Sequencing and Structure Probing of Long RNAs Using MarathonRT: A Next-Generation Reverse Transcriptase. Issue 10 (1st May 2020)

• Record Type: Journal Article
• Authors: Guo, Li-Tao; Adams, Rebecca L.; Wan, Han; Huston, Nicholas C.; Potapova, Olga; Olson, Sara; Gallardo, Christian M.; Graveley, Brenton R.; Torbett, Bruce E.; Pyle, Anna Marie
• Is Part Of: Journal of molecular biology. Volume 432:Issue 10(2020)
• Page Start: 3338
• Page End: 3352
• Publication Date: 2020-05-01
• Subjects: RT reverse transcriptase -- SHAPE-MaP selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling -- cDNA complementary DNA -- DMS-MaP dimethyl sulfate mutational profiling with sequencing -- SSII SuperScript II -- TGIRT thermostable group II reverse transcriptase -- HCV hepatitis C virus -- IRES internal ribosome entry site; RNA-Seq -- RNA structure -- DMS-MaP -- SHAPE-MaP -- RNA modifications; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2020.03.022
• Languages: English
• ISSNs: 0022-2836

Abstract

Abstract: Reverse transcriptase (RT) enzymes are indispensable tools for interrogating diverse aspects of RNA metabolism and transcriptome composition. Due to the growing interest in sequence and structural complexity of long RNA molecules, processive RT enzymes are now required for preserving linkage and information content in mixed populations of transcripts, and the low-processivity RT enzymes that are commercially available cannot meet this need. MarathonRT is encoded within a eubacterial group II intron, and it has been shown to efficiently copy highly structured long RNA molecules in a single pass. In this work, we systematically characterize MarathonRT as a tool enzyme and optimize its performance in a variety of applications that include single-cycle reverse transcription of long RNAs, dimethyl sulfate mutational profiling (DMS-MaP), selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP), using ultra-long amplicons and the detection of natural RNA base modifications. By diversifying MarathonRT reaction protocols, we provide an upgraded suite of tools for cutting-edge RNA research and clinical application. Graphical Abstract: Unlabelled Image Highlights: MarathonRT is a robust, ultra-processive RT that readily copies highly structured RNAs. Efficient end-to-end sequencing of long RNAs is now possible with MarathonRT. MarathonRT can be used for structure probing on long RNAs using SHAPE and DMS-MaP. Natural modifications in RNA can be detected and studied with MarathonRT.