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

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Patiño-Guillén, Gerardo 
Pešović, Jovan 
Panić, Marko 
Savić-Pavićević, Dušanka 


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.


Acknowledgements: U.F.K. was supported by funding from a European Research Council (ERC) consolidator grant (DesignerPores no. 647144) and an ERC-2019-PoC grant (PoreDetect no. 899538). G.P.-G. acknowledges funding from EPSRC CDT MRes/PhD Studentship in Nanoscience and Nanotechnology (NanoDTC Cambridge EP/S022953/1) and Trinity-Henry Barlow Scholarship. F.B. acknowledges research funding from the George and Lilian Schiff Foundation Studentship, the Winton Program for the Physics of Sustainability PhD Scholarship, and St John’s College Benefactors’ Scholarship. J.P., M.P., and D.S.-P. acknowledge funding from the Science Fund of the Republic of Serbia, Grant No. 7754217, READ-DM1. We thank Dr. Casey Platnich and Dr. Roger Rubio-Sánchez for critically reading the manuscript. We thank Lorenzo Peri for facilitating python-based graphic user interfaces used for analysis of data.

Funder: 1- European Research Council (ERC) consolidator grant (DesignerPores no. 647144) 2-ERC-2019-PoC grant (PoreDetect no. 899538)

Funder: Science Fund of the Republic of Serbia, Grant No. 7754217

Funder: 1-George and Lilian Schiff Foundation Studentship 2-Winton Programme, Physics of Sustainability PhD Scholarship 3-St John’s College Benefactors’ Scholarship.


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Nature Communications

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Nature Publishing Group UK
RCUK | Engineering and Physical Sciences Research Council (EPSRC) (EP/S022953/1)