White Laura K, Dobson Kezia, Del Pozo Samantha, Bilodeaux Jill M, Andersen Shelby E, Baldwin Amber, Barrington Chloe, Körtel Nadine, Martinez-Seidel Federico, Strugar Saylor M, Watt Kristin E N, Mukherjee Neelanjan, Hesselberth Jay R
Department of Biochemistry and Molecular Genetics, RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora CO 80045.
Department of Microbiology and Immunology, University of Colorado School of Medicine, Aurora CO 80045.
bioRxiv. 2024 Jul 24:2024.07.23.604651. doi: 10.1101/2024.07.23.604651.
Transfer RNAs are the fundamental adapter molecules of protein synthesis and the most abundant and heterogeneous class of noncoding RNA molecules in cells. The study of tRNA repertoires remains challenging, complicated by the presence of dozens of post transcriptional modifications. Nanopore sequencing is an emerging technology with promise for both tRNA sequencing and the detection of RNA modifications; however, such studies have been limited by the throughput and accuracy of direct RNA sequencing methods. Moreover, detection of the complete set of tRNA modifications by nanopore sequencing remains challenging. Here we show that recent updates to nanopore direct RNA sequencing chemistry (RNA004) combined with our own optimizations to tRNA sequencing protocols and analysis workflows enable high throughput coverage of tRNA molecules and characterization of nanopore signals produced by 43 distinct RNA modifications. We share best practices and protocols for nanopore sequencing of tRNA and further report successful detection of low abundance mitochondrial and viral tRNAs, providing proof of concept for use of nanopore sequencing to study tRNA populations in the context of infection and organelle biology. This work provides a roadmap to guide future efforts towards detection of RNA modifications across multiple organisms using nanopore sequencing.
转运RNA是蛋白质合成的基本衔接分子，也是细胞中最丰富且种类多样的非编码RNA分子。由于存在数十种转录后修饰，对tRNA库的研究仍然具有挑战性且较为复杂。纳米孔测序是一项新兴技术，有望用于tRNA测序和RNA修饰的检测；然而，此类研究一直受到直接RNA测序方法的通量和准确性的限制。此外，通过纳米孔测序检测完整的tRNA修饰集仍然具有挑战性。在这里，我们表明，纳米孔直接RNA测序化学方法（RNA004）的最新进展，结合我们自己对tRNA测序方案和分析工作流程的优化，能够实现tRNA分子的高通量覆盖，并对43种不同RNA修饰产生的纳米孔信号进行表征。我们分享了tRNA纳米孔测序的最佳实践和方案，并进一步报告了成功检测到低丰度线粒体和病毒tRNA，为使用纳米孔测序研究感染和细胞器生物学背景下的tRNA群体提供了概念验证。这项工作提供了一个路线图，以指导未来利用纳米孔测序检测多种生物体中RNA修饰的工作。
