|Genome Sequencing Technologies and Resources
DOE Human Genome Program Contractor-Grantee Workshop
16. Mutagenesis and Reaction Condition Studies of T7 RNA Polymerase Variants to Incorporate Deoxynucleotides
Mark Knuth, Scott Lesley, Heath
Klock, Michelle Mandrekar, Ryan Olson, James Schaefer, and Kris Zimmerman
Our aim is to alter substrate specificity in T7 RNA polymerase for efficient incorporation of dNTPs and other nucleotide analogs. Such a promoter-directed polymerase could be used for DNA sequencing and creating hybridization probes without the need for initiating primer. Previously described mutants incorporate dNTPs, but not sufficiently for practical applications. We are undertaking a combined approach of site-directed mutagenesis and evaluating reaction conditions to create an efficient polymerase with altered nucleotide specificity. Results are shown for both efforts.
For mutagenesis, we superimpose all possible substitutions of individual target sites upon the previously described mutations. Approximately 200 sites, covering a large portion of the active site, were chosen for saturation mutagenesis. Evaluation is underway, and desirable substitutions will be combined and shuffled. Our previous results indicate that dNTP incorporation is inhibited by inefficient transition from the initiation to the elongation phase. In order to screen for improvement of this property, polymerase is purified from each mutant and a fluorescent assay used to determine its ability to incorporate mixtures of r/dNTPs.
Solution conditions, such as addition of organic solvents, have been reported to enhance dNTP incorporation but result in substantial reduction of activity. We have confirmed and extended these results and note that these agents lower the apparent denaturation temperature of T7 RNAP. Mutations which increase thermostability under these conditions might offset the activity decrease, and a screen for these is being incorporated in our mutagenesis approach. We also evaluated other agents and found several which also enhanced dNTP incorporation but with a lesser thermodestabilization. Our results suggest a conformational change in the protein rather than the DNA template may be responsible for this enhancement. Using our optimal conditions, incorporation of a mixture of 3 dNTPs / 1 rATP is 300% of the 4 rNTP value. Although transcript products are somewhat shorter (most <= 200 bp) than for 4 rNTPs, we are examining their performance in dye-terminator DNA sequencing.
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