Treffer: Advancing Applications of the Expanded Genetic Alphabet: Monitoring Expanded Genetic Letters in Complex DNA Context Via a Bridge-Base Approach.
Title:
Advancing Applications of the Expanded Genetic Alphabet: Monitoring Expanded Genetic Letters in Complex DNA Context Via a Bridge-Base Approach.
Authors:
Wang H; Henan Key Laboratory of Organic Functional Molecule and Drug Innovation, NMPA Key Laboratory for Research and Evaluation of Innovative Drug, Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals, School of Chemistry and Chemical Engineering, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan Province, China., Li S; Henan Key Laboratory of Organic Functional Molecule and Drug Innovation, NMPA Key Laboratory for Research and Evaluation of Innovative Drug, Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals, School of Chemistry and Chemical Engineering, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan Province, China., Wang C; Henan Key Laboratory of Organic Functional Molecule and Drug Innovation, NMPA Key Laboratory for Research and Evaluation of Innovative Drug, Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals, School of Chemistry and Chemical Engineering, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan Province, China., Zhu A; Henan Key Laboratory of Organic Functional Molecule and Drug Innovation, NMPA Key Laboratory for Research and Evaluation of Innovative Drug, Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals, School of Chemistry and Chemical Engineering, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan Province, China., Li L; Henan Key Laboratory of Organic Functional Molecule and Drug Innovation, NMPA Key Laboratory for Research and Evaluation of Innovative Drug, Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals, School of Chemistry and Chemical Engineering, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan Province, China.
Source:
Current protocols [Curr Protoc] 2025 Nov; Vol. 5 (11), pp. e70250.
Publication Type:
Journal Article
Language:
English
Journal Info:
Publisher: John Wiley & Sons Country of Publication: United States NLM ID: 101773894 Publication Model: Print Cited Medium: Internet ISSN: 2691-1299 (Electronic) Linking ISSN: 26911299 NLM ISO Abbreviation: Curr Protoc Subsets: MEDLINE
Imprint Name(s):
Original Publication: Hoboken, NJ : John Wiley & Sons, [2021]-
MeSH Terms:
References:
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Feldman, A. W., Dien, V. T., Karadeema, R. J., Fischer, E. C., You, Y., Anderson, B. A., Krishnamurthy, R., Chen, J. S., Li, L., & Romesberg, F. E. (2019). Optimization of replication, transcription, and translation in a semi‐synthetic organism. Journal of the American Chemical Society, 141(27), 10644–10653. https://doi.org/10.1021/jacs.9b02075.
Fischer, E. C., Hashimoto, K., Zhang, Y., Feldman, A. W., Dien, V. T., Karadeema, R. J., Adhikary, R., Ledbetter, M. P., Krishnamurthy, R., & Romesberg, F. E. (2020). New codons for efficient production of unnatural proteins in a semisynthetic organism. Nature Chemical Biology, 16(5), 570–576. https://doi.org/10.1038/s41589‐020‐0507‐z.
Hirao, I., Kimoto, M., Mitsui, T., Fujiwara, T., Kawai, R., Sato, A., Harada, Y., & Yokoyama, S. (2006). An unnatural hydrophobic base pair system: Site‐specific incorporation of nucleotide analogs into DNA and RNA. Nature Methods, 3(9), 729–735. https://doi.org/10.1038/nmeth915.
Kimoto, M., & Hirao, I. (2020). Genetic alphabet expansion technology by creating unnatural base pairs. Chemical Society Reviews, 49(21), 7602–7626. https://doi.org/10.1039/d0cs00457j.
Kimoto, M., Mitsui, T., Yamashige, R., Sato, A., Yokoyama, S., & Hirao, I. (2010). A new unnatural base pair system between fluorophore and quencher base analogues for nucleic acid‐based imaging technology. Journal of the American Chemical Society, 132(43), 15418–15426. https://doi.org/10.1021/ja1072383.
