Mutant polymerases capable of 2' fluoro-modified nucleic acid synthesis and amplification with improved accuracy.

Nonnatural nucleic acids (xeno nucleic acids, XNA) can possess several useful properties such as expanded reactivity and nuclease resistance, which can enhance the utility of DNA as a biotechnological tool. Native DNA polymerases are unable to synthesize XNA, so, in recent years mutant XNA polymerases have been engineered with sufficient activity for use in processes such as PCR. While substantial improvements have been made, accuracy still needs to be increased by orders of magnitude to approach natural error rates and make XNA polymerases useful for applications that require high fidelity. Here, we systematically evaluate leading Taq DNA polymerase mutants for their fidelity during synthesis of 2'F XNA. To further improve their accuracy, we add mutations that have been shown to increase the fidelity of wild-type Taq polymerases, to some of the best current XNA polymerases (SFM4-3, SFM4-6, and SFP1). The resulting polymerases show significant improvements in synthesis accuracy. In addition to generating more accurate XNA polymerases, this study also informs future polymerase engineering efforts by demonstrating that mutations that improve the accuracy of DNA synthesis may also have utility in improving the accuracy of XNA synthesis.

Accurate and Efficient One-Pot Reverse Transcription and Amplification of 2'-Fluoro-Modified Nucleic Acids by Commercial DNA Polymerases.

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.