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Single-molecule RNA sizing enables quantitative analysis of alternative transcription termination

Author

Listed:
  • Gerardo Patiño-Guillén

    (University of Cambridge)

  • Jovan Pešović

    (Centre for Human Molecular Genetics)

  • Marko Panić

    (Centre for Human Molecular Genetics
    Vaccines and Sera “Torlak”)

  • Dušanka Savić-Pavićević

    (Centre for Human Molecular Genetics)

  • Filip Bošković

    (University of Cambridge)

  • Ulrich Felix Keyser

    (University of Cambridge)

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.

Suggested Citation

  • Gerardo Patiño-Guillén & Jovan Pešović & Marko Panić & Dušanka Savić-Pavićević & Filip Bošković & Ulrich Felix Keyser, 2024. "Single-molecule RNA sizing enables quantitative analysis of alternative transcription termination," Nature Communications, Nature, vol. 15(1), pages 1-12, December.
  • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-45968-8
    DOI: 10.1038/s41467-024-45968-8
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    References listed on IDEAS

    as
    1. Ke Liu & Chao Pan & Alexandre Kuhn & Adrian Pascal Nievergelt & Georg E. Fantner & Olgica Milenkovic & Aleksandra Radenovic, 2019. "Detecting topological variations of DNA at single-molecule level," Nature Communications, Nature, vol. 10(1), pages 1-9, December.
    2. Mihue Jang & Jong Hwan Kim & Hae Yun Nam & Ick Chan Kwon & Hyung Jun Ahn, 2015. "Design of a platform technology for systemic delivery of siRNA to tumours using rolling circle transcription," Nature Communications, Nature, vol. 6(1), pages 1-12, November.
    3. Felipe Rivas & Osama K. Zahid & Heidi L. Reesink & Bridgette T. Peal & Alan J. Nixon & Paul L. DeAngelis & Aleksander Skardal & Elaheh Rahbar & Adam R. Hall, 2018. "Label-free analysis of physiological hyaluronan size distribution with a solid-state nanopore sensor," Nature Communications, Nature, vol. 9(1), pages 1-9, December.
    4. Rajesh Kumar Sharma & Ishita Agrawal & Liang Dai & Patrick S. Doyle & Slaven Garaj, 2019. "Complex DNA knots detected with a nanopore sensor," Nature Communications, Nature, vol. 10(1), pages 1-9, December.
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