Synthesis of Sensitive Epitranscriptomically Modified RNAs

Michigan Technological University, Houghton MI

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Abstract

Project Summary/Abstract The objective of this project is to develop a solid phase technology that is universally useful for the synthesis of epitranscriptomically modified RNAs containing sensitive functional groups. Sensitive functional groups here refers to those that are unstable under the basic and nucleophilic conditions widely used for deprotection and cleavage in existing chemical RNA synthesis technologies. Many naturally occurring RNAs including tRNA, rRNA, mRNA, snRNA, snoRNA, miRNA and lncRNA contain such sensitive groups. They have important biological functions, and errors related to their installation, reading and erasing are associated with human diseases. To obtain the knowledge such as the mechanisms by which the sensitive groups modulate RNA biophysical properties and RNA-protein interactions, and the pathology of diseases involving sensitive groups, chemical synthesis of the sensitive RNAs are needed. Currently many such RNAs cannot be synthesized by any existing technologies. The objective of this project is to fill this technology gap. Using a new set of solid phase synthesis linkers and protecting groups, RNAs will be synthesized, cleaved and deprotected under mild conditions under which almost all sensitive groups found in living systems so far are stable, and thus the new technology will be capable to synthesize almost all naturally occurring sensitive RNAs. To illustrate the predicted broad impact of the sensitive RNA synthesis technology, model mRNAs containing the sensitive ac4C modification will be synthesized using the technology. Dysregulations of ac4C have been linked to many human diseases including leukemia, diabetes, obesity and neurodegenerative diseases. Recent studies found that ac4C in mRNA enhances protein synthesis, but the molecular mechanism is unknown. The ac4C-containing model mRNAs will be used to fill this knowledge gap. The new knowledge is expected to be useful in areas such as identification of new therapeutics targets for treating various human diseases, and evaluation of the potential of ac4C-containing RNAs as medicines. Beyond this specific example, with the access to a wide range of sensitive RNAs, many other biomedical projects such as the identification of proteins responsible for reading and erasing various RNA modifications, the use of RNAs containing sensitive modification as training samples for machine learning in nanopore single molecule RNA sequencing, and deciphering mechanisms by which sensitive groups regulate RNA degradation and protein synthesis, all of which are impossible or challenging to do at this time, will become possible for the biomedical research community.