High Quality Proteins with Multiple Post Translational Modifications Foundation for Applied Molecular Evolution Shuichi Hoshika ABSTRACT The proposed technology will make, by in vitro translation (IVT), proteins that hold non-canonical amino acids normally put in only by post-translational modification (PTM). The immediate deliverable will be tech- nology that delivers proteins with three PTM-AAs (acetyllysine, phosphserine, phosphotyrosine) incorporated in many, exact positions in long (300 - 1100 amino acid proteins are used) proteins with >95% occupancy. The NCI itself motivated this proposal by its calls for tools to make such proteins, which cancer researchers need throughout cancer proteomics. Today, such proteins are available only via isolation from living eukaryotic cells. These are rarely pure. Our pure PTM proteins will be used to get antibodies, identify PTM signatures of cancers, standardize quantitative immunoassays as standards, discover inhibitors and drugs for cancer-related enzymes in their drug-relevant forms, and study protein-protein interactions. These will be obtained rapidly and inexpensively in their own labs (with in vitro translation kits) or via service companies (as for antibodies). As Performance Measures, the NCI defined "useable amounts" to be "0.5 to 1 mg of protein" with "50- 80% modification at the specified site". Our technology will do better, generating 1-10 mg of protein with >95% modification for three different PTM amino acids at many specified sites. Behind this project is an ongoing revolution in the synthetic biology of DNA and RNA (xNA) that delivered expanded xNA, enhanced in 2019 by the PI. Artificial xNA looks like standard xNA. However, it adds pairs by shuffling hydrogen bonding groups, allowing expanded xNA to “write” more AA “words” in a protein “lexicon”. Consistent with an IMAT R21 format, this project will prove concepts, de-risk procedures, and take enough steps to guide the NCI as it seeks to complete this transformative technology. We will use only 8 "hachimoji" nucleotides to systematically add, in three Aims, these PTM-AAs while developing the concepts to control the interactions that must be controlled to meet this grand challenge: (i) between hachimoji codons and anticodons during translation, (ii) between synthetases and hachimoji anticodons during aminoacylation, and (iii) between orthogonal hachimoji charged tRNAs and parts of the E. coli ribosome complex. Even with this limited scope, the technology will be transformative because of the importance of these PTM-AAs. As Aims are met, our ability to meet Performance Measures will be shown by making Prohibitin 2 (7 exemplars of these 3 PTM-AAs) using enhanced IVT (eIVT) . As a long term deliverable, since 8-letter DNA can deliver 512 codons, all PTM AAs can be incorporated into proteins using this technology, a more transformative outcome. Next, as researchers advance IVT and move hachimoji DNA into living cells, even more transformative outcomes are possible in a long term vision.

High Quality Proteins with Multiple Post Translational Modifications Foundation for Applied Molecular Evolution Shuichi Hoshika NARRATIVE Central to much modern cancer research are proteomics and related epigenetic studies that require tools to distinguish proteins as they are actually present in cancer cells. Central to the development of cancer therapeutics are the proteins, as actually present in cancer cells, to screen drugs that target them. Unfortunately, such proteins are rarely composed of just the canonical 20 amino acids; they also usually include additional amino acids that influence their folding, behavior, and performance, both in cancer-driving molecular biological processes and in their interaction with anti-cancer drugs. Also unfortunately, proteins as they actually exist in cancer cells are difficult to obtain, and impossible to obtain in pure form. The proposed technology will make, rapidly and inexpensively, pure proteins in the forms that they are actually found in cancer cells. In doing so, it will revolutionize all of cancer proteomic research, speeding work to understand what cancer is, how it behaves, and how it can be stopped.