Cavendish Laboratory, University of Cambridge, Cambridge, UK.
University of Belgrade - Faculty of Biology, Centre for Human Molecular Genetics, Belgrade, Serbia.
Nat Commun. 2024 Feb 24;15(1):1699. doi: 10.1038/s41467-024-45968-8.
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.
转录是分子生物学中的一个关键过程,它在 RNA 合成方面有许多应用,包括 mRNA 疫苗和 RNA 疗法。然而,目前的 RNA 分析技术存在扩增和酶的偏向性,导致丢失了天然信息。在这里,我们介绍了一种通过 RNA 纳米技术和纳米孔来定量研究转录和 RNA 聚合酶行为的策略。首先,我们利用 T7 RNA 聚合酶转录缺乏终止序列的线性 DNA。令人惊讶的是,我们在复制起点序列中发现了替代的转录终止。接下来,我们使用没有转录终止子的环形 DNA 进行滚环转录。这使我们能够在单分子水平上深入了解 RNA 聚合酶的连续性和转录行为。我们的工作表明,RNA 纳米技术和纳米孔如何可以串联使用,用于 RNA 转录物的直接和定量分析。这种方法通过在单分子水平上研究全长 RNA 转录物,为准确的 RNA 结构图谱提供了有前途的途径。