Design and Discovery of New Combinations of Mutant DNA Polymerases and Modified DNA Substrates.
Chemical modifications can enhance the properties of DNA by imparting nuclease resistance and generating more-diverse physical structures. However, native DNA polymerases generally cannot synthesize significant lengths of DNA with modified nucleotide triphosphates. Previous efforts have identified a mutant of DNA polymerase I from Thermus aquaticus DNA (SFM19) as capable of synthesizing a range of short, 2'-modified DNAs; however, it is limited in the length of the products it can synthesize. Here, we rationally designed and characterized ten mutants of SFM19. From this, we identified enzymes with substantially improved activity for the synthesis of 2'F-, 2'OH-, 2'OMe-, and 3'OMe-modified DNA as well as for reverse transcription of 2'OMe DNA. We also evaluated mutant DNA polymerases previously only tested for synthesis for 2'OMe DNA and showed that they are capable of an expanded range of modified DNA synthesis. This work significantly expands the known combinations of modified DNA and Taq DNA polymerase mutants.
W. M. Keck Science Department, Claremont McKenna, Pitzer, and Scripps Colleges, Claremont, CA, 91711, USA.
Sydney L
SL
Rosenblum
W. M. Keck Science Department, Claremont McKenna, Pitzer, and Scripps Colleges, Claremont, CA, 91711, USA.
Aurora G
AG
Weiden
W. M. Keck Science Department, Claremont McKenna, Pitzer, and Scripps Colleges, Claremont, CA, 91711, USA.
Eliza L
EL
Lewis
W. M. Keck Science Department, Claremont McKenna, Pitzer, and Scripps Colleges, Claremont, CA, 91711, USA.
Alexie L
AL
Ogonowsky
W. M. Keck Science Department, Claremont McKenna, Pitzer, and Scripps Colleges, Claremont, CA, 91711, USA.
Hannah E
HE
Chia
W. M. Keck Science Department, Claremont McKenna, Pitzer, and Scripps Colleges, Claremont, CA, 91711, USA.
Susanna E
SE
Barrett
W. M. Keck Science Department, Claremont McKenna, Pitzer, and Scripps Colleges, Claremont, CA, 91711, USA.
Mira D
MD
Liu
W. M. Keck Science Department, Claremont McKenna, Pitzer, and Scripps Colleges, Claremont, CA, 91711, USA.
Aaron M
0000-0002-5789-9147
AM
Leconte
D004247:DNA / Q000138:chemical synthesis* / Q000737:chemistry; D004256:DNA Polymerase I / Q000737:chemistry* / Q000235:genetics; D008345:Manganese / Q000737:chemistry; D009154:Mutation; D015202:Protein Engineering; D012313:RNA / Q000138:chemical synthesis; D048348:Reverse Transcription; D019914:Taq Polymerase / Q000737:chemistry* / Q000235:genetics
D008345:Manganese; D012313:RNA; D004247:DNA; D019914:Taq Polymerase; D004256:DNA Polymerase I
DNA; DNA polymerases; modified DNA synthesis; protein engineering