PCR Amplification and Transcription for Site-Specific Labeling of Large RNA Molecules by a Two-Unnatural-Base-Pair System
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...
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| Format: | Article |
| Language: | English |
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Wiley
2012-01-01
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| Series: | Journal of Nucleic Acids |
| Online Access: | http://dx.doi.org/10.1155/2012/230943 |
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| author | Michiko Kimoto Rie Yamashige Shigeyuki Yokoyama Ichiro Hirao |
| author_facet | Michiko Kimoto Rie Yamashige Shigeyuki Yokoyama Ichiro Hirao |
| author_sort | Michiko Kimoto |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-c71e8a9f50bb464089e059be0f15eba1 |
| institution | Kabale University |
| issn | 2090-0201 2090-021X |
| language | English |
| publishDate | 2012-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | Journal of Nucleic Acids |
| spelling | doaj-art-c71e8a9f50bb464089e059be0f15eba12025-02-03T05:59:12ZengWileyJournal of Nucleic Acids2090-02012090-021X2012-01-01201210.1155/2012/230943230943PCR Amplification and Transcription for Site-Specific Labeling of Large RNA Molecules by a Two-Unnatural-Base-Pair SystemMichiko Kimoto0Rie Yamashige1Shigeyuki Yokoyama2Ichiro Hirao3RIKEN Systems and Structural Biology Center (SSBC), 1-7-22 Suehiro-cho, Tsurumi-ku, Kanagawa, Yokohama, 230-0045, JapanRIKEN Systems and Structural Biology Center (SSBC), 1-7-22 Suehiro-cho, Tsurumi-ku, Kanagawa, Yokohama, 230-0045, JapanRIKEN Systems and Structural Biology Center (SSBC), 1-7-22 Suehiro-cho, Tsurumi-ku, Kanagawa, Yokohama, 230-0045, JapanRIKEN Systems and Structural Biology Center (SSBC), 1-7-22 Suehiro-cho, Tsurumi-ku, Kanagawa, Yokohama, 230-0045, JapanFor 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.http://dx.doi.org/10.1155/2012/230943 |
| spellingShingle | Michiko Kimoto Rie Yamashige Shigeyuki Yokoyama Ichiro Hirao PCR Amplification and Transcription for Site-Specific Labeling of Large RNA Molecules by a Two-Unnatural-Base-Pair System Journal of Nucleic Acids |
| title | PCR Amplification and Transcription for Site-Specific Labeling of Large RNA Molecules by a Two-Unnatural-Base-Pair System |
| title_full | PCR Amplification and Transcription for Site-Specific Labeling of Large RNA Molecules by a Two-Unnatural-Base-Pair System |
| title_fullStr | PCR Amplification and Transcription for Site-Specific Labeling of Large RNA Molecules by a Two-Unnatural-Base-Pair System |
| title_full_unstemmed | PCR Amplification and Transcription for Site-Specific Labeling of Large RNA Molecules by a Two-Unnatural-Base-Pair System |
| title_short | PCR Amplification and Transcription for Site-Specific Labeling of Large RNA Molecules by a Two-Unnatural-Base-Pair System |
| title_sort | pcr amplification and transcription for site specific labeling of large rna molecules by a two unnatural base pair system |
| url | http://dx.doi.org/10.1155/2012/230943 |
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