Chemical labeling of DNA can be an important tool in various biological applications like polymers, biomaterials, and drug discovery. Using modified DNA (M-DNA) can provide additional benefits by being resistant to degradation by native nuclease enzymes. However, because of the structural differences, few DNA polymerases are able to accurately synthesize M-DNA that is both nuclease-resistant and has chemically reactive functional groups. The azide (N3) functional group is ideal due to its commercial availability as a modified nucleoside triphosphate (M-NTP) and the ability to be used in bioorthogonal click chemistry reactions for labeling. Here, we demonstrate the ability of two mutant DNA polymerases to accurately synthesize M-DNA oligonucleotides with three 2’ fluoro nucleotides and one 2’ azide nucleotide. Both M-DNA polymerases, SFP1 and SFM4-6, synthesize full length 47 nucleotide M-DNA with a single 2’Az M-NTP with little to no pausing. Both polymerases are also able to synthesize M-DNA with combinations of multiple 2’Az M-NTPs. In addition, we developed methodology to reverse transcribe and amplify the M-DNA synthesized to quantify the accuracy and types of errors made using high-throughput sequencing. Our results show that SFP1 and SFM4-6 successfully synthesize M-DNA with single 2’Az NTP modifications with less than 8 x 10-3 errors per base pair and less than 23 x 10-3 errors per base pair, respectively. SFP1 showed similar error rates for all 2’Az NTPs while SFM4-6 showed an increased error rate for 2’Az GTP. These results show the ability of modified DNA polymerases to accurately synthesize M-DNA with azide nucleotides, providing a robust platform for chemical labeling.

Using mutant DNA polymerases to accurately synthesize 2’Azide modified DNA

ACS Spring Meeting 2021

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