Kimoto, M., Shermane Lim, Y. W., & Hirao, I. (2019). Molecular affinity rulers: Systematic evaluation of DNA aptamers for their applicabilities in ELISA. Nucleic Acids Research, 47(16), 8362–8374. https://doi.org/10.1093/nar/gkz688.
Li, L., Degardin, M., Lavergne, T., Malyshev, D. A., Dhami, K., Ordoukhanian, P., & Romesberg, F. E. (2014). Natural‐like replication of an unnatural base pair for the expansion of the genetic alphabet and biotechnology applications. Journal of the American Chemical Society, 136(3), 826–829. https://doi.org/10.1021/ja408814g.
Malyshev, D. A., Dhami, K., Lavergne, T., Chen, T., Dai, N., Foster, J. M., Corrêa, I. R., Jr, & Romesberg, F. E. (2014). A semi‐synthetic organism with an expanded genetic alphabet. Nature, 509(7500), 385–388. https://doi.org/10.1038/nature13314.
Malyshev, D. A., Seo, Y. J., Ordoukhanian, P., & Romesberg, F. E. (2009). PCR with an expanded genetic alphabet. Journal of the American Chemical Society, 131(41), 14620–14621. https://doi.org/10.1021/ja906186f.
Morihiro, K., Moriyama, Y., Nemoto, Y., Osumi, H., & Okamoto, A. (2021). Anti‐syn unnatural base pair enables alphabet‐expanded DNA self‐assembly. Journal of the American Chemical Society, 143(35), 14207–14217. https://doi.org/10.1021/jacs.1c05393.
Morris, S. E., Feldman, A. W., & Romesberg, F. E. (2017). Synthetic biology parts for the storage of increased genetic information in cells. ACS synthetic biology, 6(10), 1834–1840. https://doi.org/10.1021/acssynbio.7b00115.
Ptacin, J. L., Caffaro, C. E., Ma, L., San, J., Gall, K. M., Aerni, H. R., Acuff, N. V., Herman, R. W., Pavlova, Y., Pena, M. J., Chen, D. B., Koriazova, L. K., Shawver, L. K., Joseph, I. B., & Milla, M. E. (2021). An engineered IL‐2 reprogrammed for anti‐tumor therapy using a semi‐synthetic organism. Nature Communications, 12(1), 4785. https://doi.org/10.1038/s41467‐021‐24987‐9.
Riedl, J., Ding, Y., Fleming, A. M., & Burrows, C. J. (2015). Identification of DNA lesions using a third base pair for amplification and nanopore sequencing. Nature Communications, 6, 8807. https://doi.org/10.1038/ncomms9807.
Seo, Y. J., Hwang, G. T., Ordoukhanian, P., & Romesberg, F. E. (2009). Optimization of an unnatural base pair toward natural‐like replication. Journal of the American Chemical Society, 131(9), 3246–3252. https://doi.org/10.1021/ja807853m.
Wang, Y., Chen, Y., Hu, Y., & Fang, X. (2020). Site‐specific covalent labeling of large RNAs with nanoparticles empowered by expanded genetic alphabet transcription. Proceedings of the National Academy of Sciences of the United States of America, 117(37), 22823–22832. https://doi.org/10.1073/pnas.2005217117.
Wang, H., Zhu, W., Wang, C., Li, X., Wang, L., Huo, B., Mei, H., Zhu, A., Zhang, G., & Li, L. (2023). Locating, tracing and sequencing multiple expanded genetic letters in complex DNA context via a bridge‐base approach. Nucleic Acids Research, 51(9), e52. https://doi.org/10.1093/nar/gkad218.
Yang, Z., Chen, F., Alvarado, J. B., & Benner, S. A. (2011). Amplification, mutation, and sequencing of a six‐letter synthetic genetic system. Journal of the American Chemical Society, 133(38), 15105–15112. https://doi.org/10.1021/ja204910n.
Zhang, Y., Ptacin, J. L., Fischer, E. C., Aerni, H. R., Caffaro, C. E., San Jose, K., Feldman, A. W., Turner, C. R., & Romesberg, F. E. (2017). A semi‐synthetic organism that stores and retrieves increased genetic information. Nature, 551(7682), 644–647. https://doi.org/10.1038/nature24659.
