Synthetic evolutionary origin of a proofreading reverse transcriptase by Jared W. Ellefson, Jimmy Gollihar, Raghav Shroff, Haridha Shivram, Vishwanath R. Iyer, and Andrew D. Ellington Science Volume 352(6293):1590-1593 June 24, 2016 Published by AAAS

Fig. 1 Evolution of a synthetic family of reverse transcriptases by RT-CSR. Evolution of a synthetic family of reverse transcriptases by RT-CSR. (A) Polymerase phylogeny depicts reverse transcription xenopolymerases (RTX) as a second, evolutionarily distinct, origin of RT function. (B) Framework for the directed evolution of hyperthermostable RT using reverse transcription compartmentalized self-replication (RT-CSR). Libraries of polymerase variants are created, expressed in Escherichia coli, and in vitro compartmentalized. During emulsion PCR, primers flanking the polymerase enable self-replication but are designed with a variable number of RNA bases separating the plasmid annealing portion from the unique recovery tag. Jared W. Ellefson et al. Science 2016;352:1590-1593 Published by AAAS

Fig. 2 Molecular checkpoints involved in template recognition. Molecular checkpoints involved in template recognition. (A) Structural heat map of mutated residues over the RT-CSR process. Conserved mutations are colored incrementally darker shades of red to indicate frequency in the polymerase pool. Amino acid residues that were mutated in more than 50% of the population are labeled. [Figure was adapted from KOD structure PDB 4K8Z.] (B) Computer modeling of KOD (gray) and RTX mutations (orange) at checkpoints responsible for DNA and RNA template recognition at R97, Y384, and E664. Free-energy changes between wild-type KOD and RTX mutations are inlet displayed. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; M, Met; N, Asn; R, Arg; V, Val; and Y, Tyr. Jared W. Ellefson et al. Science 2016;352:1590-1593 Published by AAAS

Fig. 3 RTX polymerase proofreads during reverse transcription. RTX polymerase proofreads during reverse transcription. (A) Primer extension reactions of KOD and RTX polymerases and their proofreading-deficient counterparts (exo-), on both DNA and RNA templates. Extension reactions were performed with matched 3′ primer:templates (gray) or a 3′ deoxy mismatch (orange), which must be excised before extension can proceed. The primer is denoted by a gray arrow, extended product is in green, and exonuclease-degraded primer is in red. (B) Deep sequencing of reverse transcription reaction on HSPCB gene. The overall error rate was determined by dividing the sum of base substitutions and insertions or deletions by the total number of bases sequenced. The error profile of MMLV, RTX, and RTX exo- is shown as frequency of errors per million bases sequenced. Jared W. Ellefson et al. Science 2016;352:1590-1593 Published by AAAS