DNA is a foundational tool in biotechnology and synthetic biology but is limited by sensitivity to DNA-modifying enzymes. Recently, researchers have identified DNA polymerases that can enzymatically synthesize long oligonucleotides of modified DNA (M-DNA) that are resistant to DNA-modifying enzymes. Most applications require M-DNA to be reverse transcribed, typically using a RNA reverse transcriptase, back into natural DNA for sequence analysis or further manipulation. Here, we tested commercially available DNA-dependent DNA polymerases for their ability to reverse transcribe and amplify M-DNA in a one-pot reaction. Three of the six polymerases chosen (Phusion, Q5, and Deep Vent) could reverse transcribe and amplify synthetic 2′F M-DNA in a single reaction with <5 × 10–3 error per base pair. We further used Q5 DNA polymerase to reverse transcribe and amplify M-DNA synthesized by two candidate M-DNA polymerases (SFP1 and SFM4–6), allowing for quantification of the frequency, types, and locations of errors made during M-DNA synthesis. From these studies, we identify SFP1 as one of the most accurate M-DNA polymerases identified to date. Collectively, these studies establish a simple, robust method for the conversion of 2′F M-DNA to DNA in <1 h using commercially available materials, significantly improving the ease of use of M-DNA.
Recently, several modified‐DNA (M‐DNA) polymerases have been identified which can synthesize long M‐DNAs. While these discoveries have enabled new applications of M‐DNA, these enzymes typically have poor fidelity, possessing error rates several orders of magnitude worse than native enzymes. However, to date, efforts to both quantify error rate, as well as experimental approaches towards understanding the origin of the poor fidelity of M‐DNA polymerases, have been limited. Here, we use a high‐throughput sequencing assay to characterize the error rate of leading M‐DNA polymerases, which showed that our enzyme, P1, has an error rate 1.6 times lower than SFM4‐6, the most frequently used Taq mutant for 2′M‐DNA synthesis. We also characterize the importance of individual mutations on fidelity, beginning to experimentally address the origins of fidelity, and have found that reverting even one mutation in a mutant yielded a fidelity that was 2.4 times greater than its parent, which will help us improve future M‐DNA polymerase rational design. Support or Funding Information National Science Foundation
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