Dien, V. T., Holcomb, M., Feldman, A. W., Fischer, E. C., Dwyer, T. J., & Romesberg, F. E. (2018). Progress toward a semi‐synthetic organism with an unrestricted expanded genetic alphabet. Journal of the American Chemical Society, 140(47), 16115–16123. https://doi.org/10.1021/jacs.8b08416.
Feldman, A. W., Dien, V. T., Karadeema, R. J., Fischer, E. C., You, Y., Anderson, B. A., Krishnamurthy, R., Chen, J. S., Li, L., & Romesberg, F. E. (2019). Optimization of replication, transcription, and translation in a semi‐synthetic organism. Journal of the American Chemical Society, 141(27), 10644–10653. https://doi.org/10.1021/jacs.9b02075.
Fischer, E. C., Hashimoto, K., Zhang, Y., Feldman, A. W., Dien, V. T., Karadeema, R. J., Adhikary, R., Ledbetter, M. P., Krishnamurthy, R., & Romesberg, F. E. (2020). New codons for efficient production of unnatural proteins in a semisynthetic organism. Nature Chemical Biology, 16(5), 570–576. https://doi.org/10.1038/s41589‐020‐0507‐z.
Hirao, I., Kimoto, M., Mitsui, T., Fujiwara, T., Kawai, R., Sato, A., Harada, Y., & Yokoyama, S. (2006). An unnatural hydrophobic base pair system: Site‐specific incorporation of nucleotide analogs into DNA and RNA. Nature Methods, 3(9), 729–735. https://doi.org/10.1038/nmeth915.
Kimoto, M., & Hirao, I. (2020). Genetic alphabet expansion technology by creating unnatural base pairs. Chemical Society Reviews, 49(21), 7602–7626. https://doi.org/10.1039/d0cs00457j.
Kimoto, M., Mitsui, T., Yamashige, R., Sato, A., Yokoyama, S., & Hirao, I. (2010). A new unnatural base pair system between fluorophore and quencher base analogues for nucleic acid‐based imaging technology. Journal of the American Chemical Society, 132(43), 15418–15426. https://doi.org/10.1021/ja1072383.
Kimoto, M., Shermane Lim, Y. W., & Hirao, I. (2019). Molecular affinity rulers: Systematic evaluation of DNA aptamers for their applicabilities in ELISA. Nucleic Acids Research, 47(16), 8362–8374. https://doi.org/10.1093/nar/gkz688.
Li, L., Degardin, M., Lavergne, T., Malyshev, D. A., Dhami, K., Ordoukhanian, P., & Romesberg, F. E. (2014). Natural‐like replication of an unnatural base pair for the expansion of the genetic alphabet and biotechnology applications. Journal of the American Chemical Society, 136(3), 826–829. https://doi.org/10.1021/ja408814g.
Malyshev, D. A., Dhami, K., Lavergne, T., Chen, T., Dai, N., Foster, J. M., Corrêa, I. R., Jr, & Romesberg, F. E. (2014). A semi‐synthetic organism with an expanded genetic alphabet. Nature, 509(7500), 385–388. https://doi.org/10.1038/nature13314.
Malyshev, D. A., Seo, Y. J., Ordoukhanian, P., & Romesberg, F. E. (2009). PCR with an expanded genetic alphabet. Journal of the American Chemical Society, 131(41), 14620–14621. https://doi.org/10.1021/ja906186f.
Morihiro, K., Moriyama, Y., Nemoto, Y., Osumi, H., & Okamoto, A. (2021). Anti‐syn unnatural base pair enables alphabet‐expanded DNA self‐assembly. Journal of the American Chemical Society, 143(35), 14207–14217. https://doi.org/10.1021/jacs.1c05393.
Morris, S. E., Feldman, A. W., & Romesberg, F. E. (2017). Synthetic biology parts for the storage of increased genetic information in cells. ACS synthetic biology, 6(10), 1834–1840. https://doi.org/10.1021/acssynbio.7b00115.
Ptacin, J. L., Caffaro, C. E., Ma, L., San, J., Gall, K. M., Aerni, H. R., Acuff, N. V., Herman, R. W., Pavlova, Y., Pena, M. J., Chen, D. B., Koriazova, L. K., Shawver, L. K., Joseph, I. B., & Milla, M. E. (2021). An engineered IL‐2 reprogrammed for anti‐tumor therapy using a semi‐synthetic organism. Nature Communications, 12(1), 4785. https://doi.org/10.1038/s41467‐021‐24987‐9.
Riedl, J., Ding, Y., Fleming, A. M., & Burrows, C. J. (2015). Identification of DNA lesions using a third base pair for amplification and nanopore sequencing. Nature Communications, 6, 8807. https://doi.org/10.1038/ncomms9807.
Seo, Y. J., Hwang, G. T., Ordoukhanian, P., & Romesberg, F. E. (2009). Optimization of an unnatural base pair toward natural‐like replication. Journal of the American Chemical Society, 131(9), 3246–3252. https://doi.org/10.1021/ja807853m.
Wang, Y., Chen, Y., Hu, Y., & Fang, X. (2020). Site‐specific covalent labeling of large RNAs with nanoparticles empowered by expanded genetic alphabet transcription. Proceedings of the National Academy of Sciences of the United States of America, 117(37), 22823–22832. https://doi.org/10.1073/pnas.2005217117.
Wang, H., Zhu, W., Wang, C., Li, X., Wang, L., Huo, B., Mei, H., Zhu, A., Zhang, G., & Li, L. (2023). Locating, tracing and sequencing multiple expanded genetic letters in complex DNA context via a bridge‐base approach. Nucleic Acids Research, 51(9), e52. https://doi.org/10.1093/nar/gkad218.
Yang, Z., Chen, F., Alvarado, J. B., & Benner, S. A. (2011). Amplification, mutation, and sequencing of a six‐letter synthetic genetic system. Journal of the American Chemical Society, 133(38), 15105–15112. https://doi.org/10.1021/ja204910n.
Zhang, Y., Ptacin, J. L., Fischer, E. C., Aerni, H. R., Caffaro, C. E., San Jose, K., Feldman, A. W., Turner, C. R., & Romesberg, F. E. (2017). A semi‐synthetic organism that stores and retrieves increased genetic information. Nature, 551(7682), 644–647. https://doi.org/10.1038/nature24659.
Contributed Indexing:
Keywords: NaM‐TPT3; bridge base; expanded genetic letters; isoTAT; unnatural base pair
Substance Nomenclature:
9007-49-2 (DNA)
Entry Date(s):
Date Created: 20251107 Date Completed: 20251107 Latest Revision: 20251107
Update Code:
20251107
DOI:
10.1002/cpz1.70250
PMID:
41201194
Database:
MEDLINE
Weitere Informationen
Genetic alphabet expansion by creating unnatural base pairs (UBPs) could renovate biological systems and next-generation biotechnologies. Among the expanded genetic letters, TPT3-NaM is one of the most advanced UBPs. It has been utilized in semi-synthetic organisms (SSOs) to recode therapeutic proteins. This article describes the synthesis of isoTAT and demonstrates that isoTAT can convert NaM to G, simultaneously pairing with both NaM and G as a bridging base. Additionally, the inherent base' preference for NaM leads to its transformation into T. Consequently, TPT3-NaM can be converted to C-G or A-T base pairs through simple PCR assays, enabling the first method to locate multiple sites of TPT3-NaM pairs dually. Taken together, this work presents the first general and convenient approach capable of locating, tracing, and sequencing site- and number-unlimited TPT3-NaM pairs. The data presented in this article are based on our previously published reports. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of isoTAT triphosphate Basic Protocol 2: Incorporation and extension reaction of isoTAT for a template containing NaM Basic Protocol 3: Monitor DNA containing multiple UBPs using simple PCR assays with isoTAT through Sanger sequencing.
(© 2025 Wiley Periodicals LLC.)