Comparative analysis of 43 distinct RNA modifications by nanopore tRNA sequencing.

Autor:	White LK; Department of Biochemistry and Molecular Genetics, RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora CO 80045., Dobson K; Department of Biochemistry and Molecular Genetics, RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora CO 80045., Del Pozo S; Department of Biochemistry and Molecular Genetics, RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora CO 80045., Bilodeaux JM; Department of Biochemistry and Molecular Genetics, RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora CO 80045., Andersen SE; Department of Microbiology and Immunology, University of Colorado School of Medicine, Aurora CO 80045., Baldwin A; Department of Biochemistry and Molecular Genetics, RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora CO 80045., Barrington C; Department of Biochemistry and Molecular Genetics, RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora CO 80045., Körtel N; Department of Biochemistry and Molecular Genetics, RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora CO 80045., Martinez-Seidel F; Department of Biochemistry and Molecular Genetics, RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora CO 80045., Strugar SM; Department of Biochemistry and Molecular Genetics, RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora CO 80045., Watt KEN; Department of Craniofacial Biology, University of Colorado School of Dental Medicine, Aurora CO 80045., Mukherjee N; Department of Biochemistry and Molecular Genetics, RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora CO 80045., Hesselberth JR; Department of Biochemistry and Molecular Genetics, RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora CO 80045.
Jazyk:	angličtina
Zdroj:	BioRxiv : the preprint server for biology [bioRxiv] 2024 Jul 24. Date of Electronic Publication: 2024 Jul 24.
DOI:	10.1101/2024.07.23.604651
Abstrakt:	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 de novo detection of RNA modifications across multiple organisms using nanopore sequencing. Competing Interests: Conflict of interest statement L.K.W. has received travel and accommodation expenses from Oxford Nanopore Technologies to present at scientific meetings, and has been a participant in ONT Early Access programs for SQK-RNA004. The remaining authors declare no conflicts of interest.
Databáze:	MEDLINE
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