Ribosomal Synthesis of High-Density Peptide Microarrays & Modified mRNA Applications

Peptide microarrays are an important technology for high-throughput discovery and analysis of proteinprotein binding interactions in pharmaceutical and biomedical proteomics. Characterizing these binding interactions is critical for understanding biochemical pathways within cells, and cells signaling pathways and networks, because proteins are the principal mediators of these processes. Specific applications of peptide microarrays include epitope mapping, serum immune response monitoring, enzyme activity and substrate profiling, and ligand-binding screening for biomarkers, drug candidates and diagnostics. Peptide microarrays continue to present a major synthesis challenge due to the relative complexity of peptide synthesis chemistry. The objective of this project is to develop an alternative pathway to peptide arrays that bypasses chemical peptide synthesis, and instead adapts mRNA- display technology to use ribosomes for the in situ translation of high-density RNA microarrays into peptide microarrays. The method takes advantage of the speed and simplicity of nucleic acid synthesis and the efficiency of ribosomal translation. The RNA microarrays are synthesized photolithographically using RNA phosphoramidites with photolabile 5-NPPOC protecting groups. The RNA is synthesized as one side of a branched structure, with the second branch holding a puromycin molecule on a flexible linker. After automated synthesis, an in vitro translation system is applied to the microarray. In the absence of a stop codon, the ribosome reaches the 3 end of the RNA message and stalls at the branch, remaining attached until the incorporation of the puromycin at the C-terminus. The proposed experiments will develop several alternative puromycin attachment methods, direct incorporation onto the microarray during synthesis via a novel puromycin phosphoramidite, and post synthesis addition of the puromycin branch via both click chemistry and psoralen photo-cross-linking chemistry. In addition to efficient generation of peptide microarrays, the on-array translational process itself can be a useful tool. As a part of this project, we plan to use RNA microarray-based ribosomal translation system as a very high-throughput method to optimize modified mRNA for therapeutic applications such as protein substitution for the treatment of hereditary or acquired diseases, cell reprogramming, and for stimulating immune responses against cancer or infectious disease.

The goals of the project were to develop the chemistry and enzymatic processing necessary to enable the conversion of chemically synthesized RNA microarrays to peptide microarrays through the use of a cell-fee translation system. These cell-free translation systems are extracted from bacteria or eukaryotic cells and include all of the molecular machinery, particularly ribosomes, need for peptide synthesis, plus transfer RNA and other accessory molecules. The RNA microarray synthesis approach we use was developed over many years, including through a previous FWF project, but initiated at the University of Wisconsin and at McGill University. The RNA microarrays are synthesized using a photolithographic process inspired by the similar processes used in the semiconductor microchip industry, but modified to allow the efficient synthesis of very large libraries of biomolecules. Originally, this form of biological photolithography was developed for the synthesis of peptide arrays, but then transferred to DNA synthesis due to the more challenging chemistry associated with solid phase peptide synthesis. The adaptation of DNA microarray synthesis to RNA microarray synthesis chemistry was also very challenging, but results in the creation of RNA, which has many more roles in nature, including information storage, but also in molecular machines in the cell, ribosomes, that are responsible for the synthesis of peptides and proteins. The creation of RNA microarrays thus allows us to come full circle, back to the photolithographic synthesis of peptides, but this time via RNA in the form of synthetic arrays of messenger RNA that are translated to peptides on the surface by means of ex vivo translation systems. Since we have full chemical control over the synthesis, including the ability to add non-canonical nucleotides and other nucleic acid building blocks, including branching structures, that are potentially useful in conversion to peptide arrays and testing of hypotheses regarding ribosomal translatability of non-canonical or chemically-modified RNA analogues, as well at codon-specific translation efficiency, and effects of modification to the ribosome binding sequence that provides a landing site for the ribosome to attach to the messenger RNA. The importance of non-canonical or chemically-modified RNA analogues have become much more visible to the public as a result of the Corona pandemic. All of the successful RNA-based vaccines, as well as other RNA-based therapies currently being developed, for example, against cancer and other diseases, make use of RNA molecules that have been chemically modified to avoid immediate degradation when injected, but remain translatable into proteins by cellular ribosomes. Further development in therapeutic applications of RNA will very likely involve other modifications, both ones already occurring in nature and those engineered to make the therapies even more effective.