Redesigning DNA biosynthesis: leaving group-modified nucleotides as a biosafety tool.

Artificial nucleic acids (XNA’s) are capable of storing and processing genetic information just like the natural nucleic acids DNA and RNA and can thus function as suitable genetic materials as well. Natural nucleic acid biosynthesis is catalyzed by polymerases and involves the sequential condensation of (deoxy)nucleoside triphosphate ((d)NTP) building blocks with concomitant release of pyrophosphate (PPi). Besides information storage and transfer, nucleoside triphosphates (e.g. ATP) fulfill numerous other roles in cellular metabolism. To avoid entanglement between XNA and DNA/RNA, and to prevent interference with other cellular processes, PPi should be replaced by a chemically distinct leaving group (LG) in the precursors for XNA biosynthesis. Such leaving groupmodified nucleotides must not be able to serve as substrates for natural polymerases or other cellular enzymes. Therefore, artificially-evolved polymerases need to be developed and these should no longer accept natural (d)NTPs as building blocks. This research project aims to explore the ability of different mesophilic DNA polymerases to accept a range of PPi mimics. The most promising polymerase will be artificially evolved to afford efficient incorporation of dNTP analogues carrying the optimal alternative LG. Finally, the mutant DNA polymerases will be introduced in E. coli with the aim of establishing a functional system of genomic DNA replication that relies exclusively on dNTP analogues with modified LG.