Summer Research Administrative Supplement to 1R01GM128186-01, Transforming Life Sciences: Artificial Life

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Summer Research Administrative Supplement to 1R01GM128186-01 Transforming Life Sciences: Artificial Life Foundation for Applied Molecular Evolution Steven A. Benner ABSTRACT In addition to its research, teaching, and outreach activities, the Foundation for Applied Molecular Evolution runs a program that offers opportunities for laboratory research experience to undergraduates seeking careers in medicine, biotechnology, and biomedical research. In the past few years, undergraduate trainees have gone on to MD-PhD programs (Michael Durante, Oleg Uryasev, Jennifer Le), to start biotech enterprises (Heshan Illangkoon), and to pursue Ph.D. degrees (Marc Neveu). All published papers from their undergraduate research. Here, we seek an administrative supplement for summer 2021 to support Justin Kim, an undergraduate from the University of Florida. The supplement will provide him his first experience in protein engineering in an area (DNA polymerases) that has applications not only to the project that this proposal supplements, but also can be applied in diagnostics, in the generation of therapeutic agents, and in biotechnology. The project that he will join (1R01GM128186-01) is a Director's Transformative project that seeks to create an artificial life form (a “xenobiotic” known as SEGUE). This xenobiotic will reproduce much of what we value in natural life but with a different core molecular biology. This is moving beyond “synthetic biology” and “biomimetic chemistry”. Specifically, SEGUE restructures the DNA of the living cell by adapting Watson-Crick pairing. In 4-letter standard DNA, pairing follows two complementarity rules: (a) size (large purines pair with small pyrimidines) and (b) hydrogen bonding (on purine pu and pyrimidine py ring analogs, hydrogen bond acceptors, A, pair with donors D). Rearranging D and A groups on the bases creates artificially expanded genetic information systems (AEGIS). The parent project involves creating new cells that replicate six-letter DNA, either GACTZP DNA or GACTKX DNA. This is done by recruiting E. coli as a platform, and expanding its metabolic capabilities to support six-letter replication. Replication of 6-letter DNA is done by DNA polymerases. This project requires the engineering of these polymerases to accept unnatural nucleotides, this project has led to the publication of many of these polymerases. Many of them have been thermally stable, however. While these work well in PCR, they do not perform as well at low temperature where E. coli lives. At the same time, various reverse transcriptases have been recruited that do accept six letter genetic alphabets. Thus, this background lends itself to a single Aim: Mr. Kim will use protein engineering on a diversity science platform to improve mesophilic DNA polymerases that copy DNA built from six nucleotides. These polymerases will be used in cells being engineered to manage and use expanded DNA, to be an integral part of the funded Director's Transformative Technology award.