PCR amplification and transcription for site-specific labeling of large RNA molecules by a two-unnatural-base-pair system.

IF 1.3 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY
Journal of Nucleic Acids Pub Date : 2012-01-01 Epub Date: 2012-06-26 DOI:10.1155/2012/230943
Michiko Kimoto, Rie Yamashige, Shigeyuki Yokoyama, Ichiro Hirao
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引用次数: 19

Abstract

For the site-specific labeling and modification of RNA by genetic alphabet expansion, we developed a PCR and transcription system using two hydrophobic unnatural base pairs: 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) as a third pair for PCR amplification and Ds and pyrrole-2-carbaldehyde (Pa) for the incorporation of functional components as modified Pa bases into RNA by T7 transcription. To prepare Ds-containing DNA templates with long chains, the Ds-Px pair was utilized in a fusion PCR method, by which we demonstrated the synthesis of 282-bp DNA templates containing Ds at specific positions. Using these Ds-containing DNA templates and a biotin-linked Pa substrate (Biotin-PaTP) as a modified Pa base, 260-mer RNA transcripts containing Biotin-Pa at a specific position were generated by T7 RNA polymerase. This two-unnatural-base-pair system, combining the Ds-Px and Ds-Pa pairs with modified Pa substrates, provides a powerful tool for the site-specific labeling and modification of desired positions in large RNA molecules.

Abstract Image

Abstract Image

Abstract Image

PCR扩增和转录的位点特异性标记的大RNA分子的两个非自然碱基对系统。
为了通过基因序列扩增对RNA进行位点特异性标记和修饰,我们开发了一种PCR和转录系统,使用两个疏水非天然碱基对:7-(2-噻吩基)-咪唑[4,5-b]吡啶(Ds)和2-硝基-4-丙基吡咯(Px)作为PCR扩增的第三对碱基对,以及Ds和吡咯-2-乙醛(Pa)作为修饰的Pa碱基通过T7转录将功能成分整合到RNA中。为了制备含Ds的长链DNA模板,我们利用Ds- px对进行融合PCR,在特定位置合成了282 bp的含Ds的DNA模板。利用这些含有ds的DNA模板和生物素连接的Pa底物(Biotin-PaTP)作为修饰的Pa碱基,T7 RNA聚合酶在特定位置生成260个含有生物素-Pa的RNA转录物。这两个非天然碱基对系统,结合了Ds-Px和Ds-Pa对与修饰的Pa底物,为大RNA分子中所需位置的位点特异性标记和修饰提供了强大的工具。
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来源期刊
Journal of Nucleic Acids
Journal of Nucleic Acids BIOCHEMISTRY & MOLECULAR BIOLOGY-
CiteScore
3.10
自引率
21.70%
发文量
5
审稿时长
12 weeks
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