title: Synthetic evolutionary origin of a proofreading reverse transcriptase.
abstract: Most reverse transcriptase (RT) enzymes belong to a single protein family of ancient evolutionary origin. These polymerases are inherently error prone, owing to their lack of a proofreading (3'- 5' exonuclease) domain. To determine if the lack of proofreading is a historical coincidence or a functional limitation of reverse transcription, we attempted to evolve a high-fidelity, thermostable DNA polymerase to use RNA templates efficiently. The evolutionarily distinct reverse transcription xenopolymerase (RTX) actively proofreads on DNA and RNA templates, which greatly improves RT fidelity. In addition, RTX enables applications such as single-enzyme reverse transcription-polymerase chain reaction and direct RNA sequencing without complementary DNA isolation. The creation of RTX confirms that proofreading is compatible with reverse transcription.
authors.affiliation: Center for Systems and Synthetic Biology, Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, University of Texas, 2500 Speedway, Austin, TX 78712, USA. jaredwellefson@gmail.com ellingtonlab@gmail.com.
authors.forename: Jared W
authors.initials: JW
authors.lastname: Ellefson
authors.affiliation: Center for Systems and Synthetic Biology, Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, University of Texas, 2500 Speedway, Austin, TX 78712, USA.
authors.forename: Jimmy
authors.initials: J
authors.lastname: Gollihar
authors.affiliation: Center for Systems and Synthetic Biology, Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, University of Texas, 2500 Speedway, Austin, TX 78712, USA.
authors.forename: Raghav
authors.initials: R
authors.lastname: Shroff
authors.affiliation: Center for Systems and Synthetic Biology, Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, University of Texas, 2500 Speedway, Austin, TX 78712, USA.
authors.forename: Haridha
authors.initials: H
authors.lastname: Shivram
authors.affiliation: Center for Systems and Synthetic Biology, Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, University of Texas, 2500 Speedway, Austin, TX 78712, USA.
authors.forename: Vishwanath R
authors.initials: VR
authors.lastname: Iyer
authors.affiliation: Center for Systems and Synthetic Biology, Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, University of Texas, 2500 Speedway, Austin, TX 78712, USA. jaredwellefson@gmail.com ellingtonlab@gmail.com.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Ellington
mesh_terms: D053843:DNA Mismatch Repair; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000096:biosynthesis; D019020:Directed Molecular Evolution; D019143:Evolution, Molecular; D005092:Exonucleases / Q000737:chemistry; D008958:Models, Molecular; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D020027:Pyrococcus furiosus / Q000201:enzymology; D012313:RNA / Q000737:chemistry / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000145:classification* / Q000235:genetics; D013698:Templates, Genetic; D019712:Thermococcus / Q000201:enzymology
chemical_list: D018076:DNA, Complementary; D012313:RNA; D012194:RNA-Directed DNA Polymerase; D005092:Exonucleases