Single-molecule RNA sizing enables quantitative analysis of alternative transcription termination
Authors
Patiño-Guillén, GerardoPešović, Jovan
Panić, Marko
Savić-Pavićević, Dušanka
Bošković, Filip
Keyser, Ulrich Felix
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Transcription, a critical process in molecular biology, has found many applications in RNA synthesis, including mRNA vaccines and RNA therapeutics. However, current RNA characterization technologies suffer from amplification and enzymatic biases that lead to loss of native information. Here, we introduce a strategy to quantitatively study both transcription and RNA polymerase behaviour by sizing RNA with RNA nanotechnology and nanopores. To begin, we utilize T7 RNA polymerase to transcribe linear DNA lacking termination sequences. Surprisingly, we discover alternative transcription termination in the origin of replication sequence. Next, we employ circular DNA without transcription terminators to perform rolling circle transcription. This allows us to gain valuable insights into the processivity and transcription behaviour of RNA polymerase at the single-molecule level. Our work demonstrates how RNA nanotechnology and nanopores may be used in tandem for the direct and quantitative anal...ysis of RNA transcripts. This methodology provides a promising pathway for accurate RNA structural mapping by enabling the study of full-length RNA transcripts at the single-molecule level.
Keywords:
Nanopores / RNA / RNA nanotechnology / TranscriptionSource:
Nature Communications, 2024, 15, 1, 1699-Publisher:
- Nature
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- Supplementary information: https://intor.torlakinstitut.com/handle/123456789/866
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https://intor.torlakinstitut.com/handle/123456789/866
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TorlakTY - JOUR AU - Patiño-Guillén, Gerardo AU - Pešović, Jovan AU - Panić, Marko AU - Savić-Pavićević, Dušanka AU - Bošković, Filip AU - Keyser, Ulrich Felix PY - 2024 UR - http://intor.torlakinstitut.com/handle/123456789/865 AB - Transcription, a critical process in molecular biology, has found many applications in RNA synthesis, including mRNA vaccines and RNA therapeutics. However, current RNA characterization technologies suffer from amplification and enzymatic biases that lead to loss of native information. Here, we introduce a strategy to quantitatively study both transcription and RNA polymerase behaviour by sizing RNA with RNA nanotechnology and nanopores. To begin, we utilize T7 RNA polymerase to transcribe linear DNA lacking termination sequences. Surprisingly, we discover alternative transcription termination in the origin of replication sequence. Next, we employ circular DNA without transcription terminators to perform rolling circle transcription. This allows us to gain valuable insights into the processivity and transcription behaviour of RNA polymerase at the single-molecule level. Our work demonstrates how RNA nanotechnology and nanopores may be used in tandem for the direct and quantitative analysis of RNA transcripts. This methodology provides a promising pathway for accurate RNA structural mapping by enabling the study of full-length RNA transcripts at the single-molecule level. PB - Nature T2 - Nature Communications T1 - Single-molecule RNA sizing enables quantitative analysis of alternative transcription termination IS - 1 SP - 1699 VL - 15 DO - 10.1038/s41467-024-45968-8 ER -
@article{ author = "Patiño-Guillén, Gerardo and Pešović, Jovan and Panić, Marko and Savić-Pavićević, Dušanka and Bošković, Filip and Keyser, Ulrich Felix", year = "2024", abstract = "Transcription, a critical process in molecular biology, has found many applications in RNA synthesis, including mRNA vaccines and RNA therapeutics. However, current RNA characterization technologies suffer from amplification and enzymatic biases that lead to loss of native information. Here, we introduce a strategy to quantitatively study both transcription and RNA polymerase behaviour by sizing RNA with RNA nanotechnology and nanopores. To begin, we utilize T7 RNA polymerase to transcribe linear DNA lacking termination sequences. Surprisingly, we discover alternative transcription termination in the origin of replication sequence. Next, we employ circular DNA without transcription terminators to perform rolling circle transcription. This allows us to gain valuable insights into the processivity and transcription behaviour of RNA polymerase at the single-molecule level. Our work demonstrates how RNA nanotechnology and nanopores may be used in tandem for the direct and quantitative analysis of RNA transcripts. This methodology provides a promising pathway for accurate RNA structural mapping by enabling the study of full-length RNA transcripts at the single-molecule level.", publisher = "Nature", journal = "Nature Communications", title = "Single-molecule RNA sizing enables quantitative analysis of alternative transcription termination", number = "1", pages = "1699", volume = "15", doi = "10.1038/s41467-024-45968-8" }
Patiño-Guillén, G., Pešović, J., Panić, M., Savić-Pavićević, D., Bošković, F.,& Keyser, U. F.. (2024). Single-molecule RNA sizing enables quantitative analysis of alternative transcription termination. in Nature Communications Nature., 15(1), 1699. https://doi.org/10.1038/s41467-024-45968-8
Patiño-Guillén G, Pešović J, Panić M, Savić-Pavićević D, Bošković F, Keyser UF. Single-molecule RNA sizing enables quantitative analysis of alternative transcription termination. in Nature Communications. 2024;15(1):1699. doi:10.1038/s41467-024-45968-8 .
Patiño-Guillén, Gerardo, Pešović, Jovan, Panić, Marko, Savić-Pavićević, Dušanka, Bošković, Filip, Keyser, Ulrich Felix, "Single-molecule RNA sizing enables quantitative analysis of alternative transcription termination" in Nature Communications, 15, no. 1 (2024):1699, https://doi.org/10.1038/s41467-024-45968-8 . .