Our goal is to evolve enzymes that synthesize DNA and RNA containing unnatural nucleobases using phage display technology. Our objective is to expand the genetic alphabet and to create novel, stable, replicable biopolymers useful in biotechnical applications.
Specific Aim I involves evolving DNA polymerases that incorporate unnatural nucleobases. First, a library of mutant polymerases for the Stoffel fragment DNA polymerase will be generated (Specific Aim, I. a). This library will then be displayed on phage and used to select for efficient incorporation, extension, and fidelity of unnatural deoxynucleobases with a template attached to the same phage particle (Specific Aim I.b). Concurrently, for Specific Aim II similar studies will be conducted for transcription with RNA polymerases. An assay will be developed to determine the kinetic capabilities of T7 RNA polymerase (Specific Aim II.a). The directed evolution selection process will then be employed for non-canonical nucleotides that do not show ease of incorporation and extension by the wild-type enzyme. First, T7 RNA polymerase will be displayed on phage (Specific Aim II.b). Once successful, a mutant library of T7 polymerase will be generated for display (Specific Aim II.c). Finally, a selection with the mutant library will be executed for incorporation of two types of unnatural ribonucleotides (Specific Aim II.d): One set comprised of sugar modified nucleotides, and another set composed of modified nucleobases.
