements are poised to expedite fundamental research, functional evolution, and translational applications of FANA-based XNA agents. They also have the potential to inspire explorations of a broader range of non-natural nucleic acids that can be routinely studied in laboratories, ultimately expanding the repertoire of nucleic acid-based biomedicine and biotechnology. Scheme of the amplification of DNA-FANA chimera via single-fNTP substitutional PCR, the synthesis of RNA from DNA-FANA chimeric templates via IVT, followed by the activation of the fluorescent spinach aptamer upon the addition of DFHBI. |
| WOS关键词 | GENETIC POLYMERS |
| 资助项目 | Hangzhou Institute of Medicine, Chinese Academy of Sciences and Soochow University ; Young Scientists Fund of National Natural Science Foundation of China[22107083] |
| WOS研究方向 | Chemistry |
| 语种 | 英语 |
| WOS记录号 | WOS:001260041300001 |
| 出版者 | ROYAL SOC CHEMISTRY |
| 资助机构 | Hangzhou Institute of Medicine, Chinese Academy of Sciences and Soochow University ; Young Scientists Fund of National Natural Science Foundation of China |
| 源URL | [http://ir.hfcas.ac.cn:8080/handle/334002/136689]  |
| 专题 | 中国科学院合肥物质科学研究院 |
| 通讯作者 | Wang, Yajun |
| 作者单位 | 1.Soochow Univ, Coll Pharmaceut Sci, Suzhou 215123, Peoples R China 2.Chinese Acad Sci, Hangzhou Inst Med HIM, Hangzhou 310000, Zhejiang, Peoples R China 3.Univ Chinese Acad Sci, Canc Hosp, Zhejiang Canc Hosp, Hangzhou 310022, Zhejiang, Peoples R China |
| 推荐引用方式 GB/T 7714 | Liu, Yingyu,Wang, Jun,Wu, Yashu,et al. Advancing the enzymatic toolkit for 2′-fluoro arabino nucleic acid (FANA) manipulation: phosphorylation, ligation, replication, and templating RNA transcription[J]. CHEMICAL SCIENCE,2024. |
| APA | Liu, Yingyu,Wang, Jun,Wu, Yashu,&Wang, Yajun.(2024).Advancing the enzymatic toolkit for 2′-fluoro arabino nucleic acid (FANA) manipulation: phosphorylation, ligation, replication, and templating RNA transcription.CHEMICAL SCIENCE. |
| MLA | Liu, Yingyu,et al."Advancing the enzymatic toolkit for 2′-fluoro arabino nucleic acid (FANA) manipulation: phosphorylation, ligation, replication, and templating RNA transcription".CHEMICAL SCIENCE (2024). |
入库方式： OAI收割
来源：合肥物质科学研究院
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