Sequencing and Structure Probing of Long RNAs Using MarathonRT: A Next-Generation Reverse Transcriptase.

Autor: Guo LT; Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520, USA., Adams RL; Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520, USA., Wan H; Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520, USA., Huston NC; Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520, USA., Potapova O; Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520, USA., Olson S; Department of Genetics and Genome Sciences, Institute for Systems Genomics, UConn Health, Farmington, CT 06030-6403, USA., Gallardo CM; The Scripps Research Institute, La Jolla, CA 92037, USA., Graveley BR; Department of Genetics and Genome Sciences, Institute for Systems Genomics, UConn Health, Farmington, CT 06030-6403, USA., Torbett BE; The Scripps Research Institute, La Jolla, CA 92037, USA., Pyle AM; Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA; Department of Chemistry, Yale University, New Haven, CT 06520, USA. Electronic address: anna.pyle@yale.edu.
Jazyk: angličtina
Zdroj: Journal of molecular biology [J Mol Biol] 2020 May 01; Vol. 432 (10), pp. 3338-3352. Date of Electronic Publication: 2020 Apr 04.
DOI: 10.1016/j.jmb.2020.03.022
Abstrakt: 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.
Competing Interests: Declaration of Competing Interest A patent application on MarathonRT has been filed by Yale University.
(Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
Databáze: MEDLINE
Externí odkaz: