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.

Examining Sources of Error in PCR by Single-Molecule Sequencing

“…For very accurate polymerases, such as Q5 DNA polymerase, the rate of base substitution errors approaches the background error rate of the assay. For other next-generation sequencing-based fidelity assays, the lower threshold for determining polymerase error rates by barcoded Illumina sequencing was reported as 1 × 10 −6 errors/base, and attributed to barcode fidelity [32]. Single-molecule sequencing has lowered the threshold for error rate determination, yet as engineered DNA polymerases become more accurate, accurately measuring the error rates of extremely high fidelity polymerases still remains challenging.…”

Identification of tumor-reactive B cells and systemic IgG in breast cancer based on clonal frequency in the sentinel lymph node

“…These cells were subjected to paired BCR-Seq (17,18), a process by which the VH and VL antibody transcripts from a single B-cell antibody receptor (BCR) are reverse transcribed and physically linked (VH:VL) using emulsion-based overlap-extension RT-PCR. The RT-PCR reaction integrates reverse transcription xenopolymerase (RTX), an enzyme that exhibits both reverse transcriptase and polymerase activity (19), to enhance the fidelity and efficiency of the reaction. The VH:VL library was deep sequenced using paired-end Illumina MiSeq, bioinformatically filtered to remove low-confidence sequences, and the annotated antibody sequences were separated by isotype.…”

“…Briefly, the flow focusing device was used to coemulsify single cells with lysis buffer (100 mM Tris pH 7.5, 500 mM LiCl, 10 mM EDTA, 1% lithium dodecyl sulfate, and 5 mM DTT) and oligo d(T) 25 magnetic beads (New England Biolabs). The magnetic beads were washed, resuspended in a customized high-yield RT-PCR solution (19) (SI Table 1), emulsified, and subjected to overlap-extension RT-PCR under the following conditions: 30 min at 68°C followed by 2 min at 94°C; 25 cycles of 94°C for 30 s, 60°C for 30 s, 68°C for 2 min; 68°C for 7 min; held at 4°C. The RT-PCR solution contained a multiplex set of VH and VL primers (SI Table 2) designed to physically stitch the two antibody chains into a single amplicon.…”

Compartmentalized Self‐Replication for Evolution of a DNA Polymerase

“…Depending on the nature of the non-standard substrates and the DNA polymerase activity desired, nonstandard nucleotide residues may be incorporated at the 3'-ends of primers (Loakes et al, 2009), in the 5'-'tag' regions of the primers (Laos et al, 2013), or between the 5'-tag and the 3'-annealing regions of the primers (Ellefson et al, 2016) (Fig. Depending on the nature of the non-standard substrates and the DNA polymerase activity desired, nonstandard nucleotide residues may be incorporated at the 3'-ends of primers (Loakes et al, 2009), in the 5'-'tag' regions of the primers (Laos et al, 2013), or between the 5'-tag and the 3'-annealing regions of the primers (Ellefson et al, 2016) (Fig.…”