A photo-crosslinking platform for the construction of chemically modified and biofunctional RNA molecules

Thesis

Several key developments in medicine have been made by exploiting the varied biological roles of RNA, as highlighted by the recent applications of mRNA vaccines for SARS-CoV-2 and RNA-guided Cas9 gene editing. These technologies are further enhanced by advances in nucleic acid chemistry that allow for highly targeted and extensive modification of oligonucleotides to improve or alter their functions. Despite the new opportunities presented by RNA technologies, several key challenges remain in their application. These include limitations to the length of chemically synthesized oligonucleotides and a lack of adequate methods to control the spatial-temporal activity of oligonucleotide-based medicines. The work described in this thesis attempts to address these issues with carbazole-derived nucleosides which undergo site-specific reversible crosslinking when irradiated with UV light. Crosslinking nucleosides are first applied to construct Cas9 guide RNAs (gRNAs) from shorter starting RNAs. This approach provides a simple and cost-effective strategy to construct individual gRNAs or libraries and is demonstrated to be compatible with extensive chemical modification. Crosslinked gRNAs are functional both in vitro and in cells, with comparable activity to full-length control gRNAs. Several methods of conjugating large DNA cargos to gRNAs are then explored which are compatible with this crosslinking strategy. DNA cargos encoding expressible gene cassettes are applied to attempt to report the transcriptional status of target sites as directed by dCas9. Lastly, the reversible crosslinking of carbazole nucleosides is explored as a platform to control the translation of exogenous mRNAs in cells. Short carbazole-oligonucleotide blockers are applied to inhibit the translation of mRNA in a crosslinking-dependent and sequence-specific manner. Upon reversing the crosslink by UV irradiation, translation is initiated. The blockers are optimized to maximize the dynamic range of activation and are then applied to novel mRNA vectors that cannot be controlled with existing light-regulation strategies. The work of this thesis underscores the extensive versatility of carbazole nucleosides in the manipulation of synthetic RNAs.

Authors: Largey, B

• DOI: 10.5287/ora-2agnp1kvw
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: mRNA; nucleic acid chemistry; RNA; photochemistry; CRISPR Cas9
• Subjects: Chemical Biology